the development of improved diagnostics for acute and...

Development of Improved Diagnostics for Acute and

Persistent Chlamydia trachomatis Infections

Trudi Anne Armitage

B. App. Sc. (Hons)

Submitted to Queensland University of Technology

in fulfilment of the requirements for the

degree of Doctor of Philosophy

August 2007

ABSTRACT

The asymptomatic nature of chlamydial infection renders the differential diagnosis

of acute and chronic infection difficult. An untreated Chlamydia trachomatis

infection can become chronic, result in disease sequelae such as salpingitis and

pelvic inflammatory disease (PID), and ultimately culminate in tubal occlusion and

infertility. Diagnostic tests for C. trachomatis such as nucleic acid amplification

testing (PCR), antigen detection and serological methods have variable performance

capabilities with respect to sensitivity, specificity and stage of infection. The use of

PCR as a diagnostic tool is somewhat limited, as specimen collection is routinely

sampled from the lower genital tract; hence, infections in the fallopian tube where

inflammatory damage is most significant, escape detection. Furthermore, PCR can

only detect selected Chlamydia DNA sequences from readily accessible sites of the

genital tract, and therefore cannot differentiate between acute and chronic infection.

Other serological assays aim to discriminate the various stages of C. trachomatis

infection through identification of key antigens. The efficacy of these assays

however is impeded due to cross-reactivity between chlamydial species and the

subsequent antibody response against the target antigen is not restricted to patients

with a specific stage of infection.

To identify antibody responses capable of differentiating various states of

chlamydial infection, samples were collected from both men and women given the

variability of immune responses between the two genders. Samples were assigned

to a patient group according to infection status and then probed against protein

extracts of HEp-2 cells infected with C. trachomatis serovar L2 and HEp-2 cells pre-

treated with IFN-γ and infected with C. trachomatis serovar L2. (persistence cell

culture) Serological analysis revealed the presence of five antigens (denoted bands

A, B, C, D and M) which were shown to be differential between patient groups.

Identification of bands B and C by N-terminal sequencing provided two possible

candidates for each antigen, ie. CT727 and CT396 (band B) and CT157 and CT423

(band C). In contrast, band M which was unique to males was a PmpB (probable

outer membrane protein B) fragment. The four target antigens (CT157, CT423,

CT727 and CT396) were expressed as recombinant proteins using autoinduction

media and were subsequently probed by both male and female sera to evaluate their

iii

diagnostic potential. Results showed that two chlamydial antigenic targets (CT157

and CT727) have the potential to discriminate between acute and chronic C.

trachomatis infection. However, since only a small number of samples (n = 3) were

used for this aspect of the study, the findings should simply be viewed as

preliminary. In females, sensitivity and specificity values were derived using

various combinations of the four target antigens into a panel format for the purpose

of detecting chronic C. trachomatis infections. The preferred format was B + C with

a sensitivity and specificity of 80% and 84% respectively. Using the IFN-γ-

mediated persistence model, only two of the five antigenic targets were shown to be

differentially expressed. PmpB in males and CT157 (the most likely band C

candidate) in females were shown to be up-regulated to varying degrees in samples

across the patient groups. We also demonstrated that no other chlamydial antigens

are up-regulated during a persistent C. trachomatis infection. In conclusion,

although combinations of bands A, B, C, D and M differentiate between male and

female patient groups under normal chlamydial growth conditions, during IFN-γ-

induced persistence, only bands C (CT157) and M (CT413 - PmpB) are up-regulated

thus suggesting a potential role in chronic C. trachomatis infection.

Keywords: Chlamydia trachomatis, pelvic inflammatory disease, chronic infection,

persistence, diagnostic test, autoinduction, differential banding, sensitivity,

specificity.

iii

TABLE OF CONTENTS

Page

TITLE PAGE i

ABSTRACT ii

TABLE OF CONTENTS iv

LIST OF FIGURES x

LIST OF TABLES xiii

LIST OF ABBREVIATIONS xvi

STATEMENT OF ORIGINAL AUTHORSHIP xviii

PUBLICATIONS AND PRESENTATIONS xix

ACKNOWLEDGEMENTS xx

CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW

1.0 Introduction 2

1.1 Taxonomy – Classification of Chlamydia 2

1.2 Chlamydia – The Pathogen 4

1.3 Human Genital C. trachomatis Infections and Spectrum of Disease 7

1.3.1 Pelvic Inflammatory Disease 7

1.3.1.1 Aetiology/Prevalence 7

1.3.1.2 Upper Genital Tract Infection 8

1.3.1.3 Inflammatory Response to Chlamydial Infection 10

1.3.1.4 Associated PID Sequelae 10

1.3.1.4.1 Infertility 10

1.3.1.4.2 Ectopic Pregnancy 12

1.3.1.4.3 Chronic Pelvic Pain 13

1.3.1.5 PID Risk Factors 14

1.3.1.6 Treatment 16

1.3.2 Cervicitis 19

1.3.3 Urethritis, Epididymitis and Sexually Reactive Arthritis 19

1.3.4 Lymphogranuloma Venereum 20

1.4 Chlamydial Immune Response and Pathogenesis 21

iv

1.4.1 Role of Heat Shock Proteins in the Pathogenesis of Chlamydial Disease 27

1.4.2 Role of Antibody to Chlamydial HSP60 27

1.4.3 Cell-mediated Responses to HSP 29

1.5 Hormonal Influences 30

1.6 Chlamydial Persistence 31

1.6.1 Nutrient Deficiency – Induced Persistence 32

1.6.2 Antibiotic-Induced Persistence 33

1.6.3 Cytokine-Induced Persistence 35

1.7 Chlamydial Diagnostics 37

1.8 Objectives and Aims of this Study 42

CHAPTER 2: FEMALE & MALE PATIENT RECRUITMENT

2.0 Introduction 48

2.1 Materials and Methods 52

2.2 Female Cohorts 54

2.3 Male Cohorts 60

CHAPTER 3: SEROLOGICAL IDENTIFICATION OF POTENTIAL

DIAGNOSTIC MARKERS IN FEMALES

3.0 Introduction 64


3.1.1 Strategy Used to Identify Novel Diagnostic Markers 67

3.1.2 Patient Groups Analysed 68

3.1.3 C. trachomatis Cell Culture 68

3.1.4 Identification of Reactive C. trachomatis Proteins in Patient Samples 68

3.1.5 Immunoprecipitation of Chlamydia-Specific Antibodies 69

3.1.6 SDS-PAGE/Western Blot of Immunoprecipitated Chlamydial Proteins 70

3.1.6.1 N-Terminal Sequence Analysis 70

3.1.7 Antigenic Target Identification and Verification via Mass

Spectrometry 70

v

3.1.8 Antibody Reactivity to Recombinant MOMP 72

3.1.9 Species and Serovar Specificity of the Identified Novel Markers 72

3.1.10 Diagnostic Potential of Antigenic Targets 72

3.2 Results 74

3.2.1 Immunoreactivity Patterns of Females with C. trachomatis Genital

Infections 74

3.2.2 Identification and Verification of Chlamydial Antigenic Targets 78


3.2.4 Species and Serovar Specificity of Target Antigens 83

3.2.5 Diagnostic Potential of the Four Newly Identified Antigens 90

3.2.6 Antibody Response of Identified Antigens during Different Stages of

C. trachomatis Infection 92

3.3 Discussion 94

CHAPTER 4: OPTIMISATION OF EXPRESSION OF ANTIBODY-REACTIVE

CANDIDATE PROTEINS

4.0 Introduction 102


4.1.1 Pilot Study using IPTG-induction for Protein Expression 106

4.1.2 A Comparison of Expression of Candidate Proteins by IPTG and

Autoinduction 109

4.1.2.1 IPTG Induction of Target Proteins 110

4.1.2.2 Autoinduction of Target Proteins 110

4.1.3 Fractionation of Proteins Expressed via IPTG or Autoinduction 108

4.1.4 SDS-PAGE Determination of Protein Solubility of Each Candidate

Protein 111

4.1.5 Level of Protein Expression in Autoinduced Cultures 111

4.1.6 Densitometric Evaluation of Autoinduced and IPTG-Induced Protein

Expression and Solubility 112

4.1.7 Rare Codon Usage Determination of Each Candidate Protein 112

vi

4.1.8 Preliminary Determination of Antibody Reactivity of Newly

Expressed Recombinant Proteins 112

4.2 Results 113

4.2.1 Induction Method and Temperature Comparison of Candidate Protein

Expression 113

4.2.2 Verification of Protein Expression using Anti-GST 117

4.2.2.1 Codon Usage and Expression of Candidate Proteins 119

4.2.3 Antigenicity of Recombinant Proteins for the Potential Diagnosis of

Infection by C. trachomatis 122

4.3 Discussion 124

CHAPTER 5: THE DIAGNOSTIC POTENTIAL OF NOVEL ANTIGENS IN FEMALE SAMPLES




5.1.2 Production of Recombinant Chlamydial Proteins 133

5.1.3 Evaluation of Recombinant Proteins Diagnostic Potential by Western

Blotting 133

5.1.3.1 Banding Intensity Grading System for Recombinant Protein

Western Blots 134

5.2 Results 135

5.2.1 Evaluation of Candidate Antigens Diagnostic Potential via Western

Blotting with Patient Samples 135

5.2.2 Antibody Response of Recombinant Antigens during Stages of C.

trachomatis Infection 143

5.3 Discussion 145

CHAPTER 6: VARIATION IN SEROLOGICAL RESPONSES TO C. TRACHOMATIS INFECTION IN MALES AND FEMALES



vii

6.1.1 Patients Groups Analysed for Initial Western Blot Screen 155


6.1.3 Identification of Immunoreactive C. trachomatis Proteins by Western

Blotting 155

6.1.4 Antigen Identification via N-terminal Sequencing 155

6.1.4.1 N-terminal Sequence Analysis 156

6.1.4.2 Isolation of Untrypsinised Proteins from Cell Culture 156


6.1.6 Species and Serovar Specificity of the Identified Novel Marker 157

6.1.7 Production of Recombinant Chlamydial Proteins 157

6.1.8 Western Blotting of Recombinant Chlamydial Proteins with Patient

Samples 157


Western Blots 158

6.2 Results 159

6.2.1 Immunoreactivity Patterns of Males with C. trachomatis Genital

Infections 159

6.2.2 Identification of Novel Marker via N-terminal Sequencing 162

6.2.2.1 Confirmation of Novel Protein Identity 164


6.2.4 Species and Serovar Specificity of Novel Male Marker 166

6.2.5 Blotting of Recombinant Proteins with Patient Samples to Confirm the

Identity of the Immunoreactive Target Antigens 170

6.2.6 A Comparison of the Temporal Response of Recombinant Antigens

during Stages of C. trachomatis Infection in Males and Females 176

6.3 Discussion 179

CHAPTER 7: CHLAMYDIAL PERSISTENCE MARKERS IN SAMPLES OF MALES AND FEMALES



viii



7.1.3 IFN-γ Persistence Cell Culture 190

7.1.4 Identification of Reactive C. trachomatis Proteins in Patient Samples

by Western Blotting 190

7.2 Results 191

7.2.1 Identification of Antigens Differentially Expressed in the IFN-γ

Persistence Model in Males and Females 191

7.2.1.1. Female Patient Samples 191

7.2.1.2 Male Patient Samples 194

7.3 Discussion 197

CHAPTER 8: GENERAL DISCUSSION

8.0 General Discussion 202

CHAPTER 9: LITERATURE CITED

References 211

ix

LIST OF FIGURES Page Figure 1.1. Old and new taxonomy the Chlamydiales. 3

Figure 1.2. Developmental cycle of Chlamydia. 5

Figure 1.3. Ventro-dorsal schematic of the female reproductive organs and

constituent structures. 8

Figure 1.4. Innate and adaptive immunity resultant of acute chlamydial

infection. 22

Figure 1.5. Primary and secondary humoral immune responses. 23

Figure 1.6. The inflammatory response during infection. 26

Figure 3.1. Western blots of screened patient samples. 75

Figure 3.2. Prediction of trypsin cleavage sites in proteins CT157, CT423 and

CT727. 81

Figure 3.3. Western blot of uninfected cells, infected cells and recombinant

MOMP probed with a single patient sample. 82

Figure 3.4. Species and serovar comparison of the four antigenic targets. 84

Figure 3.5. Protein alignments of C. trachomatis serovars D and L2, and

C. pneumoniae for target antigens (a) CT157 and (b) CT423. 87

Figure 3.6. Protein alignment of C. trachomatis serovars D and L2, and

C. pneumoniae for target antigen CT727. 88

Figure 3.7. Protein alignment of C. trachomatis serovars D and L2, and C.

pneumoniae for target antigen CT396. 89

Figure 3.8. Comparison of female antibody responses to Bands A, B, C and D

for C. trachomatis-infected patient groups (I – IV). 92

Figure 4.1. Comparison of protein expression by IPTG or autoinduction. 114

Figure 4.2. Verification of target protein expression by western blot using

Anti-GST. 118

Figure 4.3. Western blot of recombinant proteins probed with a patient sample. 123

x

Figure 5.1. Reactivity of recombinant candidate proteins to patient samples –

part A 137

Figure 5.2. Reactivity of recombinant candidate proteins to patient samples –

part B 138

Figure 5.3. Reactivity of samples from negative control groups to the

recombinant candidate proteins. 139

Figure 5.4. Banding intensity grading scale for each of the four recombinant

proteins. 139

Figure 5.5. Band intensity levels for recombinant proteins CT157 and CT423. 141

Figure 5.6. Band intensity levels for recombinant proteins CT727 and CT396. 142

Figure 5.7. Antibody responses to the four recombinant antigens. 144

Figure 6.1. Western blots of male samples probed against uninfected and

infected whole cell extracts. 160

Figure 6.2. Western blot and Coomassie-stained gel comparison. 163

Figure 6.3. Confirmation of novel band M identity by western blot. 165

Figure 6.4. Comparison of size of recombinant MOMP and band C. 166

Figure 6.5. Species and serovar comparison of the novel 19kDa marker

between patient groups MI and MII. 167

Figure 6.6. Protein sequence alignments of C. trachomatis serovars L2 and D,

and C. pneumoniae 19kDa PmpB fragment. 170

Figure 6.7. Western blots of recombinant proteins probed with samples from

male patients. 172

Figure 6.8. System used to grade intensity of bands on western blots probed

with male samples. 173

Figure 6.9. Band intensity levels for patient samples from groups MI –MIII

when probed against recombinant proteins CT157 and CT423. 174

Figure 6.10. Band intensity levels for patient samples from groups MI –MIII

when probed against recombinant proteins CT727 and CT396. 175

xi

Figure 6.11. Male antibody responses to CT157 and CT423 in comparison

with females in the same defined patient groups. 177

Figure 6.12. Male antibody responses to CT727 and CT396 in comparison

with females in the same defined patient groups. 178

Figure 7.1. Western blots showing immunoreactivity of female patient samples

to proteins from IFN-γ-induced persistence cell culture. 192

Figure 7.2. Western blots showing immunoreactivity of male patient samples

to proteins from IFN-γ-induced persistence cell culture. 195

xii

LIST OF TABLES Table 1.1. An overview of the four Chlamydial species. 6

Table 1.2. Treatment guidelines for parenteral antibiotic therapy for acute PID. 17

Table 1.3. Treatment guidelines for oral antibiotic therapy for acute PID. 18

Table 1.4. An overview of various diagnostic methods associated with

C. trachomatis detection. 39

Table 2.1. Female groups I and II showing clinical history, C. trachomatis

infection and C. pneumoniae IgG, and CHSP60 for each sample. 56

Table 2.2. Female groups III and IV showing clinical history, C. trachomatis


Table 2.3. Female infertile control group V showing clinical history and results

for C. trachomatis and C. pneumoniae IgG and HSP60 for each sample. 58

Table 2.4. Female control groups VI and VII clinical history and results for C.

trachomatis and C. pneumoniae IgG, and CHSP60 for each sample. 59

Table 2.5. Male groups MI and MII showing clinical history, C. trachomatis


Table 2.6. Male control groups III and showing clinical history, C. trachomatis


Table 3.1. Individual patient profiles of differential bands A, B, C and D for

groups I - IV. 76

Table 3.2. Individual patient profiles of differential bands A, B, C and D for

groups V – VII. 77

Table 3.3. The percentage prevalence and estimated MW bands designated

A, B, C and D. 78

Table 3.4. Major and minor amino acid sequences for each sample obtained via

N-terminal sequencing. 79

Table 3.5. Identification of bands A, B and C by N-terminal sequencing or

or mass spectrometry. 80

xiii

Table 3.6. Serovar and species specificity for patient samples from groups I, II and

IV C. trachomatis serovars L2, D and K, and C. pneumoniae. 85

Table 3.7. Assessment of the diagnostic potential of different panel formats for the

diagnosis of acute and chronic chlamydial infection. 91

Table 4.1. Molecular weights of target genes with and without the GST fusion tag. 106

Table 4.2. Evaluation of autoinduction and IPTG expression levels for each

candidate protein. 115

Table 4.3. Summary of optimised conditions of induction and the levels of

expression and solubility. 116

Table 4.4. The number of rare codons in the genes encoding the candidate

proteins 120

Table 4.5. Molecular weights of observed and predicted truncated fragments of

the candidate proteins. 121

Table 5.1. Reactivity of individual patient samples against the recombinant

candidate proteins. 140

Table 6.1. Individual patient sample profiles of differential bands A, B, C, D and

M on western blots. 161

Table 6.2. The percentage prevalence of all differential bands A, B, C, D and M. 162

Table 6.3. NCBI BLASTP alignments of the most significant N-terminal

sequence matches to amino acid residues. 163

Table 6.4. In silico tryptic digest of the entire PmpB protein. 164

Table 6.5. Serovar and species specificity and serology for sera probed against

C. trachomatis serovars L2, D and K, and C. pneumoniae. 168

Table 6.6. Species and serovar specificity of band M in infected male samples. 169

Table 6.7. Banding intensities of recombinant proteins when probed by samples

from groups MI, MII and MIII. 173

xiv

Table 7.1. Individual patient profiles of differential bands A, B, C and D after

sample probe against IFN-γ-mediated persistence culture. 193

Table 7.2. Individual patient profiles of differential bands A, B, C, D and M after

sample probe against IFN-γ-mediated persistence culture. 196

xv

LIST OF ABBREVIATIONS

CMI Cell-mediated immunity

CI Confidence interval

DFA Direct fluorescent antibody

DNA Deoxyribose nucleic acid

EB Elementary body

EIA Enzyme immunoassay

ELISA Enzyme-linked immunosorbent assay

GM-CSF Granulocyte-macrophage colony-stimulating factor

GRO-α Growth oncogene-α

GST Glutathionine S-transferase

HSP60 Heat shock protein 60

IDO Indoleamine 2,3-dioxygenase

IFN-γ Interferon gamma

IL-1α Interleukin 1α

IL-2 Interleukin 2

IL-6 Interleukin 6

IL-8 Interleukin 8

IPTG Isopropyl - β - D thiogalactoside

LGV Lymphogranuloma venereum

LPS Lipopolysaccharide

MIF Microimmunofluorescence

MOMP Major outer membrane protein

xvi

MS Mass spectrometry

NAAT Nucleic acid amplification testing

NGU Non-gonococcal urethritis

NK Natural killer

NPV Negative predictive value

OCP Oral contraceptive pill

OR Odds ratio

PID Pelvic inflammatory disease

PmpB Polymorphic outer membrane protein B

PPV Positive predictive value

RB Reticulate body

RNA Ribose nucleic acid

SARA Sexually-acquired reactive arthritis

SF Synovial fluid

TFI Tubal factor infertility

TNF-α Tumour necrosis factor α

xvii

STATEMENT OF ORIGINAL AUTHORSHIP

The work contained in this thesis has not been previously submitted for a degree or

diploma at any other higher education institution. To the best of my knowledge and

belief, this thesis contains no material previously published or written by another person

except where due reference is made.

Trudi Anne Armitage

xviii

PUBLICATIONS AND PRESENTATIONS

REFEREED CONFERENCE ABSTRACTS

Armitage T, Macnaughton T, Debattista J, Walsh T and Timms P. (2006) Identification

of novel markers for acute and chronic Chlamydia trachomatis infections. In: The

Eleventh International Symposium on Human Chlamydial Infections. p603-606.

PATENTS

Armitage T, Macnaughton T, Timms P, Walsh T (2006) Diagnostic methods and kits

useful for same. Full patent lodged on 13th September, 2006, PCT Au 20060001346.

PRESENTATIONS

CRC for Diagnostics Annual Retreat, Caloundra (November, 2004).

Brisbane International Chlamydia Congress (July, 2005).

CRC for Diagnostics Annual Retreat, Melbourne (November, 2005).

xix

ACKNOWLEDGEMENTS

My sincerest thanks go to my principal supervisor, Professor Peter Timms for his

wisdom, support and guidance throughout my PhD. Thank you also to my associate

supervisors Dr. Tom Macnaughton, Associate Professor Terry Walsh and Dr. Joe

Debattista. Tommie, what would I have done without you?!! Your infinite wisdom and

guidance has been invaluable and I sincerely thank you. Terry, your input over the

course of my project has been invaluable, so have the coffees! To Joe, your continuing

enthusiasm and support of me and this project has been sincerely appreciated.

My sincerest thanks to the Wesley Hospital, Brisbane Sexual Health Clinic, Red Cross

Blood Bank, Queensland Medical Laboratory and the Brisbane Family Planning Clinic

for all your time and efforts in patient sample collection over the course of this project.

Many thanks to the CRC for Diagnostics, in particular Dr. Ron Epping and Associate

Professor Phil Morris for their support and faith in my abilities. Also a big thank you to

the CRC and Faculty of Science for my scholarship.

Thank you to the staff and students in the School of Life Sciences and to members of

the Chlamydia Group for making my PhD journey a little easier.

To Beck (Rebecca Byrnes) and Claire-Bear (Claire Sullivan), we’ve come a long way

since QIMR days! All my love, hugs, kisses and big thank you’s for your continued

love and support and copious amounts of red wine. All of which have helped to keep

me sane!

To Toney Toney Bo Boney. I’m glad we’ve travelled this road together. It wouldn’t

have been the same without you.

xx

To my ma, Lesley Miller. What ever could I say that would even come close to telling

you just how much I have appreciated your love and support all these years, except to

say I love you ma and thank you.

Finally, to the love of my life, Chris Collet, your endless support and love has given me

such strength as I have journeyed along this path. I love you and thank you.

xxi

Chapter 1: Literature Review

- 1 -

CHAPTER 1

Introduction and Literature Review


- 2 -

1.0 INTRODUCTION

The order Chlamydiales is comprised of several closely-related pathogenic species

(Schachter, 1999) which are distinctive prokaryotic organisms that diverged

approximately 2 billion years ago from other groups within the domain Bacteria

(Weisburg et al., 1986; Everett et al., 1999). Common to chlamydiae is their unique

developmental cycle and innate ability to infect various avian, marsupial and

mammalian species. Furthermore, as obligate intracellular bacteria, chlamydiae are

amongst the most prosperous bacterial pathogens and are associated with a number of

chronic human diseases (Paavonen and Eggert-Kruse, 1999; Schachter, 1999; Bas et al.,

2001; Chen et al., 2006). The World Health Organisation estimates 90 million

chlamydial infections worldwide are detected annually (Gerbase et al., 1998).

Approximately 55% of infections are a consequence of Chlamydia trachomatis

(Wagenlehner et al., 2006) which is recognised as the most frequent sexually

transmitted bacteria in humans (Schachter et al., 1975; Groseclose et al., 1996). C.

trachomatis infections of the genital tract often result in urethritis, cervicitis and

sequelae which include pelvic inflammatory disease (PID), ectopic pregnancy, tubal

occlusion, chronic pelvic pain, epididymitis and reactive arthritis (Schachter, 1999).

The general asymptomatic nature of C. trachomatis infection in men and women

renders discrimination between acute and chronic infection difficult. Research has

focused on the biology of the organism, chlamydia-host cell interactions and immune

responses, chronic sequelae such as PID, hormonal influences and diagnostic

advancement. As such, this review focuses on these vital areas which are relevant to

the proceeding research.

1.1 TAXONOMY – CLASSIFICATION OF CHLAMYDIA

The Chlamydia genus was recognised in 1966 and subdivided into two species,

Chlamydia trachomatis and Chlamydia psittaci (Moulder, 1966). Chlamydia

pneumoniae and Chlamydia pecorum, formerly recognised as strains of C. psittaci, were

nominated as distinct species in 1989 and 1992 respectively (Grayston et al., 1989;


- 3 -

Fukushi and Hirai, 1992). In 1999, a novel taxonomy was proposed that increases the

number of species in the Chlamydiaceae family from four to nine and classifies them

into two genera (Everett et al., 1999) (Figure 1.1). This review will not utilise the

amended taxonomy as controversy regarding this issue has not been resolved.

OLD TAXONOMY NEW TAXONOMY

Order: Chlamydiales

Family: Chlamydiaceae

Genus: Chlamydia Chlamydia Chlamydophila

Species: C. trachomatis C. trachomatis C. pneumoniae

C. pneumoniae C. muridarum C. psittaci

C. psittaci C. suis C. pecorum

C. pecorum C. caviae

C. abortus

C. felis

Figure 1.1. Old and new taxonomy the Chlamydiales (modified from Everett et al.,

1999).


- 4 -

1.2 CHLAMYDIA – THE PATHOGEN

Chlamydiae are obligate intracellular organisms that undergo a biphasic developmental

cycle within a eukaryotic host cell (Byrne, 1978). Subsequent to internalisation,

chlamydiae develop inside the intracellular vacuole or inclusion, where the infectious

and metabolically-inert elementary bodies (EBs) differentiate into the metabolically-

active replicative form, or reticulate body (RB). Differentiation occurs in the first few

hours following primary infection with the RBs replicating by binary fission.

Approximately 20 hours post-infection, increasing numbers of RBs convert back into

EBs (Zhang and Stephens, 1992). The main mechanism is via lysis (Abdelrahman and

Belland, 2005) although a second mechanism involving exocytosis (Kiseley et al.,

2007) has also recently been described (Figure 1.2a). Chlamydiae are prokaryotic

Gram-negative bacteria. They possess a trilaminar outer membrane containing

lipopolysaccharides (LPS) and several membrane proteins, yet lack peptidoglycan, a

common cell membrane component inherent in related bacterial species and one that

provides structural integrity and osmotic stability (Hatch, 1996). Instead, the

extracellular chlamydial form, the EB has extensive disulfide cross-linking between

cysteine residues within and surrounding the outer membrane proteins (Newhall et al.,

1983; Hatch et al., 1986). The intracellular fate of a chlamydial infection is reliant upon

three factors: (i) the infecting biovar or species, (ii) the target cell type, and (iii) the

mechanism by which the biovar enters the host cell (ie. receptor-mediated endocytosis

or microfilament-dependent phagocytosis) (Prain and Pearce, 1989).

Three of the four chlamydial species have been linked with various human diseases

(Table 1.1). The most important diseases caused by chlamydiae in humans are

trachoma, PID, epididymitis and reactive arthritis (C. trachomatis), upper respiratory

tract infections and pneumonia (C. pneumoniae), and psittacosis due to C. psittaci

(Schachter, 1988; Sheehy et al., 1996). In animals, C. psittaci and C. pecorum give rise

to a wide variety of conditions including pneumonia, enteritis, polyarthritis,

encephalomyelitis, conjunctivitis and spontaneous abortion (Storz, 1988).


- 5 -

Figure 1.2. Developmental cycle of Chlamydia. (a) Chlamydial infection is initiated

by attachment of the infectious EB to a suitable host cell. Once internalised within the

inclusion, EBs transform into RBs and become metabolically active. Differentiation

continues until chlamydial numbers within the eukaryotic cell increase, exocytosis or

cell lysis is initiated, thus releasing further infectious EBs into the host. (b) Persistence

is instigated following host secretion or addition of chlamydial growth inhibitory factors

e.g. interferon gamma (IFN-γ) which induce non-replicating inclusions that contain

reticulate-like aberrant bodies. Continuation of the chlamydial developmental cycle is

achieved upon removal of the inhibitory factor, therefore enabling the resumption,

growth and further release of infectious EBs within the eukaryotic host (adapted from

Rottenberg et al., 2002).

EB

Nucleus

RB

Inclusion EB attachment and

invasion (endocytosis) Primary differentiation

EB to RB

+ IFN-γ

- IFN-γ

Aberrant bodies

Growth (binary fission)

Secondary differentiation RB to EB

Exocytosis or host cell lysis

a. Normal Replication Cycle b. Persistence


- 6 -

Table 1.1. An overview of the four Chlamydial species, their respective biovars and associated diseases.

SPECIES BIOVARS METHOD OF INFECTION NATURAL HOST ASSOCIATED HUMAN DISEASES

C. trachomatis

LGV (L1, L2, L3) Trachoma (A, B, Ba, C) (B, Ba, D-K) Mouse pneumonitis

Sexual transmission Hand to eye fomites, flies Sexual transmission, neonatal Aerosol

Human Human Human Mouse

Lymphogranuloma venereum Ocular trachoma PID, urethritis, epididymitis, reactive arthritis, infant pneumonia None

C. pneumoniae TWAR

Aerosol

Human

Bronchitis, pneumonia, coronary artery disease (?) and asthma

C. psittaci Many

Aerosol, sexual transmission (in animal hosts)

Avian, sheep, cats

Pneumonia, endocarditis, abortions

C. pecorum Bo/E8, BE, H3, DP

Sexual transmission

Bovine, koala, sheep, swine, goats

None


- 7 -

1.3 HUMAN GENITAL C. TRACHOMATIS INFECTIONS AND SPECTRUM

OF DISEASE

1.3.1 Pelvic Inflammatory Disease

PID results from persistent and/or multiple C. trachomatis infections in the upper

genital tract of women. Moreover, it is attributed to the ascension of micro-organisms

from the vagina and endocervix (lower genital tract) to the endometrium, fallopian

tubes or contiguous structures (Figure 1.3). PID is considered a principal cause of

chronic pelvic pain, ectopic pregnancy, tubal occlusion and infertility (Sweet, 1987;

Groseclose et al., 1996). Although the terms acute PID and acute salpingitis (fallopian

tube inflammation) are often interchanged, salpingitis is the most important element of

the PID spectrum. By definition, PID is an acute infectious process, whereas chronic

refers to the sequelae of the acute process such as adhesions, scarring and tubal

occlusion leading to infertility. PID is one of the most frequent infections observed in

nulliparous reproductive aged women (Black, 1997).

1.3.1.1 Aetiology/Prevalence

The aetiology of PID is polymicrobial in nature and various organisms have been

recovered from the upper genital tract of women with acute PID (Westrom, 1980). Such

organisms include C. trachomatis, Neisseria gonorrhoeae, genital Mycoplasma,

anaerobic and aerobic bacteria from the endogenous vaginal flora, and aerobic

Streptococci. Although these organisms were found in the urogenital tract of PID-

affected women, studies have indicated C. trachomatis to be the prime source of the

disease (Mardh et al., 1977; Sweet, 1987; Rice and Schachter, 1991). The prospect of

C. trachomatis-infected women developing PID ranges from 10 - 40% (Simms and

Stephenson, 2000). Analyses of PID estimates undertaken between 1985 and 2001 by

the National Center for Health Statistics, the National Hospital Discharge Survey and

the National Ambulatory Medical Care Survey showed a 68% decline in hospitalised

PID patients in the United States (Sutton et al., 2005). Given the latent nature of C.


- 8 -

trachomatis infections and, as a consequence, the difficulties of diagnosing the exact

number of women suffering from PID may not truly be exemplified by these figures.

Figure 1.3. Ventro-dorsal schematic of the female reproductive organs and constituent

structures. The lower genital tract is comprised of the vagina and cervix, whilst the

upper genital tract consists of the remaining reproductive structures (adapted from

www.mydr.com.au).

1.3.1.2 Upper Genital Tract Infection

The endocervical canal and cervical mucous plug serve as the major barriers providing

protection for the upper genital tract against organisms present in the vagina and cervix

Endometrium

Upper genital tract

Lower genital tract

Fallopian tube Body of uterus Fundus

Ovary Fimbriae

Myometrium

Cervix

Vagina


- 9 -

(Rice and Schachter, 1991). Repeated infection of the cervix with C. trachomatis may

result in damage to either of these structures, allowing the organism to infiltrate the

upper genital tract. Hormonally-driven changes in cervical mucous and uterine

contractility may facilitate C. trachomatis ascension and subsequent upper genital tract

infection. Rice and Schachter (1991) proposed that the mechanism of retrograde

menstruation could propel infectious micro-organisms from the endometrial cavity into

the fallopian tubes leading to partial or total tubal blockage, and ultimately tubal

infertility. Retrograde menstruation is well established as a possible method by which

endometrial debris can be disseminated into the pelvic cavity (Halme et al., 1984;

Salamanca and Beltran, 1995). Retrograde uterine contractions were shown in three

separate studies to be common during various phases of the menstrual cycle in non-

pregnant women and those suffering from endometriosis (Bulletti et al., 2000; 2001;

2002). Furthermore, the frequency of contractions increased the degree of retrograde

uterine bleeding (Bulletti et al., 2002). In a recent study investigating the functionality

and physiology of transport in the female reproductive tract, Zervomanolakis et al.

(2007) showed the uterus and fallopian tubes function synergistically as a peristaltic

pump to provide the necessary pressure gradient for spermatozoa transport from the

lower to upper genital tract. Since both retrograde menstruation and retrograde uterine

contractions are common amongst women and the innate action of the uterus is one of

upward transport, it is not unreasonable to conclude that the ascension of genital C.

trachomatis infections can be further assisted by the inherent nature of the female

reproductive tract.

Another mechanism by which C. trachomatis infection can proliferate throughout the

female genital tract is via human sperm. Toth et al. (1982) proposed that C.

trachomatis attaches to human spermatozoa which may then act as a vector for the

transference of micro-organisms, including C. trachomatis into the upper genital tract.

The attachment of C. trachomatis to sperm, however, was not demonstrated. In support

of this hypothesis, Knox et al. (2003) showed that the bacteria Ureaplasma remained

adhered to the surface of spermatozoa despite triplicate washings prior to assisted

reproductive techniques. The latter study provides strong evidence that sperm can


- 10 -

indeed function as transporters for various micro-organisms, thus gaining greater access

to the female upper genital tract.

1.3.1.3 Inflammatory Response to Chlamydial Infection

Activation of both cell-mediated and humoral immune responses occurs upon

chlamydial infection. Although the host immune system protects against infection by

means of rapid and efficient removal of bacteria, resultant inflammatory responses may

lead to tissue damage or persistent infection (Pal et al., 1998; Darville et al., 2003).

Development of an active cell-mediated immune response is demonstrated by the

proliferative response ratio of peripheral blood lymphocytes to chlamydial EBs. In

association with the inflammatory process, there is concurrent tissue repair comprising

the removal of dead cells and the ingrowth of fibroblasts leading to scar formation and

impairment of fallopian tube function (Rice et al., 1992). All three tubal layers,

interstitial, muscular and serosal, may be implicated in scar formation. If the serosal

layer is involved in the chlamydial infection, an inflammatory exudate presents on the

peritoneal surface adhering adjacent surfaces together. As a result of the inflammatory

response, deciliation of tubal epithelium, intraluminal adhesions, tubal occlusion and

peritubal adhesions occur (Sweet, 1987). These anatomical changes lead to infertility

(Sweet, 1991), ectopic pregnancy (Brunham et al., 1992) and possible spontaneous

abortion (Oakeshott et al., 2002).

1.3.1.4 Associated PID Sequelae

1.3.1.4.1 Infertility

One of the most important and common long-term complications of acute PID is tubal

factor infertility (TFI) (Westrom, 1980; Westrom et al., 1992; Patton et al., 1994).

Westrom et al. (1992) conducted a study on a large cohort of 2501 women treated for

suspected PID between 1960 and 1984 at the University Hospital in Lund, Sweden.

Laparoscopic investigation revealed 1844 women had acute PID, 657 had no sign of the


- 11 -

disease (control group), and the reproductive events of 1732 PID patients and 601

controls were followed. There were 1309 (75.6%) cases and 451 (75.0%) controls that

attempted to fall pregnant during the follow-up period. Of those monitored, 16% (209)

cases and 2.7% (12) of control patients failed to conceive. Confirmed TFI was

established in 141 patients with PID and nil found in the control group. The rate of

infertility was directly associated with the number and severity of PID infections.

Moreover, every subsequent episode of PID roughly doubled the rate of TFI, increasing

from 8% with one C. trachomatis infection, to 19.5% with two exposures resulting in

infection and 40% with three or more episodes. Interestingly, whilst all PID cases were

laparoscopically confirmed, only symptomatic patients were included in the study and

hence the incidence rates presented by Westrom et al. (1992) may not be a realistic

representation of the overall frequency of TFI in women suffering the disease. An

investigation of tubal infertility rates as a consequence of C. trachomatis or N.

gonorrhoeae–induced PID conducted by the World Health Organisation showed C.

trachomatis-induced PID was associated with a higher incidence of tubal infertility

compared to PID contracted from N. gonorrhoeae (World Health Organisation, 1995).

To evaluate the incidence of infertility subsequent to PID and factors associated with

tubal damage, Pavletic et al. (1999) obtained long term follow-up data from women

with previously documented acute PID. Of the 58 women included in the study, results

demonstrated a high rate of post-infection infertility. Furthermore, infertile women

were older (P = 0.02), more likely to have partial or total tubal occlusion (P = 0.03),

peritubal adhesions (P = 0.007) or perihepatic adhesions (P = 0.02) as evidenced by

laparoscopy.

Numerous studies have demonstrated serological evidence of prior C. trachomatis

infection is associated with TFI (Gijsen et al., 2002; Veenemans and van der Linden,

2002; den Hartog et al., 2005, 2006; Tiitinen et al., 2006). In a recent study evaluating

C. trachomatis heat shock protein 60 (cHSP60) – specific antibody and cell-mediated

responses as a predictor for TFI, Tiitinen et al. (2006) demonstrated C. trachomatis-

specific IgG antibodies were more common in TFI patients (43.2%) than in the control


- 12 -

group (13.5%). Furthermore, cHSP60-induced lymphocyte responses were shown to be

45.5% and 30.7% in the TFI and control groups respectively.

1.3.1.4.2 Ectopic Pregnancy

Post-PID damage to the fallopian tube is an established cause of ectopic pregnancy

(Westrom, 1991). In a group of eight women who had suffered from ectopic

pregnancy, initial laparoscopic investigation revealed no visible pelvic inflammation or

disease (Cumming et al., 1988). Upon removal of the fallopian tube (salpingectomy)

however, histological examination showed evidence of ongoing, low-grade post-

inflammatory endosalpingeal disorganisation (salpingitis) in areas away from the

ectopic pregnancy site. These findings suggest that surgical diagnosis does not exclude

microscopic fallopian tube inflammation or infection in women where a disease-free

pelvis has previously been established. Westrom et al. (1992) were able to demonstrate

that ectopic pregnancy occurred in the first pregnancy after the index laparoscopy in

9.1% of PID cases, compared to 1.4% of control patients (p<0.0001). In a large British

contingent of 1355 cases and 10507 controls, Buchan et al. (1993) reported that women

diagnosed with PID had a 10-fold increased risk of being admitted to hospital with an

ectopic pregnancy when compared to the controls. These results are comparable to

those demonstrated by Westrom et al. (1992). In another study, the presence and

metabolic status of C. trachomatis in the fallopian tubes of women presenting with

ectopic pregnancy was examined by polymerase chain reaction (PCR) and reverse

transcription-PCR (Gerard et al., 1998). Interestingly, of the 10 women tested, 70%

were positive for C. trachomatis DNA with viable and metabolically active Chlamydia.

In addition, all but one of the positive tissue samples showed moderate to severe

fallopian tube inflammation when subjected to histological analysis. Similarly, Barlow

et al. (2001) using PCR, in situ hybridisation and Southern blotting, demonstrated the

presence of chlamydial DNA in 56% and 67% of patients presenting with ectopic

pregnancy from the United Kingdom and Trinidad respectively. Moreover, the

detection of C. trachomatis DNA in the control groups from India and the United

Kingdom was a combined total of 32% by one or more of the methods employed.


- 13 -

Unlike Barlow et al. (2001) whose total study population was 97 (women presenting

with ectopic pregnancy n = 33; women undergoing surgery for TFI n = 14; negative

control patients n = 50), the small number of specimens (n = 10) collected by Gerard et

al. (1998) and lack of negative control samples severely limits the efficacy of the study.

Epidemiological analysis of ectopic pregnancy over a 28 year period (1970 - 1997) in

Sweden demonstrated a >2 fold increase of ectopic pregnancy, with the greatest

incidence in women ≥ 25 years of age (Kamwendo et al., 2000). Furthermore, a

reduction in the frequency of PID was strongly associated with a decline of ectopic

pregnancy.

1.3.1.4.3 Chronic Pelvic Pain

Although chronic pelvic pain is a common consequence of chronic chlamydial

infection, frequency of occurrence and severity of acute PID has yet to be fully

explored. The cause of chronic pelvic pain is directly linked to the presence of pelvic

adhesions that form subsequent to the inflammatory response of acute PID. In the large

Swedish cohort studied by Westrom et al. (1992), it was observed that chronic pelvic

pain lasting longer than six months was present in 18% of patients surgically confirmed

to have PID compared with 4% of controls. A comparable study by Safrin et al. (1992)

concluded that 24% of cases had chronic pelvic pain longer than six months following

primary hospitalisation. Evidence suggests that the severity and number of PID

episodes are directly proportional to the incidence of chronic pelvic pain witnessed in

PID-affected women (Westrom et al., 1992). In a longitudinal study to assess the risk

profile for chronic pelvic pain subsequent to PID, multivariate logistic regression was

employed to analyse the data obtained from 780 predominately black women with

clinically suspected PID (Haggerty et al., 2005). The authors concluded being married

(odds ratio (OR), 2.17; 95% confidence interval (CI), 1.02 – 4.18), a low mental health

composite score (OR, 2.71; 95% CI, 1.69 -4.34), ≥2 prior PID episodes (OR, 2.84; 95%

CI, 1.07 – 7.54) and smoking (OR, 1.65; 95% CI, 1.01 – 2.71) were independent

predictors of chronic pelvic pain. Interestingly, Haggerty et al. (2005) showed

histologically confirmed endometritis or evidence of endometrial C. trachomatis or N.


- 14 -

gonorrhoeae infection was negatively associated with chronic pelvic pain. A similar

study also reported the strong association of chronic pelvic pain with several

gynecological (PID – P <0.01) and psychosocial factors (Latthe et al., 2006).

1.3.1.5 PID Risk Factors

The spread of disease can be minimised by evaluating which factors increase

transference of pathogen to host. PID has several risk factors, including: age,

socioeconomic status, sexual behaviour, contraceptive use, health care behaviour,

douching and menses.

Age appears to be inversely related to PID rates. Westrom et al. (1992) reported that

nearly 70% of women with acute salpingitis were under 25 years and thirty-three

percent of these experienced their first infection prior to 19 years of age, with 75%

nulliparous. The adolescent population may have a greater risk of contracting C.

trachomatis-induced PID as the number of sexual partners is often high and there is a

failure to use appropriate contraceptive methods to protect against the transmission and

development of chlamydial infection (Westrom, 1980; Anon, 2000). Furthermore,

anovulatory cycles are common in adolescent females, thus it has been suggested that

the high, continued production of endogenous oestrogen may permit easier cervical

penetration and subsequent upper tract ascension. Cervical ectopy, which presents large

areas of columnar epithelium allowing greater surface areas for C. trachomatis

attachment and infection, is also frequent in adolescents (Hewitt and Brown, 2000). In

a study evaluating various risk factors of PID amongst young, single and sexually active

women, investigators recruited 1170 participants, aged 13 - 36 at high risk for

chlamydial infection and monitored them for 3 years for incidence of PID (Ness et al.,

2006). Based on multivariate analysis, the authors showed that age at initial sexual

intercourse (≤15 years), current cervicitis, history of PID, smoking, sex during menses,

Depo-Provera use and an annual income less than $20 000 were all risk factors for PID.

Although in the high-risk category for PID, adolescents appear to have less PID-

associated sequelae such as infertility compared with affected older women since


- 15 -

infertility remains undiagnosed as opposed to older women who are actively trying to

conceive. Generally, it is at this point, where tubal damage in older females is

consequently diagnosed.

Several aspects of sexual behaviour may be associated with an increased risk of PID

including: (i) multiple sexual partners, (ii) high frequency of sexual intercourse, (iii)

rate of acquisition of new sexual partners within the previous thirty days, and (iv) age at

first sexual intercourse encounter (Lee et al., 1988; Wolner-Hanssen et al., 1990;

Jossens et al., 1996). The rationale of sexual behaviour and PID risk is linked to the

increased chance of acquiring multiple C. trachomatis infections thereby elevating the

likelihood of PID and associated sequelae.

Sexual intercourse during menstruation may also be a significant risk factor in the

development of PID. In a case-control study of 234 women with PID and 122 controls

that assessed potential PID-associated risk factors, Jossens et al. (1996) was able to

demonstrate a significant association between PID and previous sexual intercourse

during menses. Given that retrograde menstruation or backflow of uterine menstrual

blood into the fallopian tubes and peritoneal cavity affects between 70% - 90% of all

women (Blumenkrantz et al., 1981), it is reasonable to conclude the ascension of

infectious bacteria could be further disseminated and introduced into the upper genital

tract. Moreover, abnormal chlamydial deposition and growth is unimpeded in the

peritoneal cavity thereby perpetuating further infection and infertility (Rice and

Schachter, 1991).

The use of various contraceptive methods can increase the risk of developing C.

trachomatis-induced PID (Washington et al., 1991). The case-control study of Jossens

et al. (1996) identified a lack of contraception was quite a significant PID risk factor. In

a recent study, Gareen and colleagues (2001) demonstrated a relative risk of 3.3 (95%

CI 2.1 – 5.3) in women using intrauterine devices (IUDs). Interestingly, the risk of PID

appears to be limited to the first three months post-IUD insertion due to the initial

uterine introduction of micro-organisms (Fairley et al., 1992; Eschenbach et al., 1997).


- 16 -

Previous studies have reported that oral contraceptive pill (OCP) usage is associated

with a reduced incidence of PID and has a protective role against infection (Wolner-

Hanssen et al., 1985, Svensson et al., 1987; Wolner-Hanssen et al., 1990; Kimani et al.,

1996). In contrast, the PID Evaluation and Clinical Health (PEACH) study showed

hormonal and barrier contraceptive methods did not reduce the risk of upper genital

tract disease in women with clinical PID however, the authors did demonstrate a

reduction of clinical disease severity in the study participants (Ness et al., 2001).

Scrutinisation of women’s personal hygiene habits has led investigators to propose

vaginal douching as another potential risk factor for PID (Forrest et al., 1989; Wolner-

Hanssen et al., 1990; Washington et al., 1991). In a case-control study (234 PID cases

versus 122 matched controls) examining the risk of PID-associated douching, a two-

fold increased risk was noted (Jossens et al., 1996). Other case-control studies have

similarly reported a two-fold higher incidence of ectopic pregnancy associated with

vaginal douching (Chow et al., 1985, 1990; Daling et al., 1991). Conversely, neither a

randomised field trial nor a prospective study conducted over a five year period

demonstrated an association between vaginal douching and PID (Rothman et al., 2003;

Ness et al., 2005). In an attempt to explain the causal relationship of douching and

potential PID risk, it has been suggested that regular douching removes inherent vaginal

and uterine flora which thereby alters the normal vaginal pH and permits microbial

progression and proliferation. To date, this theory has not been proven.

1.3.1.6 Treatment

The therapeutic goal in the management of PID is the prevention of PID, PID sequelae

and potential recurrent infection. Early diagnosis and treatment are vital to the

preservation of fertility. Animal models of acute PID have confirmed that a short

window of approximately 5-6 days of disease onset exists within which a good fertility

outcome can be achieved by antibiotic treatment (Swenson and Schachter, 1984). In

humans, fertility outcomes are further enhanced if antibiotic treatment is prescribed

within 48 hours of symptom onset (Hillis et al., 1993) although in the majority of cases,


- 17 -

the subclinical nature of C. trachomatis infection hinders a timely response. Treatment

guidelines of C. trachomatis-generated PID as published by the Centers for Disease

Control and Prevention 2006, is generally accomplished via a course of antibiotics

taken orally or administered intravenously (Tables 1.2 and 1.3) (Walker and

Wiesenfeld, 2007).

Table 1.2. Recommended parenteral antibiotic therapy for acute PID from the Centers

for Disease Control and Prevention of sexually transmitted diseases treatment

guidelines 2006.

RECOMMENDED PARENTERAL REGIMEN A

TREATMENT DOSAGE Cefotetan 2g iv every 12 hours

OR

Cefoxitin 2g iv every 6 hours PLUS

Doxycycline 100mg po or iv every 12 hours

RECOMMENDED PARENTERAL REGIMEN B

TREATMENT DOSAGE Clindamycin 900mg iv every 8 hours

PLUS

Gentamicin iv or im (2mg/kg body weight), followed by maintenance dose (1.5mg/kg) every 8 hours

ALTERNATIVE PARENTERAL REGIMENS

TREATMENT DOSAGE 1. Levofloxacin 500mg iv once daily

OR Ofloxacin 400mg iv every 12 hours

WITH OR WITHOUT

Metronidazole 500mg iv every 8 hours

2. Ampicillin – sulbactam 3g iv every 6 hours

PLUS

Doxycycline 100mg po or iv every 12 hours iv: intravenously; im: intramuscularly; po: orally


- 18 -

The effectiveness of various treatment strategies and patient status for women with PID

was assessed in a randomised multicentre trial of 831 females presenting with mild-to-

moderate PID (Ness et al., 2005). Inpatients were initially dosed with intravenous

cefoxitin and doxycycline compared to outpatient treatment which consisted of one

intramuscular injection of cefoxitin and the administration of doxycycline orally. In

addition, over a mean follow-up period of 84 months, the investigators compared

treatment groups for the incidence of pregnancy, live births, time to conception,

infertility, chronic pelvic pain, ectopic pregnancy and PID recurrence. Interestingly,

outpatient therapy did not negatively impact the number of women in relation to the

various issues and conditions examined by Ness et al. (2005).

Table 1.3. Recommended oral antibiotic therapy for acute PID from the Centers for

Disease Control and Prevention of sexually transmitted diseases treatment guidelines

2006.

RECOMMENDED ORAL REGIMEN A

TREATMENT DOSAGE Levofloxacin 500mg once daily for 14 days

OR

Ofloxacin 400mg once daily for 14 days WITH OR WITHOUT

Metronidazole 500mg twice daily for 14 days

RECOMMENDED ORAL REGIMEN B

TREATMENT DOSAGE Ceftriaxone 250mg im in a single dose

OR

Cefoxitin 2g im in a single dose; and probenecid, 1g orally in a single dose

OR

Cephalosporin Not indicated

PLUS Doxycycline 100mg twice daily for 14 days

WITH OR WITHOUT

Metronidazole 500mg twice daily for 14 days im: intramuscularly


- 19 -

Successful therapy for the cessation of associated disease progression relies heavily

upon early detection and rapid treatment with antibiotics. Administration of antibiotics

is essential, however certain penicillin derivatives can induce chlamydial persistence in

vivo (Phillips et al., 1984; Dreses-Werringloer et al., 2000). Treatment must occur

within the initial stage of infection however paradoxically, the asymptomatic nature of

chlamydial infection belies early detection.

1.3.2 Cervicitis

Epidemiological evidence has indicated that approximately 70% of women with an

endocervical infection display no symptomology or have only mild symptoms such as

vaginal discharge or itching, bleeding, mild abdominal pain or dysuria (Cates and

Wasserheit, 1991). The prevalence of mucopurulent cervicitis appears to be age related,

with women less than 25 years of age at greater risk of infection when compared to an

older age group (Marrazzo et al., 2002). In addition, the same study concluded that

non-white races were associated with a two-fold increase in the detection of cervical

infection.

1.3.3 Urethritis, Epididymitis and Sexually Reactive Arthritis

Urethritis and sexually reactive arthritis (SARA) are not gender specific, however a

higher incidence has been observed in males (Keat et al., 1983; Taylor-Robinson et al.,

1988; Pearlman and McNeeley, 1992; Kvien et al., 1994; Hopkinson, 2001; Lindberg,

2003). The incubation period for symptomatic chlamydial urethritis is usually between

7 and 14 days and the presenting signs are dysuria and a clear, white or grey urethral

discharge. A study by Hopkinson et al. (2001) showed urethritis to be a precursor to

epididymitis and SARA. Epididymitis usually manifests itself as tenderness, redness

and swelling of the scrotum (Ostaszewska et al., 2000), although, in some instances, it

is accompanied by abdominal pain and fever. Typically, the infection is unilateral and

symptom development is rapid. Research aimed at analysing signs and symptoms


- 20 -

associated with acute C. trachomatis epididymitis uncovered two risk factors

(Ostaszewska et al., 2000). Firstly, chlamydial epididymitis was more prevalent and of

longer duration in a younger age group. Secondly, urethral discharge had a higher

incidence in Chlamydia-infected patients, although epididymis oedema and scrotum

erythema or redness was rarely observed. SARA appears to be an immune-mediated

inflammatory response to infection (Bas et al., 1999), however the exact cause is not

well understood. Subsequent to an infection by C. trachomatis, very few individuals

presenting with non-gonococcal urethritis (NGU) develop reactive arthritis (Keat et al.,

1983; Kvien et al., 1994; Bas et al., 1999). Arthritic symptoms emerge from peripheral

joints but mandibular, shoulder, elbow, wrist and hand joints may also be involved in

the disease (Kvien et al., 1994) and synovitis may develop in various parts of the body

one month prior to other symptoms. The majority of SARA cases recover within a 2-6

month time frame (Kousa et al., 1978) although approximately 15% of patients with

Reiter’s syndrome (characterised by arthritis, NGU and inflammation of the middle

layer of the eye) have an annual recurrence of symptoms.

1.3.4 Lymphogranuloma Venereum

Lymphogranuloma Venereum (LGV), caused by C. trachomatis serovars L1, L2 and

L3, tends to be restricted to Africa, India, Southeast Asia, South America and the

Caribbean (Mabey and Peeling, 2002), however LGV is increasing in frequency in

Europe, North America and Australia (Laar et al., 2004; Blank et al., 2005; Stark et al.,

2007; Ward et al., 2007). In contrast to other genital C. trachomatis strains, LGV

disseminates through the lymphatic system and can elicit severe pain and inflammation

(Quinn et al., 1981). LGV has three distinct stages (Bolan et al., 1982). Firstly, the

formation of a primary lesion on the genital mucosa or adjacent skin ulcerates and heals

rapidly without scarring. Secondly, days or weeks subsequent to the primary lesion,

swelling of the inguinal and/or femoral lymph nodes occurs. Thirdly, due to the

extensive inflammatory response particularly within the infected lymph nodes, the

surrounding tissue forms an inflammatory mass and pus-filled abscesses develop.

Moreover, although relatively uncommon, evidence has established LGV to be a cause


- 21 -

of rectal strictures (Papagrigoriadis and Rennie, 1998) and rectovaginal fistulas (Lynch

et al., 1999).

1.4 CHLAMYDIAL IMMUNE RESPONSE AND PATHOGENESIS

The hallmark of chlamydial infection is an inflammatory process that is exacerbated by

reinfection and culminates in fibrosis, tissue damage, scarring and may elicit blindness,

tubal infertility, ectopic pregnancy, epididymitis, reactive arthritis and chronic pelvic

pain (Cumming et al., 1988; Cates and Wasserheit, 1991; Cohen et al., 1999; Mabey et

al., 2003). Numerous immunological host responses which are activated as a result of

bacterial invasion synergistically function to resolve acute infection. In reaction to

chlamydial entry, innate immune responses at the mucosal level occur within 1-2 days

post-infection (Williams et al., 1981) (Figure 1.4). Moreover, the primary host immune

response sees the antigenic stimulation and activation of naïve B cells which

differentiate into antibody-secreting cells that produce immunoglobulins eg. IgG, IgA

and IgM to neutralise the offending antigen (Figure 1.5). Intense inflammation and

predominant mucosal infiltration of phagocytes, neutrophils and macrophages, which

ingest and attempt to destroy the pathogen (Williams et al., 1981), can result in clinical

conditions such as mucopurulent cervicitis and endometritis in women, and non-

gonococcal urethritis in men (Brunham and Rey-Ladino, 2005). In conjunction with the

release and activation of phagocytic cells at immune inductive sites, others, critical to

infection resolution, are also deployed (Igietseme et al., 2004). Natural killer (NK)

cells are activated in direct response to chlamydial infection or indirectly by

macrophagic stimulation of IL-12, a powerful NK cell-triggering cytokine. Following

activation, NK cells produce IFN-γ which stimulates macrophages and further promotes

phagocytosis, thus providing an early defensive mechanism against chlamydial

infection. Innate immunity may impede infection, however the inaccessibility of

Chlamydia to circulating antibodies whose purpose is elimination necessitates the

immune effector mechanisms of both cell-mediated and humoral immunity. In a cell-

mediated immune response, T cells, which are functionally differentiated into Th1 or


- 22 -

Th2 subtypes based upon their cytokine secretion profiles, also accrue at the infection

site (Williams et al., 1981; Rank et al., 1985; Stephens, 2003).

Figure 1.4. Innate and adaptive immune responses as a result of acute chlamydial

infection. The innate immune response to chlamydial invasion consists primarily of

phagocytes and NK cells, and various cytokines (IL-12 and IFN-γ). The typical

adaptive immune response is directed by cell-mediated immunity. T cells activate

phagocytes to eliminate the pathogen. In general, depletion or blockage of the

numerous effector mechanisms at different stages of infection results in unrestrained

chlamydial growth (dashed lines). (Adapted from Cellular and Molecular Immunology,

fifth ed. 2003).

IL-12 IFN-γ

T cells

Macrophages Neutrophils

NK cells

Macrophages

IFN-γ

0 7 14

Days after infection

Num

ber

of v

iabl

e ba

cter

ia

Control of Infection

Eradication of Infection

Depletion of NK cells; IL-12 and IFN-γ inhibition Depletion of T cells

Innate Immunity Adaptive Immunity


- 23 -

Figure 1.5. Primary and secondary humoral immune responses. The initial immune

response, stimulated by antigen activated naïve B cells, differentiate and produce IgG

and IgM antibodies specific for the eliciting antigen. Long-lived B cells are also

produced during the primary response. A secondary immune response that generates a

more rapid proliferation, production and differentiation of larger quantities of specific

antigen compared to the primary response occurs when the same antigen stimulates the

memory B cells. (Adapted from Cellular and Molecular Immunology, fifth ed. 2003).

First Infection

Repeated Infection

Memory B cell

Naïve B cell

Activated B cells

Memory B cell

Antibody-secreting cell

Antibody-secreting cells in peripheral lymphoid tissue

Long-lived plasma cells in bone marrow

Low-level antibody production

IgM

IgG

IgG

10 3 >30 >30// //

Am

ount

of A

ntib

ody

Days after antigen exposure Days after antigen exposure

7

Primary Antibody Response

Secondary Antibody Response


- 24 -

Furthermore, interaction of Chlamydia with the cytokine network is a key component in

infection resolution and one in which a cytokine response is elicited either through the

direct infection of epithelial cells (Rasmussen et al., 1997), or by interaction with cells

of the host immune system (Fitzpatrick et al., 1991). The vital role of T cells and

subsequent influence over infection has been exemplified in nude mice studies.

Investigators demonstrated that nude mice cannot control chlamydial infection, however

adoptive transfer of CD4+ or CD8+ Chlamydia-spp.- specific T cell lines, permitted the

mice to successfully manage the infection (Rank et al., 1985; Ramsey et al., 1991).

Type 1 cells typically produce pro-inflammatory cytokines such as interferon gamma

(IFN-γ) and interleukin – 2 (IL-2). In contrast, type 2 cells secrete IL-4, IL-5, IL-9, IL-

10, IL-13 and inhibit IFN-γ production (Constant and Bottomly, 1997). Several animal

models of C. trachomatis infection have shown the activation of Th1 cells and the

secretion of IFN-γ is required for protection against re-infection (Su and Caldwell,

1995; Perry et al., 1997; van Voorhis et al., 1997). In addition, IFN-γ induces delayed

chlamydial development thereby limiting chlamydial infection. In vitro infection of

cervical epithelial cells with C. trachomatis or C. psittaci has been shown to induce

epithelial production of pro-inflammatory cytokines IL–8, growth related oncogene –

alpha, granulocyte-macrophage colony-stimulating factor, IL–6 and IL-1α (Rasmussen

et al., 1997). However, unlike the rapid but transient cytokine response instigated upon

entry into the host cell by other bacteria, chlamydial invasion did not produce such a

response until 20 – 24 hours post-infection (Eckmann et al., 1993; Rasmussen et al.,

1997). This finding suggests intracellular chlamydial development is a prerequisite for

activation of the immune response.

The humoral defense of mucosal surfaces is provided by antibodies, principally of the

IgA isotype, which are selectively transported into external secretions (Mestecky et al.,

1991). In humans, secretory IgA (IgAs) dominate and are an intrinsic characteristic of

the mucosal immune response (Figure 1.6). IgAs has two subclasses, IgA1 and IgA2

which vary in protein and carbohydrate structures, body fluid distribution and effector

functions (Mestecky et al., 1989). Specific antibodies against viral antigens originate

from the IgA1 subclass, whilst anti-LPS are most commonly found in the IgA2


- 25 -

subclass. Interestingly, IgA1 and IgA2 ratios and the prevalence of polymeric IgA

(pIgA) in cervical secretions show that the majority of IgA is resultant from local

production not plasma (Kutteh et al., 1988). In addition IgM are produced by

differentiated naïve B cells in response to infection. Moreover, the different immune

responses ie. innate and adaptive, systematically co-ordinate their efforts to ultimately

resolve chlamydial infection. As previously discussed, reaction to a C. trachomatis

infection by the host’s immune system manifests as a Th1-like response (Ward et al.,

1999). In contrast however, this form of immune response also has the ability to induce

persistent infection and evoke tissue damage thus promoting the development of

associated adverse sequelae.


- 26 -

Figure 1.6. The inflammatory response to chlamydial infection is characterised by the infiltration of macrophages and neutrophils and the formation of immune inductive sites in the submucosa. These sites contain both B and T cells, dendritic cells and macrophages which co-ordinate the instigation of an adaptive immune response. Secretory IgA and polymeric IgA are also deployed during the initial stages of the immune response.

Macrophage

Lamina propria

B cell

Dendritic cell

T cell

Epithelial cell

Fallopian tube

Endometritis

Salpingitis INFECTED MUCOSANORMAL MUCOSA

Vagina

Mucopurulent cervicitis

Ovary

Uterus

Neutrophil

Immune inductive site

pIgA

Chlamydial inclusion

sIgA


- 27 -

1.4.1 Role of Heat Shock Proteins in the Pathogenesis of Chlamydial Disease

HSPs are highly conserved ubiquitous molecules, and are categorized into four groups

based upon their molecular weight: (i) HSP60, (ii) HSP70, (iii) HSP90, and (iv) small

HSP. HSPs are chaperone proteins that fulfill a plethora of functions including

cytoprotection, intracellular assembly, folding and translocation of oligomeric proteins

(Hightower, 1991). The reversal of polypeptide unfolding and prevention of protein

aggregation, especially during periods of stress are also chief functions of this group of

proteins (Craig et al., 1993; Becker and Craig, 1994; Hartl, 1996). In normal or

nonstressed cells, HSPs are present in low concentrations while in stressed cells they

accumulate at high levels. Ward (1999) proposed that immune responses to chlamydial

HSPs, in particular HSP60 through antigenic mimicry, initiate an autoimmune response

against related human HSPs. Beatty et al. (1994) showed that although treatment with

IFN-γ of C. trachomatis-infected cells hindered chlamydial development, HSP60 was

still expressed. In addition, continued HSP60 expression (secondary to IFN-γ action)

activates the chronic inflammatory responses connected with chlamydial disease

sequelae (Brunham et al., 1994; Pockley et al., 2000). Accordingly, Debattista et al.

(2003) showed reduced production of IFN-γ in response to chlamydial HSP60

(cHSP60) in women with chlamydial PID and those with a history of multiple C.

trachomatis infections compared to women infected once and those with endometriosis.

1.4.2 Role of antibody to Chlamydial HSP60

Several studies have revealed an association between chlamydial HSP and antibodies in

chronic complications such as ectopic pregnancy, infertility, PID and coronary artery

disease (Wagar et al., 1990; Brunham et al., 1992; Eckert et al., 1997; Peeling et al.,

1997; Fong et al., 2002). Chlamydial HSP60 antibody levels correlated with the

presence of C. pneumoniae antigen in atheromas (Fong et al., 2002), thus implying a

potential role in the pathogenesis of atherosclerosis and cardiovascular disease. Since

host immune response to HSP60 antibodies has been implicated in trachoma

pathogenesis (Peeling et al., 1998), studies have examined mucosal immune responses


- 28 -

to HSP60 in infected populations (Hessel et al., 2001). Tears and sera from Nepalese

villagers were reacted against HSP60 and the major outer membrane protein (MOMP).

The level of tear anti-chlamydial HSP60 antibody was significantly associated with

instances of inflammation and scarring, indicating HSP60 antibodies may not only be a

marker of infection, but also a risk factor for disease progression (Hessel et al., 2001).

The theory that immune response to HSP60 is important in the development of chronic

disease is reinforced by the finding that 62/90 males suffering chronic NGU displayed

antibodies to C. trachomatis HSP60 (Horner et al., 1997).

Numerous studies have demonstrated a correlation between antibody responses to

chlamydial HSP60 and pathologic sequelae in women (Brunham et al., 1992; Toye et

al., 1993; Dieterle and Wollenhaupt, 1996; Eckert et al., 1997). A significant

association was demonstrated between the presence of antibodies to chlamydial HSP60

and PID (Eckert et al., 1997; Peeling et al., 1997; Witkin et al., 1998). Similarly, an

association of antibodies to chlamydial HSP60 and tubal factor infertility has been

reported (Toye et al., 1993; Arno et al., 1995). Money et al. (1997) showed women

suffering from perihepatitis-salpingitis displayed both an increased prevalence of pelvic

adhesions and elevated titres of antibodies to chlamydial HSP60 relative to a control

group. A study of 129 women with laparoscopically-verified PID tested an association

between humoral immunity to unique and conserved epitopes of C. trachomatis HSP60,

and reactivity to human HSP60 (Domeika et al., 1998). Approximately half the cases

that had antibodies to human HSP60 also exhibited cross-reactive antibodies with

chlamydial HSP60 peptide 260-271. Antibodies to chlamydial peptide 260-271 were

strongly associated with anti-chlamydial IgG and IgA. It was proposed that an

autoimmune response to human HSP60 could develop following a C. trachomatis upper

genital tract infection, possibly as a result of the host’s immune response to an epitope

of chlamydial HSP60 that was cross-reactive with human HSP60 (Domeika et al.,

1998). This result is not surprising as the HSP60 peptide sequence is highly conserved

between chlamydial and human species. Nevertheless, these findings may simply be

the result of hyper-immunisation due to chronic chlamydial infection.


- 29 -

Host genotype appears to have an integral role in determining disease severity post-

infection (Ward, 1999). Cohen et al. (2000) have shown an association between the

DQA010 and DQB0501 alleles of the HLA class II locus, tubal infertility and infection

by C. trachomatis. Among the 47 infertile women, DQA0101 and DQB0501 alleles

were associated with C. trachomatis tubal infertility. In addition, the authors

hypothesised that the DQ locus may alter propensity to and pathogenicity of chlamydial

infection. Gaur et al. (1999) also investigated HLA class II alleles and demonstrated

that the presence of HLA type II alleles HLA DQA10401 and DQB10402 were

associated with increased prevalence and level of antibody to chlamydial HSP60.

1.4.3 Cell-Mediated Responses to HSP

Chlamydiae elicit predominantly T cell-mediated immune responses (Hassell et al.,

1993; Johansson et al., 1997). Atherosclerotic plaque tissue contains high levels of T

cells (Mach et al., 1998), hence Curry et al. (2000) examined whether T lymphocyte-

mediated immune responses to C. pneumoniae antigens within atherosclerotic plaque

contributes to the pathogenesis of the disease. Curry and colleagues concluded that

plaque-derived T cell lines were capable of recognising chlamydial antigens and local

antigens such as HSP60-activated T cells. This finding supports a possible pathogenetic

role for HSP60. Interestingly, elevated IgG and IgA antibody titres and a strong T

lymphocyte proliferative response to whole EB antigens of C. pneumoniae was

associated with coronary heart disease in males compared to females and controls

(Halme et al., 1997). These results also reinforce the potential role of various immune

mechanisms such as T lymphocytes and HSP60 in coronary atherosclerotic

pathogenesis.

Bailey et al. (1995) examined lymphocyte proliferative responses to selected

chlamydial and common recall antigens in 26 Gambian subjects whose clinical signs of

trachoma persisted over a six month duration. Results were compared to 21 subjects

whose disease resolved spontaneously within the same time frame. No difference was

evident in IFN-γ levels between both groups. Conversely, the seven-day


- 30 -

lymphoproliferative responses to Chlamydia and HSP60, but not common recall

antigens, were significantly elevated in those whose disease resolved spontaneously.

These results support the view that cell-mediated immune responses are essential in the

clearance of chlamydial ocular infection and potentially for chlamydial infection in

general.

1.5 HORMONAL INFLUENCES

The mucosal immune system in the female reproductive tract is of great importance as it

is the first site of immunological contact against bacterial infection (Ogra et al., 1981;

Underdown and Schiff, 1986). As previously stated, defense at the mucosal level is

mediated by both humoral and cell-mediated immunity. Initiation of an immune

response requires the internalisation of an exogenous antigen which is subsequently

processed and returned to the cell surface for recognition by CD4+ T cells (Weinberger

et al., 1981). Endocrine balance in the preliminary phase of genital tract infection may

enhance or suppress mucosal immune protection (Prabhala and Wira, 1995). In

addition, steroid hormones and the stage of the reproductive cycle regulate uterine cell

antigen presentation (Prabhala and Wira, 1995; Kaushic et al., 2000) thereby further

influencing the immune response. Interestingly, Prabhala and Wira (1995) also

demonstrated that at proestrus, when oestradiol levels are highest, uterine antigen

presentation is elevated. In addition, antibody levels, specifically IgG and IgA present

in the female reproductive tract, vary as a result of anatomical location and in

accordance with the phase of the menstrual cycle (Wira et al., 1992). Using a rat model

to investigate the effects of dihydrotestosterone and oestradiol on IgA and secretory

components in the immune system of males, the authors showed that androgens and

oestrogens act at selected target sites and play a role in maintaining secretory

component levels, thus indicating that the movement of IgA from tissues into secretions

is hormonally driven (Stern et al., 1992).


- 31 -

In addition to the influences of oestrogen over infection progression, progesterone,

secreted during various stages of the menstrual cycle, has been shown to enhance C.

trachomatis infection in the genital tract (Kaushic et al., 1998). In a study to determine

the effects of progesterone and oestradiol on susceptibility and early immune responses

to C. trachomatis in the female reproductive tract, Kaushic et al. (2000) demonstrated

that ovariectomised rats, pre-treated with oestradiol, had complete protection from

chlamydial infection with no visible signs of uterine or vaginal inflammation. In

contrast, rats that were exclusively administered progesterone became heavily infected

which was accompanied by an acute inflammatory response. Interestingly, animals

concurrently treated with oestradiol and progesterone were also heavily infected,

however none demonstrated an inflammatory response. This suggests that both steroid

hormones have distinctive individual effects on susceptibility to infection and

inflammation.

1.6 CHLAMYDIAL PERSISTENCE

In addition to acute infection, C. trachomatis has the ability to exist in a chronic or

persistent state (Figure 1b). Persistence is defined as a long-term association between

the pathogen and host in which the organism remains viable in a culture-negative or

non-infectious form without overt growth or replication (Beatty et al., 1994). The

limited metabolic capacity of Chlamydia in the persistent phase may influence the

antigenic and biochemical properties of the bacteria thus potentially rendering them

undetectable via conventional diagnostic means. Factors that favour persistent infection

include: (i) action of penicillin, (ii) IFN-γ, and (iii) deprivation of amino acids which are

essential for chlamydial replication. Persistent infection is characterised by enlarged,

aberrant chlamydial bodies within the inclusion. Although in a latent phase, chlamydial

viability is maintained and recovery of infectious progeny is possible subsequent to

IFN-γ removal and other inhibitory conditions (Beatty et al., 1993).


- 32 -

Moulder (1991) originally suggested the existence of persistent intracellular Chlamydia

in a morphologically altered state. Both mouse fibroblasts (L cells), and the C. psittaci

strain were preserved indefinitely in the absence of conventional inclusions. This

unidentified form of the bacteria was termed a cryptic body. Further investigation

demonstrated that cryptic bodies are related to solitary, inclusion-encased, dense oval

bodies as witnessed in L cell cultures infected with C. trachomatis (Moulder, 1991).

Numerous studies have reported anomalous RBs whereby their growth by binary fission

and secondary differentiation into EBs is arrested (Allan et al., 1985; Coles et al., 1993;

Dreses-Werringloer et al., 2000, 2001, 2003). Reversal of this developmental

abnormality is generally accomplished upon removal of the inhibitory factor.

1.6.1 Nutrient Deficiency – Induced Persistence

Omission of essential nutrients in cell culture medium temporarily halts replication of

both Chlamydia and the eukaryotic host cells until optimal cyclic conditions are

restored (Beatty et al., 1991). Inhibition of host cell protein synthesis is achieved

through the addition of cycloheximide that allows Chlamydiae unobstructed access to

the cell’s nutrient pools. Depletion of cysteine severely retarded differentiation of RBs

to EBs in cycloheximide treated McCoy cells (Allan et al., 1985). However, cysteine

deprivation did not irreparably inhibit bacterial development in the 10 strains of C.

trachomatis once cysteine was reintroduced into the cell medium. The effect appears

specific to cysteine as omission of other amino acids had minimal or no effect on

chlamydial differentiation.

Coles et al. (1993) examined the intracellular development of C. trachomatis serovar L2

in McCoy cells lacking all amino acids and found anomalous growth in almost every

inclusion concomitant with a reduced infectivity rate. Normal chlamydial development

was restored after reintroduction of the missing amino acids. In contrast to previous

studies, depletion of any of the 20 amino acids from culture with the exception of valine

produced aberrant chlamydial growth. To fully assess the morphological changes

associated with amino acid deprivation in a eukaryotic host, Chlamydiae were grown in


- 33 -

medium with amino acids maintained at normal blood plasma concentrations (Harper et

al., 2000). This study demonstrated that amino acid levels could directly influence and

promote abnormal chlamydial development in vivo.

Investigation of McCoy cells infected with C. trachomatis strains A-C showed that

tryptophan was required for normal growth, whilst serotypes D-I exhibited no such

growth prerequisite (Allan and Pearce, 1983). The findings reported by Allan and

Pearce (1983) were not replicated in the study by Fehlner-Gardiner et al. (2002) as all

serotypes were inhibited in the absence of tryptophan. Furthermore, only genital

serovars D-K were able to produce tryptophan from exogenous indole (a tryptophan

precursor) under conditions of starvation and continue the chlamydial reproductive

cycle. Interestingly, due to mutations or truncations within the trpA gene, C.

trachomatis serovars A-C do not have the ability to rescue tryptophan. Thus, this

finding may indicate a possible mechanism behind disparity in the pathogenesis of C.

trachomatis serovars (Shaw et al., 2000).

A reduction of other nutrients can also generate persistence in vivo. Iron is a mediator

of virulence in several bacterial pathogens eg. Chlamydia and Campylobacter. An iron-

chelating reagent, deferoxamine mesylate, was used to minimise iron availability in

cells infected with C. trachomatis serovar E strain resulting in adverse chlamydial

development and a significant reduction in EB infectivity (Raulston, 1997).

Microscopic examination revealed no difference in absolute numbers of C. trachomatis

inclusions that formed in an iron-restricted versus an iron-rich environment; however

development of inclusions and ensuing EB-infectivity was greatly reduced.

Importantly, there is an increased synthesis of HSP60 upon iron deficiency. This

antigen provokes an inflammatory response in the human host, high levels are

maintained in chlamydial persistence and the protein is associated with destructive

sequelae such as PID, TFI and ectopic pregnancy. C. trachomatis serovar L2

propagated in McCoy cells in the absence of glucose, displayed reversible atypical

persistent chlamydial growth with analogous effects on morphology and infectivity as

observed in amino acid-deprived cells (Harper et al., 2000). Since Chlamydia is reliant


- 34 -

upon host nutrients for growth and reproduction, it is not surprising that either a partial

or total reduction in nutrient levels has a dramatic impact on its normal developmental

cycle.

1.6.2 Antibiotic-Induced Persistence

Standard treatment for acute PID is an oral fourteen day course of doxycycline and

ceftriaxone or intravenous injections of cefotetan (Haggerty and Ness, 2007), however

penicillin-derived antibiotics are capable of inducing a state of chlamydial persistence

in vivo (Phillips et al., 1984). Consequently, chlamydial binary fission is arrested and

the formation of enlarged reticulate-like aberrant bodies contained within the inclusions

occurs (Dreses-Werringloer et al., 2000, 2001, 2003). The effect of erythromycin

which reduces ribosome activity and subsequent protein synthesis in the chlamydial

reproductive cycle was examined via electron microscopy. Prior to 12 hours post-

infection, conversion of EBs to RBs was halted (Clark et al., 1982). Also,

supplementation of 10µg/mL of erythromycin into the McCoy cell culture at 18 hours

or 24 hours post-infection hindered glycogen production, inhibited further

differentiation of EBs to RBs and produced RBs twice the diameter of the untreated

control cells. Similarly, persistence of C. trachomatis was induced in vitro by

ciprofloxacin and ofloxacin in HEp-2 cells, and the development of anomalous

inclusions and reduced infectivity were again observed (Dreses-Werringloer et al.,

2000). The formation of small or “miniature” RBs and inclusions developing from and

within enlarged chlamydial bodies is another feature of penicillin-induced persistence

(Dreses-Werringloer et al., 2000). An in vivo study identified similar characteristics in

ultrastructural analyses of C. trachomatis infected murine oviducts as those witnessed in

penicillin treated cells (Phillips et al., 1984).

Tetracyclines, which inhibit protein synthesis by reversibly binding to the 30S

ribosomal subunit, have also been shown to hinder intracellular development of

Chlamydia (Samra et al., 2001; Lenart et al., 2001). A concentration of 3µg/mL of

tetracycline in cell medium caused 100% of all inclusions to contain morphologically-

altered RBs (Lenart et al., 2001). Normal RBs and a productive developmental cycle


- 35 -

were observed subsequent to tetracycline removal. Folic acid antagonists, or

sulfonamides, also alter chlamydial morphology and the reproductive cycle in vitro

(Hammerschlag, 1982; Hammerschlag and Vuletin, 1985). When increasing

concentrations of trimethoprim and sulfamethoxazole were added to C. trachomatis-

infected McCoy cells, scant, undersized and pyknotic inclusions were detected under

microscopic examination (Hammerschlag, 1982). Normal growth and morphological

development was induced following the transference of cells containing the atypical

inclusions to a drug-free medium.

Overall, the studies discussed above demonstrate the dose-dependent inhibitory effect

of various antibiotics on chlamydial differentiation and replication at various stages of

the reproductive cycle in vitro. The effects of antibiotics are dependent upon

concentration and the stage in which the drug is introduced into the developmental

cycle of the infected cells. In addition, continued inappropriate antibiotic treatment may

permit Chlamydia to persevere in vivo.

1.6.3 Cytokine-Induced Persistence

Chlamydial infection invokes cytokine responses via direct infection of host cells, and

interaction with cells of the host immune system. The use of cytokines such as IFN-γ to

generate a state of persistence of chlamydial infections in vitro provides a novel method

for understanding possible in vivo events. There is evidence that in vivo IFN-γ initiates

persistence by interfering with the replicative capacity of Chlamydia. HeLa 229 cells

infected with C. trachomatis serovar A were treated with various concentrations of IFN-

γ to assess its effects on growth and differentiation (Beatty et al., 1993). An IFN-γ

concentration of 2ng/mL totally hindered chlamydial maturation, whilst persistence was

established at a 0.2ng/mL concentration suggesting the effect of IFN-γ to be dose-

dependent. The effect of IFN-γ may be strain dependent as Rasmussen et al. (1996)

showed treatment of C. trachomatis-infected HeLa cells with IFN-γ generated aberrant

RBs and non-infectious progeny in serovars A and B, while serovar L2 produced


- 36 -

normal morphology with decreased inclusion size and a substantial reduction in

infectivity.

In vitro, IFN-γ depresses Chlamydia development through the induction of the host cell

tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) (Jones et al., 2001).

IDO catabolises intracellular pools of L-tryptophan, essentially starving chlamydial

bacteria, and thus prevents secondary RB differentiation, infectivity and cell-to-cell

transmission. Jones et al. (2001) demonstrated the concentration of IFN-γ was

inversely proportional to the level of tryptophan. In addition, the degree of abnormality

observed within chlamydial-infected HeLa cells increased as IFN-γ levels rose. It has

been reported that infectivity of all human C. trachomatis serovars was arrested in

tryptophan-depleted medium (Fehlner-Gardiner et al., 2002). By supplementing

exogenous indole to the medium of genital serovars D-K, L1-L3, with the exception of

ocular serovars A-C and Ba, infectivity was restored. Further investigation revealed

only the genital C. trachomatis serovars D, I or L2 in HeLa cells were able to rescue

and utilise exogenous indole after tryptophan depletion (Caldwell et al., 2003).

Evidence suggests inhibition of tryptophan metabolism to be the major activator of IFN-

γ mediated persistence in vitro, however nitric oxide induction and intracellular iron

deprivation also appear to have an integral role in the persistence mechanism.

Investigation into the action of IFN-γ on C. trachomatis using a human epithelial RT4

cell line revealed nitric oxide induction, tryptophan catabolism and iron deprivation

contribute to cytokine-mediated inhibition of chlamydial growth and reproduction

(Igietseme et al., 1998). Nitric oxide accounted for 20% of the entire inhibition

witnessed, tryptophan catabolism 30%, while iron deprivation proved to be the least

efficient at inhibiting chlamydial growth. In combination, however, all three

mechanisms produced a 60% inhibition of chlamydial development (Igietseme et al.,

1998). The role of the inducible nitric oxide synthase (iNOS) pathway in controlling

chlamydial infection in vivo was analysed using a chlamydial-specific murine T-cell

clone in the presence of an iNOS inhibitor (Igietseme, 1996). Results revealed the

clones’ ability to infect was partly aided by the induction of nitric oxide as the capacity


- 37 -

to clear a genital mouse pneumonitis infection was inhibited. These findings support

the hypothesis that the IFN-γ iNOS pathway is involved in chlamydial persistence in

vivo. The studies above demonstrate that chlamydial growth and development in vitro

and in vivo appear to be influenced by IFN-γ levels. As a consequence, chlamydial

progression may be dependent upon the interaction between IFN-γ, NO, tryptophan

catabolism and iron deprivation.

Accompanying these factors is the possible combined effects of another cytokine,

tumour necrosis factor-alpha (TNF-α). In C. trachomatis-infected HEp-2 cells,

chlamydial growth was arrested upon addition of TNF-α and supplementation with

200ng/mL of the cytokine maintained inhibition up to 12 hours post-infection (Shemer-

Avni et al., 1988). In contrast to these findings, no morphological differences or

cessation of the C. trachomatis developmental cycle was observed either in mouse or

guinea pig animal models upon in vivo treatment with TNF-α (Darville et al., 2000).

The cytokine IL-1α may also play a role in persistence. Carlin and Weller (1995)

demonstrated that combined treatment of IFN-γ and IL-1α in human macrophage

cultures induced significant IDO activity and inhibited chlamydial growth at a 10-fold

lower IFN-γ concentration upon comparison to cultures treated with IFN-γ alone. Not

surprisingly, as IDO activity increased, so did the inhibitory effects on chlamydial

growth and maturation. Evident from these studies is the important interaction between

NO, tryptophan catabolism, iron deprivation, IDO, TNF-α, IL-1α and IFN-γ and their

combined role in chlamydial persistence.

1.7 CHLAMYDIAL DIAGNOSTICS

Diagnosis of chlamydial infection is paramount in the prevention of chronic disease and

associated adverse sequelae. Despite the asymptomatic nature of C. trachomatis

infection and the inherent difficulties associated with diagnosis such as infection status,

numerous diagnostic tests are currently available (Table 1.4). Importantly, the

performances of diagnostic tests differ in high and low risk populations. Moreover, as


- 38 -

prevalence varies, so does the positive predictive value (Earle and Hebert, 1996).

Traditionally, chlamydial detection consisted of cell culture which was viewed by many

as the definitive test due to the ability to witness chlamydial intracellular growth and

development. Whilst the culture method had the advantage of chlamydial preservation

in vitro, poor sensitivity (65% - 85%) (Schachter et al., 1994; Wiesenfeld et al., 1994;

Lee et al., 1995), high expense, delay in acquiring results (3 – 7 days) and the need for

technical expertise (Black, 1997) limited the test’s efficacy. Given most infected

individuals who do not receive treatment during the initial stages of infection will

develop a measurable antibody and cell-mediated immune response (Hanna et al.,

1979), serological assays were developed to aid chlamydial detection. Once such test,

the microimmunofluorescence (MIF) assay which was considered the gold standard

(Dowell et al., 2001), initially had EB antigens of each C. trachomatis serotype

incorporated into the matrix (Wang et al., 1970). Future versions of the MIF test

reduced the number of antigens to the single broadly reacting C. trachomatis L2

serotype (Treharne et al., 1977), however cross-reactivity between C. pneumoniae and

C. trachomatis greatly reduced its diagnostic efficiency (Freidank et al., 1997; Gijsen et

al., 2001). Cross-reactivity tends to elicit a higher rate of false-positives. Moreover,

serological cross-reactivity with conserved proteins also found in other bacteria (e.g.

Acinetobacter) increases the rate of false-positives (Brade and Brunner, 1979). To

combat this problem, LPS, a common component of the outer membrane of all

Chlamydia, was removed thus providing a species-specific test. MIF still has a number

of limitations however, as a high degree of expertise is required to correctly interpret

the fluorescence data given the reading is observer-dependent. Furthermore, MIF

testing is labour intensive and variation between laboratories is significant (Peeling et

al., 2000).

The need for a cheaper and more rapid diagnostic test for Chlamydia led to the

development of antigen detection methods. The two tests most commonly used are

direct fluorescent antibody (DFA) and enzyme immunoassay (EIA). DFA staining

shows an increased sensitivity rate of detection of 80 - 85%, and a specificity of >99%

upon comparison to cell culture approaches (Schachter et al., 1992; Black, 1997;


- 39 -

Marrazzo and Stamm 1998). Despite high efficiency, the labour and skill needed to

correctly perform the test preclude its use with large specimen numbers (Black, 1997).

Sensitivity for the EIA method is between 60% and 80%, considerably lower than the

DFA method (Schachter et al., 1992; Black, 1997). Cross-reactivity is once again

problematic as antibodies to chlamydial LPS may cross-react with the same protein of

other Gram-negative bacteria eliciting false-positives and as a consequence, decrease

test specificity (Black, 1997).

Table 1.4. An overview of various diagnostic methods associated with C. trachomatis

detection (adapted from Caul and Herring 2001).

Diagnostic Test Detection of Advantages Disadvantages

Sole test for medical/legal work

Requires skilled labour and viable EBs

Specific Reduced sensitivity compared with NAATs Culture

Viable EBs

within specimen Viable Chlamydiae preserved for subsequent screening

Prolonged waiting period for test results

Simple Requires confirmation LPS detected in low EB copy numbers

Sensitivity variation EIAs LPS

Standardisation potential

Direct Genus specific LPS epitopes

Determination of specimen quality

Limited throughput

Immunofluorescence Species specific

MOMP epitopes

Confirmatory test Quality of results reliant upon technician skills

Rapid High risk of contamination Standardisation potential Expensive

Easy preparation Inability for antibiotic resistance monitoring

NAATs DNA and RNA

Extremely sensitive and specific

Occasional problems with reproducibility


- 40 -

Both antigen detection methods have a false-positive rate of 2 - 3% (Black, 1997).

Although the sensitivity and specificity of these tests are higher than previously

advocated methods, the positive antigen predictive value is reduced in low-prevalence

populations. For example, in a population with a prevalence of 2 - 3%, a positive result

by antigen detection will prove correct in only 50% of instances (Black, 1997). To

evaluate the discriminative power of chlamydial antibody titers for the diagnosis of

tubal pathology, a meta-analysis of various studies compared Chlamydia antibody titres

with findings presented at laparoscopy (Mol et al., 1997). In this critical review of

previous research, the authors demonstrated that screening for tubal subfertility by C.

trachomatis IgG antibody testing yielded heterogeneous results. Furthermore, the

predictive value of chlamydial antibody testing was poor given the variable sensitivities

(30 - 80%) and specificities (45 - 100%). A more recent study using laparoscopy as a

reference, compared the performance of five commercially available serological

antibody tests and their ability to predict tubal factor subfertility (Land et al., 2003).

The tests included in the comparative analysis were the standard MIF (Biomerieux, the

Netherlands) in which C. trachomatis L2 was the group antigen; a species-specific MIF

(Labsystems, Finland) with incorporated C. pneumoniae, C. trachomatis and C. psittaci

EBs as antigens; and three enzyme-linked immunosorbent assays (ELISAs) which were

based on a MOMP-derived synthetic peptide (Labsystems, Finland; Medac, Germany),

and C. trachomatis species-specific peptides derived from ‘different’ serotypes

(Savyon, Israel). Previously, ELISA tests have been attributed with high sensitivity and

specificity, however this was not supported by Land et al. (2003). Of the five tests

evaluated, Labsystems’ MIF demonstrated the best overall performance (OR 15.7) in

diagnosing tubal subfertility, whilst of the remaining ELISA tests, the pELISA by

Medac was found to be superior (OR 8.2). Interestingly, stepwise logistic regression

analysis showed that performance of Labsystems’ MIF test could not be enhanced by

the addition of a second test. Considering a consecutive cohort of 315 subfertile women

was used for this study, the differences in test performance can be accredited to the

innate differences in antigenic composition of each test.


- 41 -

Since several studies have shown a correlation between pathologic sequelae in women

and antibody responses to cHSP60 (Brunham et al., 1992; Toye et al., 1993; Dieterle

and Wollenhaupt, 1996; Eckert et al., 1997), a commercial ELISA screening test based

upon cHSP60 (Medac, Germany) has been developed. Two studies subsequently

evaluated the diagnostic potential of the cHSP60 ELISA test (Bax et al., 2004; Gazzard

et al., 2006). Bax et al. (2004) demonstrated an 11% increased anti-cHSP60 antibody

response in women with tubal pathology compared to women without tubal pathology

(16%) and the control group comprising pregnant women (4.8%). In contrast, a more

recent study showed a decreased incidence (8%) of cHSP60 reactivity in the PID/Tubal

Damage group compared to 28% in acute patients (Gazzard et al., 2006), thus indicating

the expression and subsequent antibody reactivity to cHSP60 is not limited to patients

with chronic disease. In a case-control study to determine the diagnostic potential of

cHSP10 and/or cHSP60 for the detection of TFI, 54 women suffering primary or

secondary infertility and 90 nulliparous women were evaluated for the seroprevalence

of C. trachomatis antibodies (Dadamessi et al., 2005). The authors showed the

detection of C. trachomatis-associated anti-cHSP10 or anti-cHSP60 antibodies

collectively permitted diagnosis, with a sensitivity of 57.4% and specificity of 75.5%, of

TFI in Cameroon women. Whilst these figures for the diagnosis of TFI are suggestive

of a viable test for chronic pathology, the study participants may in fact not be a true

representation of the general population. At present, C. trachomatis IgG antibody

testing by MIF or ELISA is of limited use in infertile women for predicting associated

sequelae given the significant variation in diagnostic accuracy between commercially

available tests. Whilst IgG antibodies are definitive indicators of infection, they do not

reflect, nor discriminate accurately with regards to previous infection or adverse

chlamydial-induced pathology.

Nucleic acid amplification testing (NAAT) is sensitive and highly specific (Black,

1997). Various studies have shown NAATs to have greater diagnostic capabilities than

either culture or antigen detection methods (Schachter et al., 1994; Wiesenfeld et al.,

1994; Black, 1997; Rabenau et al., 2000). The most widely employed NAAT approach

is polymerase chain reaction (PCR) which targets nucleotide sequences on the plasmid


- 42 -

of Chlamydia present in multiple copies within each EB. Other NAATs such as ligase

chain reaction (LCR) and strand displacement assay from Becton Dickinson are also

available. At present, PCR is the favoured approach to chlamydial diagnostics,

however this test (and LCR) are substantially more expensive than previous chlamydial

detection methods. A commercial LCR assay (LCx) developed by Abbott Laboratories

for use on urogenital samples, was employed to evaluate its viability in the detection of

chlamydial DNA in synovial fluid (SF) of patients with reactive arthritis (Bas et al.,

1997). SF samples, previously verified C. trachomatis-positive in a prior study by at

least two different commercial DNA amplification kits (Bas et al., 1995), were

diagnosed negative by the LCx (Abbott Laboratories) test. In another study, the

presence of inhibitors, a causative factor of false-negative results in a commercial C.

trachomatis gap-filling LCR was assessed in urine specimens (Berg et al., 1997).

Results clearly demonstrated inhibitors present in urine samples greatly reduced the

efficacy of the test. The effect of inhibitors inherent in clinical samples is not restricted

to false-negatives, rather NAATs are also at risk of generating false-positive results

(Loeffelholz et al., 1992; Pasternack et al., 1997; Peterson et al., 1997; Toye et al.,

1998) thereby decreasing the overall value of the test method.

1.8 OBJECTIVES AND AIMS OF THIS STUDY

Chlamydiae are intracellular eubacteria that exhibit a propensity for urogenital,

conjunctival and respiratory epithelia. This evolutionary distinct pathogen causes

widespread infection in humans and in addition to acute infection, has the ability to

exist in a chronic or persistent state. Persistence is defined as the long-term association

between pathogen and host in which the organism remains viable in a culture-negative

or non-infectious form without overt growth or replication (Beatty et al., 1994). A

prolonged persistent infection, which can be induced by exposure to IFN-γ, nutrient

deprivation and penicillin-derived antibiotics (Harper et al., 2000; Fehlner-Gardiner et

al., 2002; Caldwell et al., 2003; Dreses-Werringloer et al., 2003), can lead to severe

inflammation, fibrosis, extensive tissue damage and scarring. Diseases caused by


- 43 -

Chlamydia are based on acute and chronic inflammation elicited and maintained by

subsequent reinfection or persistent infection. Furthermore, chlamydial infections that

remain untreated can lead to various disease states such as salpingitis (fallopian tube

inflammation), PID, tubal occlusion, epididymitis, arthritis, and infertility.

C. trachomatis, the most common global sexually transmitted pathogen, has an

estimated 3 million new infections reported annually in the United States alone

(Groseclose et al., 1996). Currently in Australia, the reported incidence of genital C.

trachomatis infections and chronic disease in both men and women are steadily

increasing (Chen et al., 2006; Stark et al., 2007). A study by Kang et al. (2006)

investigating the prevalence of C. trachomatis infection amongst high risk young people

(14 -25 year) in New South Wales showed that 84.1% were sexually active and had a

mean number of sexual partners (over the preceding 3 months) of 1.4. Of those that

were sexually active, one quarter failed to ever use a condom. A similar result was

demonstrated by Williams et al. (2003) who showed that a recent change of partner

(OR4.5 (985% CI 1.5 to 13.8)) was the strongest associated independent risk factor for

urogenital C. trachomatis infection. Clearly, the rising incidence of C. trachomatis

infection can to some extent be attributed to the number and change of sexual partners

and the failure by young adults to take proper precautions during sexual relations.

Whilst the exact prevalence of chronic disease remains undefined, recent figures

indicate that 10 - 40% of women with a previous or recurrent chlamydial infection will

subsequently develop PID (Westrom et al., 1992; Simms and Stephenson, 2000).

Furthermore, the prevalence of C. trachomatis infection in asymptomatic males ranges

from 4 - 10% (McNagny et al., 1992; Rietmeijer et al., 1991) and between 15 - 20% in

men attending STD clinics (Stamm et al., 1984). In 1990, the direct medical costs of

PID and two of its adverse side effects, infertility and ectopic pregnancy were estimated

at USD$2.7 billion (Washington and Katz, 1991). Moreover, additional estimates

showed that by the year 2000, direct and indirect costs associated with PID increased by

a staggering USD$7.3 billion. The principal challenge related to the control of C.

trachomatis infection is that 70 - 80% of women and up to 50% of men who are

suffering from an infection are asymptomatic (Schachter et al., 1983; Stamm and Cole,


- 44 -

1986; Zelin et al., 1995). As a consequence, diagnosis and treatment are often

problematic.

Current diagnostics encompass numerous methods however, their sensitivities and

specificities are considerably varied. Whilst it is true that a C. trachomatis infection can

be detected by various diagnostic tests, at present, none have the ability to discriminate

between an acute or chronic infection. Moreover, although there is a wealth of

knowledge regarding Chlamydia-host interactions, intrinsic immune responses and

hormonal influences in acute and persistent phases, chlamydial antigenic targets are yet

to be fully defined. As a consequence, the ultimate goal of this project was the

serological identification of proteins that were differentially expressed during acute and

chronic C. trachomatis infection stages and to utilise these antigens for the development

of a simple, cost-effective and discriminatory diagnostic assay.

The specific aims of this project were:

1. To identify chlamydial antigens that are differential in acute versus chronic

C. trachomatis infections in male and females

2. To identify novel antigens and express as recombinant proteins for

subsequent serological screening and

3. Formulate selected antigens into a test format and evaluate with established

male and female patients groups

This thesis contains eight chapters and is structured as follows:

• Chapter 1 is a general introduction into chlamydial biology, Chlamydia-

host cell interactions, host immune responses, chronic disease such as PID,

hormonal influences on C. trachomatis infection, diagnostic methods

currently employed and the aims and structure of this thesis.


- 45 -

• Chapter 2 provides detail on the male and female study participants

including selection criteria and subsequent groupings, sample collection

methods and diagnosis at time of blood collection.

• Chapter 3 presents the initial screening process of female samples using

Western blot methods, the preliminary identification of differential

antigens, the potential sensitivities and specificities of these antigens in

various combinations for the basis of a serological diagnostic test and a

species and serovar comparison of the identified antigens.

• Chapter 4 details the comparison of protein expression using autoinduction

media or IPTG-induction for the production of the identified target

antigens.

• Chapter 5 describes the recombinant protein screen of the differential

antigens when probed against samples from the established female patient

groups and the diagnostic potential of these proteins for the discrimination

of chronic disease.

• Chapter 6 presents the initial Western blot screening process in males and

the identification of a novel 17.3kDa protein.

• Chapter 7 details the expression of the previously identified antigens in

men and women in a C. trachomatis IFN-γ cell culture model of

persistence.

• Chapter 8 discusses the findings and significance of the project in general.


- 46 -

Chapter 2: Female and Male Patient Recruitment

CHAPTER 2

Female and Male Patient Recruitment

- 47 -


2.0 INTRODUCTION

Chlamydia trachomatis can elicit asymptomatic and infrequently, symptomatic

infections in both men and women. Chlamydial infection is characterised by an

inflammatory immune response which is exacerbated upon reinfection and can thereby

result in adverse pathology such as tissue damage and scarring (Cumming et al., 1988;

Cates and Wasserheit, 1991; Cohen et al., 1999; Mabey et al., 2003). Clinical

manifestations, resultant from acute C. trachomatis infection, involve mucopurulent

cervicitis and urethritis which can on occasion, progress to adverse sequelae such as

salpingitis and tubal occlusion in females and more rarely, epididymitis in males.

Outcomes from such pathology include ectopic pregnancy and infertility (Sweet, 1991;

Rice and Schachter, 1991; Brunham et al., 1992; Schachter, 1999). In women, the most

important and common long-term complication of acute PID is TFI (Westrom, 1980;

Westrom et al., 1992; Patton et al., 1994). In males, non-gonococcal urethritis, caused

by C. trachomatis, is the most common sexually transmitted bacterial disease in young

men (Lindberg, 2003). Moreover, research has shown urethritis to be a precursor to

epididymitis and sexually-reactive arthritis (Hopkinson, 2001).

As discussed in Chapter 1, traditional laboratory methods for the routine diagnosis of C.

trachomatis infection include cell culture or antigen detection. NAAT, such as LCR

and PCR, have revolutionised standard diagnostic practices. The associated costs,

inability of antibiotic resistance monitoring and occasional reproducibility problems are

however a limitation of these particular diagnostic methods (Cook et al., 2005).

Currently, attempts to diagnose chronic chlamydial infection and associated pathology

have proven difficult. As a consequence, several studies have examined the diagnostic

potential of various chlamydial antigens associated with chronic disease (Mol et al.,

1997; Veenemans and van der Linden, 2002; Dadamessi et al., 2005; Tiitinen et al.,

2006) and the reliability of presently available commercial tests, such as Medac’s

cHSP60 ELISA (Land et al., 2003; Bax et al., 2004; Gazzard et al., 2006).

Interestingly, none have the demonstrated ability, or high level of sensitivity or

specificity required to successfully discriminate between acute and chronic C.

- 48 -


trachomatis infection. Furthermore, current serological diagnostic assays merely

diagnose the presence of chlamydial infection; they do not distinguish resolved, current,

recurrent or persistent infections. Importantly, since the incidence of C. trachomatis

urogenital infection is increasing and acute chlamydial infection has the ability to

become chronic and therefore potentially result in severe disease and sequelae, the

development of a test that can distinguish acute from chronic infection is essential.

Moreover, a diagnostic test such as this would permit decisive treatment thus

minimising disease progression and further tissue damage. As a consequence, this

project aimed to address this issue by identifying chlamydial antigens that are

differentially expressed during acute, chronic and recovery stages of C. trachomatis

infection and to utilise these antigens to form the basis of a serological diagnostic assay

for the purpose of discriminating acute versus chronic C. trachomatis infection.

To accomplish this objective, male and female patient cohorts were recruited and

assigned to one of the following categories:

(i) first-time C. trachomatis infection (diagnosed by PCR) estimated to

have been acquired within the preceding 4 months

(ii) history of single C. trachomatis infection (diagnosed by PCR) and

treated more than 12 months previously

(iii) history of two C. trachomatis infections (diagnosed by PCR), with

the most recent estimated to have been acquired within the preceding

4 months (females only)

(iv) females with current presumptively diagnosed pelvic inflammatory

disease based on clinical history, a medical history and with a current

diagnosis of C. trachomatis by PCR or serological evidence of

previous C. trachomatis infection

(v) C. trachomatis-negative females diagnosed with infertility due to

other causes (laparoscopically confirmed endometriosis), with no

previous history or positive C. trachomatis serology (females only)

- 49 -


(vi) uninfected adult males or females who were serologically C.

trachomatis-negative and

(vii) uninfected male or female children (<16yo) who were serologically

negative for C. trachomatis.

In response to chlamydial infection, humoral immunity, in the form of secretory IgA

antibodies, primarily targets MOMP, the major immunogen of the infectious elementary

body in an attempt to opsonise and destroy the pathogen. Failure by the humoral

immune response to resolve the infection further facilitates chlamydial infiltration

whereby current antibody responses alone are insufficient to clear what is now an acute

infection. The host immune response occurs within 1-2 days and is characterised by

inflammation and predominant mucosal infiltration of phagocytes, neutrophils, T-cell

lymphocytes and macrophages (Williams et al., 1981). Accessible EBs are

phagocytosed by neutrophils whilst control of the infection by T cells occurs via cell-

mediated destruction of the infected host cells (Barteneva et al., 1996). Whilst an acute

chlamydial infection can generally be resolved within ~14 days post infection (pi), the

majority of circulating antibodies generated from the primary immune response can be

serologically detected four months later. In contrast, single C. trachomatis infections

that have been cleared more than 12 months previously produce waning antibody

responses as antigens are no longer continually presented to the host immune system for

humoral destruction. Upon comparison to an acute infection, multiple chlamydial

infections generate a more rapid proliferation, production and differentiation of larger

quantities of antibodies. This heightened inflammatory immune response, like chronic

infection which is resultant from either exogenous or endogenous influences, can elicit

PID and further incite inflammatory damage and adverse pathological sequelae (Pal et

al., 1998; Darville et al., 2003). To demonstrate specificity and exclusivity for chronic

chlamydial infection, women with endometriosis, an established chronic cause of

infertility due to other origins, were included in this study as infertile controls. In

addition, male and female children under 16 years of age were used as negative controls

given the increased confidence of a nil history for C. trachomatis. In conclusion, the

- 50 -


remainder of this chapter fully describes the male and female patient cohorts and tests

used to assign study participants to their various groups.

- 51 -


2.1 MATERIALS AND METHODS

A. Whole Blood Collection

Samples used for this project were collected over two periods: (1) 1998 – 2002 (batch

#1) and (2) 2006 (batch #2). During the 1998 – 2002 period, whole blood was collected

in heparin coated vials by peripheral venipuncture from participants attending the

Brisbane Sexual Health Clinic who had a current or previous diagnosis for C.

trachomatis and from patients attending the Wesley Hospital IVF Clinic for infertility

investigations. During the 2006 period (batch #2), peripheral whole blood was

collected in serum tubes from patients attending the Brisbane Family Planning Clinic

for routine sexual health checks. Specimens were centrifuged at 1500rpm for 10

minutes at 4°C and the serum removed.

B. Sample Storage

All samples were stored at -80°C. Batch #2 samples were aliquoted and frozen,

however batch #1 samples were not sub-aliquoted. As a result, many were

freeze/thawed several times (5 – 10).

C. Analysis of Antibodies to C. trachomatis

All patient samples from batch #1 were assayed for C. trachomatis IgG using

commercial EIA (Labsystems Chlamydia trachomatis IgG EIA) with a 1/10 dilution as

per manufacturer’s instructions. A positive result was based on a ratio comparing

sample absorbance and the designated cut-off value (<1.0 negative, <1.4 equivocal,

<2.5 positive and >2.5 high positive). Batch #2 samples were classified as positive with

a titre of 64.

- 52 -


D. Analysis for Antibodies to C. pneumoniae

C. pneumoniae serology was determined using a commercial MIF assay (Focus

Diagnostics (USA) Chlamydia MIF IgG) according to the manufacturer’s instructions.

Positive titres for C. pneumoniae for batch #1 patients ranged from 256 – 1024, and

batch #2 patients were designated positive with a titre of 64.

E. Testing for Chlamydial HSP60

Serology for anti-cHSP60 antibodies was performed using the Medac cHSP60-IgG-

ELISA (Medac Hamburg, Germany) commercial assay according to the manufacturer’s

instructions. The sample/cutoff ratio of < 0.9 was designated negative, 0.9 – 1.1 was

equivocal and > 1.1 was classified positive.

F. PCR Testing for the Presence of C. trachomatis

Blood specimens obtained from patients attending the Brisbane Sexual Health Clinic

and Family Planning Clinic were only collected where a confirmed diagnosis of C.

trachomatis (by PCR) was available or where a previous diagnosis of C. trachomatis

(by PCR or prior to 1997, by DFA) was recorded in the patient’s medical history. PCR

testing (Roche, Amplicor) was performed according to the manufacturer’s instructions.

G. Assignment of Patients to Groups

Samples were assigned to various groups based on patient clinical histories for

chlamydial infection (Sections 2.2 and 2.3). Uncertainty as to the time of infection

and/or diagnosis resulted in the patient sample being omitted from the study. In

addition, patient groups containing less than three participants were also excluded from

study participation.

- 53 -


H. Project Ethics

Informed consent was obtained from all participants and the study was approved by the

human ethics committees of the Queensland University of Technology, the Wesley

Hospital Brisbane, Queensland, the Prince Charles Hospital Brisbane, Queensland and

the Family Planning Clinic Brisbane, Australia.

2.2 Female Cohorts

Individuals were assigned to one of seven groups according to the following criteria:

• Group I – C. trachomatis positive females diagnosed (by PCR) with first-time C.

trachomatis infection estimated to have been acquired within the preceding 4

months (n = 10). All were treated (by antibiotics) following PCR diagnosis.

• Group II – C. trachomatis positive females diagnosed (by PCR) with first-time

C. trachomatis infection estimated to have been acquired more than 12 months

previously (n = 12). All were treated following PCR diagnosis.

• Group III – C. trachomatis positive females diagnosed (by PCR) with a history

of two C. trachomatis infections, with the most recent estimated to have been

acquired within the preceding 4 months (n = 5). All were treated (by antibiotics)

following PCR diagnosis.

• Group IV - C. trachomatis positive (by PCR) PID females with presumptively

diagnosed pelvic inflammatory disease based on clinical history, a confirmed

medical history and serological evidence of previous C. trachomatis infection (n

= 11). All were treated (by antibiotics) following PCR diagnosis.

- 54 -


- 55 -

• Group V – C. trachomatis negative females visually diagnosed with

endometriosis due to other causes, with no previous history and negative C.

trachomatis serology (n = 18).

• Group VI – C. trachomatis uninfected adult females who were serologically C.

trachomatis negative (n = 13).

• Group VII - C. trachomatis uninfected female children (<16 yo) who were

serologically negative for C. trachomatis (n = 8).

Groups 1 – IV patient samples were obtained from women attending either the Brisbane

Sexual Health Clinic for first-time or follow-up diagnosis and management of C.

trachomatis infections diagnosed by urine or swab-based Amplicor PCR (Roche

Diagnostics), or from the Queensland Family Planning Clinic (Brisbane, Australia).

Group V samples were obtained from women presenting to the Wesley Hospital

Department of Reproductive Medicine (Brisbane, Australia) for laparoscopic and

falloposcopic investigation of infertility with diagnosis based on visual findings.

Group VI samples were collected from patients attending the Brisbane Red Cross Blood

Bank.

Group VII samples were obtained from female children attending the Queensland

Medical Laboratory (Brisbane, Australia).

Tables 2.1 – 2.4 summarise patient group assignment, clinical history and

symptomology and C. trachomatis PCR, HSP60 and serological status for each study

participant.


Table 2.1. Summary of assignment of female patients to groups I and II. All were PCR positive for C. trachomatis.

SEROLOGICAL STATUS PATIENT

GROUP PATIENT

I.D.

C. trachomatis INFECTION

(months)(1) C. tr IgG (EIA)

C. pn IgG (MIF)

HSP60 (Medac)

CLINICAL HISTORY and SYMPTOMOLOGY

13578 1 +ve -ve -ve Asymptomatic 4081 1 -ve -ve -ve On HRT (2), previous ectopic pregnancy? 13817 1 +ve +ve -ve Asymptomatic contact, mild cervix discharge 8015 1 -ve +ve -ve Asymptomatic contact 13785 1 +ve -ve -ve Symptomatic, no OCP (3)

2361176 1 +ve +ve -ve Asymptomatic 2248900 1 -ve -ve -ve Asymptomatic 2180122 1 +ve +ve -ve Asymptomatic 1996360 1 -ve -ve -ve Asymptomatic

GR

OU

P I

QUTNP12* 1 +ve +ve -ve Asymptomatic 1580 51 +ve +ve -ve Symptomatic, OCP at time of diagnosis 10622 24 +ve -ve +ve Dysuria, abdominal pain, OCP 339 14 -ve +ve -ve Asymptomatic, current thrush, OCP 14697 156 +ve +ve -ve Symptomatic, depo (4), ?PID no C.tr isolated 10306 36 +ve -ve +ve Thrush, depo 18456 120 +ve +ve +ve Diagnosed 10 years ago 13619 96 -ve -ve -ve Current BV(5), discharge, HSV(6) Hx 10553 12 -ve -ve -ve Symptomatic, on OCP 11036 12 -ve +ve -ve No OCP, abdominal pain 17096 12 -ve -ve -ve Severe abdominal pain 11796 12 -ve +ve -ve Vaginal discharge, OCP

GR

OU

P II

QUTNP007* >12 -ve +ve -ve Asymptomatic

(1)Duration of C. trachomatis infection in months at time of blood collection (2) Hormone replacement therapy (3) Oral contraceptive pill (4) Depo provera (5) Bacterial vaginosis (6) Herpes simplex virus * sample collected 2006

- 56 -


Table 2.2. Summary of assignment of female patients to groups III and IV. All were PCR positive for C. trachomatis.


GROUP PATIENT

I.D.


(months)(1) C. tr IgG (EIA)

C. pn IgG (MIF)

HSP60 (Medac)


4020 <1 +ve -ve +ve Dx Ctr 11 yrs ago, re-exposed 5 days ago, abdominal pain 1 yr ago

13108 <1 +ve -ve -ve Dx 1/99 and 1/00, symptomatic, HSV, no OCP 9908 <1 +ve -ve -ve Dx 10/97 and 7/99, symptomatic, no OCP 1132 <1 +ve +ve -ve Symptomatic, repeated infections G

RO

UP

III

QUTNP11* <1 +ve -ve +ve Asymptomatic, multiple infections

11011 >12 +ve -ve -ve Past hx C.tr, PID on several occasions, currently pregnant

13114 >12 +ve +ve -ve PID at 18 yrs of age

113 >12 +ve +ve -ve Symptomatic, repeated infections over 3 yrs, presumptive PID

12581 14 +ve -ve -ve Hx PID last 12 mths, symptomatic for 1 yr,OCP IVF012 >12 +ve +ve +ve (R) tube damage, Hx miscarriage IVF013 >12 +ve -ve -ve Severe bilateral tubal damage, C.tr in FT IVF017 >12 +ve -ve -ve History of PID IVF029 >12 +ve +ve +ve History of PID, normal tubes IVF031 >12 -ve +ve -ve Adhesions and bilateral tubal scarring IVF032 >12 +ve -ve -ve Hx of PID, severe bilateral damage & adhesions

GR

OU

P IV

QUTNP004* >12 -ve +ve -ve PID, pelvic pain (1)Duration of C. trachomatis infection in months at time of blood collection * sample collected 2006 FT = Fallopian tube

- 57 -


Table 2.3. Summary of assignment of female patients to group V. All were PCR negative for C. trachomatis.

SEROLOGICAL

STATUS PATIENT GROUP

PATIENT I.D.

C. tr PCR

(Roche) C. tr IgG (EIA)

C. pn IgG (MIF)

HSP60 (Medac)


IVF008 -ve -ve +ve -ve Infertile due to endometriosis IVF019 -ve -ve +ve +ve Infertile due to endometriosis IVF018 -ve -ve -ve -ve Infertile due to endometriosis IVF022 -ve -ve +ve -ve Infertile due to endometriosis IVF034 -ve -ve -ve -ve Infertile due to endometriosis IVF010 -ve -ve -ve -ve Infertile due to endometriosis

IVF040 -ve -ve -ve -ve Infertile due to endometriosis IVF009 -ve -ve -ve -ve Infertile due to endometriosis IVF016 -ve -ve -ve -ve Infertile due to endometriosis IVF021 -ve -ve +ve -ve Infertile due to endometriosis IVF027 -ve -ve -ve -ve Infertile due to endometriosis IVF034 -ve -ve -ve -ve Infertile due to endometriosis IVF018 -ve -ve -ve -ve Infertile due to endometriosis IVF026 -ve -ve +ve -ve Infertile due to endometriosis IVF035 -ve -ve -ve -ve Infertile due to endometriosis IVF041 -ve -ve +ve -ve Infertile due to endometriosis IVF020 -ve -ve +ve -ve Infertile due to endometriosis

GR

OU

P V

IVF011 -ve -ve +ve -ve Infertile due to endometriosis

- 58 -


- 59 -

Table 2.4. Summary of assignment of female patients to groups VI and VII. All were PCR negative for C. trachomatis.


GROUP PATIENT

I.D.

C. tr PCR

(Roche) C. trachomatis IgG (EIA)

C. pneumoniae IgG (MIF)

HSP60 (Medac)


4120081 -ve -ve +ve -ve negative control 4120335 -ve -ve +ve +ve negative control Mary -ve -ve -ve -ve negative control 4120647 -ve -ve +ve -ve negative control 4131367 -ve -ve -ve -ve negative control 4131329 -ve -ve -ve -ve negative control

4374044 -ve -ve -ve -ve negative control 4373968 -ve -ve -ve -ve negative control 4120057 -ve -ve -ve -ve negative control 4374317 -ve -ve +ve -ve negative control 4131135 -ve -ve -ve -ve negative control 4373969 -ve -ve -ve -ve negative control

GR

OU

P V

I

4374766 -ve -ve -ve -ve negative control CF1 -ve -ve -ve -ve negative control CF2 -ve -ve -ve -ve negative control CF3 -ve -ve -ve -ve negative control CF4 -ve -ve -ve -ve negative control CF5 -ve -ve -ve +ve negative control CF6 -ve -ve +ve -ve negative control CF7 -ve -ve +ve -ve negative control G

RO

UP

VII

CF8 -ve -ve -ve -ve negative control


- 60 -

2.3 Male Cohorts

Individuals were assigned to one of four groups according to the following criteria:

• Group MI – C. trachomatis-positive males diagnosed (by PCR) with first-time

C. trachomatis infection estimated to have been acquired within the preceding 4

months (n = 5). All were treated (by antibiotics) following PCR diagnosis.

• Group MII – C. trachomatis-positive males diagnosed (by PCR) with first-time


previously (n = 12). All were treated (by antibiotics) following PCR diagnosis.

• Group MIII – C. trachomatis uninfected male controls who were uninfected and

serologically C. trachomatis negative (n = 4).

• Group MIV – C. trachomatis uninfected male children (<16 yo) who were

serologically negative for C. trachomatis (n = 12).

Groups MI – MIII samples were obtained from men attending either the Brisbane

Sexual Health Clinic for first-time or follow-up diagnosis and management of C.

trachomatis infections diagnosed by urine or swab-based Amplicor PCR (Roche

Diagnostics), or from the Queensland Family Planning Clinic (Brisbane, Australia).

Group IV samples were collected from male children attending the Queensland Medical

Laboratory (Brisbane, Australia).

Tables 2.5 and 2.6 summarise patient groups, clinical history and symptomology and

status for C. trachomatis PCR, HSP60 and serology status for each study participant.


Table 2.5. Summary of assignment of male patients to groups MI and MII. All were PCR positive for C. trachomatis.

SEROLOGICAL


PATIENT I.D.


(months)(1) C. t IgG (EIA)

C. pn IgG (MIF)

HSP60 (Medac)


7292 1 -ve +ve -ve Symptomatic, HSV 13986 <1 +ve +ve -ve Asymptomatic 14483 1 -ve -ve -ve Symptomatic 12390 1 -ve -ve -ve Low fertility - immotile sperm, asymptomatic

GR

OU

P M

I

QUTNP005* 1 +ve +ve +ve Symptomatic 1237 25 -ve -ve -ve Symptomatic 13664 12 -ve +ve +ve Symptomatic 8956 19 +ve +ve -ve Asymptomatic, previous genital warts 10019 15 -ve +ve -ve Asymptomatic 7803 23 -ve +ve -ve Asymptomatic 13651 >12 -ve +ve -ve Hx of Ct and genital warts, current HSV 2305 24 +ve +ve -ve Asymptomatic 8963 20 -ve +ve -ve Asymptomatic 14035 168 -ve +ve -ve Asymptomatic 11917 12 -ve -ve -ve Symptomatic 12367 12 -ve -ve -ve Asymptomatic

GR

OU

P M

II

QUTNP017* 28 -ve +ve +ve Asymptomatic (1)Duration of C. trachomatis infection in months at time of blood collection * sample collected 2006

- 61 -


Table 2.6. Summary of assignment of male patients to groups MIII and MIV. All were PCR negative for C. trachomatis.

SEROLOGICAL


PATIENT I.D.

C. tr PCR

(Roche) C. tr IgG (EIA)

C. pn IgG (MIF)

HSP60 (Medac)


Control 1 -ve -ve +ve -ve negative control Control 2 -ve -ve +ve -ve negative control Control J -ve -ve +ve +ve negative control

GR

OU

P M

III

Oras179 -ve -ve +ve -ve negative control CM1 -ve -ve -ve -ve negative control CM2 -ve -ve -ve -ve negative control CM3 -ve -ve -ve -ve negative control CM4 -ve -ve -ve -ve negative control CM5 -ve -ve -ve -ve negative control CM6 -ve -ve +ve -ve negative control CM7 -ve -ve +ve -ve negative control CM8 -ve -ve -ve -ve negative control CM9 -ve -ve -ve -ve negative control CM10 -ve -ve +ve -ve negative control CM11 -ve -ve -ve -ve negative control

GR

OU

P M

IV

CM12 -ve -ve +ve -ve negative control

- 62 -

Chapter 3: Serological Identification of Potential Diagnostic Markers in Females

- 63 -

CHAPTER 3

Serological Identification of Potential Diagnostic Markers in

Females


- 64 -

3.0 INTRODUCTION

The asymptomatic nature of chlamydial infection renders diagnosis difficult,

particularly when attempting to differentiate acute and chronic infection. An untreated

Chlamydia trachomatis infection can become chronic, result in disease sequelae such as

salpingitis and pelvic inflammatory disease (PID), and ultimately culminate in tubal

occlusion and infertility (Cates and Wasserheit, 1991; Cohen and Brunham, 1999).

Current diagnostic tests for C. trachomatis such as nucleic acid amplification testing,

antigen detection and serological methods have variable performance capabilities with

respect to sensitivity, specificity and disease/infection stages. The use of PCR as a

diagnostic tool is to some extent limited as PCR is restricted to detecting selected

Chlamydia DNA sequences from readily accessible sites of the genital tract. Moreover,

specimen collection is routinely sampled from the lower genital tract, hence infections

in the fallopian tube where inflammatory damage is most significant, escape detection.

Importantly, since a definitive chlamydial marker of chronic urogenital C. trachomatis

infection has yet to be identified, this method does not have the ability to differentiate

between acute and chronic infection. Other serological assays aim to discriminate the

various stages of C. trachomatis infection through identification of key antigens (eg.

HSP60), however their efficacy is impeded due to cross-reactivity between chlamydial

species (Land et al., 2003) and the subsequent antibody response against the target

antigen is not restricted to patients with a specific stage of infection (Gazzard et al.,

2006). Consequently, several enzyme-linked immunosorbent assays have been

developed that incorporate recombinant C. trachomatis antigens to improve species

specificity (Labsystems Research Laboratory, Helsinki, Finland; Medac, Hamburg,

Germany). The Labsystems ELISA employs synthetically derived C. trachomatis-

specific epitopes of MOMP, whilst the Medac ELISA utilises a recombinantly produced

Chlamydia-specific fragment of LPS. The Labsystems MOMP and Medac LPS tests

have the ability to diagnose C. trachomatis infection but lack the ability to identify the

infection status ie. resolved, acute, recurrent or chronic. Furthermore, the Medac LPS

assay has been reported to have low sensitivity and high cross-reactivity between

chlamydial species (Bas et al., 2001).


- 65 -

Numerous studies have demonstrated a correlation between antibody responses to

chlamydial heat shock protein 60 (cHSP60) and pathologic sequelae in women

(Brunham et al., 1992; Dieterle and Wollenhaupt, 1996; Eckert et al., 1997), including a

significant association between the presence of antibodies to cHSP60 and PID (Eckert

et al., 1997; Peeling et al., 1997; Witkin et al., 1998). An association of antibodies to

cHSP60 and tubal factor infertility has also been reported (Arno et al., 1995; Claman et

al., 1997; Freidank et al., 1997). The identification of a potential C. trachomatis

infection stage discriminator led to the development of a commercial ELISA screening

test based on cHSP60 (Medac, Hamburg, Germany). Two studies subsequently

evaluated the diagnostic potential of the Medac cHSP60 ELISA test and demonstrated

conflicting results (Bax et al., 2004; Gazzard et al., 2006). Bax et al. (2004) showed an

11% increase in anti-cHSP60 antibody response in women with tubal pathology (27%)

compared to women without tubal pathology (16%) and the control group comprising

pregnant women (4.8%). In contrast, a more recent study showed an 8% decreased

incidence of cHSP60 reactivity in the PID/Tubal Damage group (20%) compared to

28% in acute patients (Gazzard et al., 2006). Consequently, the ability of the cHSP60-

based assay to distinguish various C. trachomatis infection stages may be limited.

Whilst all these tests have the capacity to identify chlamydial infection, none can

reliably differentiate between acute and chronic C. trachomatis infection.

As a result of the inability of previous tests to reliably discriminate between stages of

infection and disease caused by C. trachomatis infection, this study aimed to identify

antigens capable of differentiating various states of chlamydial infection ie. acute versus

chronic C. trachomatis infection. To identify differential antigens, samples obtained

from seven patient groups with various histories of chlamydial infection (uninfected,

acute, post acute, multiply infected and those with disease sequelae), were used to probe

protein extracts of HEp2 cells infected with C. trachomatis serovar L2 (I) and non-

infected HEp-2 cells (UI) using standard Western blotting techniques. Four chlamydial

antigens were identified that showed differential banding patterns between the C.


- 66 -

trachomatis-infected patient groups which in various combinations, could potentially be

used to discriminate acute and suspected chronic C. trachomatis infections.


- 67 -


3.1.1 Strategy Used to Identify Novel Diagnostic Markers

The overall strategy employed to identify various antibody responses to specific

chlamydial antigens capable of differentiating between stages of C. trachomatis

infection in women. Samples from seven patient groups were collected:

• female patients diagnosed by Amplicor PCR and treated for recently acquired,

first-time C. trachomatis infection within the previous 4 months

• female patients previously diagnosed by Amplicor PCR and treated for first-time


previous

• female patients with a history of presumptive PID or infertility and evidence of

prior C. trachomatis infection through serological and Amplicor PCR diagnosis

• female patients undergoing infertility management and visually diagnosed with

endometriosis with no past history of C. trachomatis infection

• uninfected female adults with no serological evidence of C. trachomatis infection

and

• uninfected female children (<16 yo) with no serological evidence of C.

trachomatis infection.

Using Western blotting methods, samples from each group were used to probe crude

protein extracts of HEp2 cells infected with C. trachomatis serovar L2, with

differentiating chlamydial proteins visualised and subsequently identified via

immunoprecipitation, N-terminal sequencing and Mass Spectrometry (MS).


- 68 -

3.1.2 Patient Groups Analysed

Samples from all seven female patient groups were analysed for this aspect of the

project, however only those samples collected from 1998 – 2002 (batch #1) were

screened (Chapter 2, Section 2.2).

3.1.3 C. trachomatis Cell Culture

HEp-2 cells were cultured in DMEM supplemented with 5% heat-inactivated foetal calf

serum (FCS), 0.002% gentamycin, 10µL cycloheximide (1g/mL), 5% CO2 and upon

90% confluency, cells were infected with 2mL of C. trachomatis L2 strain. Six hours

post infection (pi), the infection status was observed under light microscopy (Leica). It

has previously been shown that C. trachomatis inclusions can be relatively easily

visualised unstained, under light microscopy. This visualisation method was used to

determine the infection levels of the cultures. The medium was then discarded and

10mL of fresh 5% FCS-DMEM was added. At 30 hours pi, Chlamydia plus host cells

were extracted as follows. Trypsinised cells were resuspended in 48mL phosphate

buffered saline (PBS), centrifuged at 1000 rpm for 5 minutes at 4°C and pelleted. The

pellet was resuspended in 3mL of PBS, repelleted for 15 seconds, and resuspended in

3mL 2X sample buffer (0.09M Tris-HCl pH 6.8, 20% glycerol, 2% SDS, 0.1M DTT

and 0.02% bromophenol blue) and stored at -20ºC until required for use in SDS-

PAGE/Western blot experiments. In addition to the infected culture (I), HEp-2 only

cells (UI) were maintained in 5% FCS-DMEM, and proteins extracted as described

above.

3.1.4 Identification of Reactive C. trachomatis Proteins in Patient Samples by

Western Blotting

To identify potential diagnostic chlamydial antigens, individual patient samples from

each group were probed against standardised protein extracts of HEp2 cells infected

with C. trachomatis serovar L2 (I) and non-infected HEp-2 cells (UI). Briefly, 10μg of


- 69 -

each protein extract was loaded onto 12.5% polyacrylamide SDS-PAGE gels and

electrophoresed at 110 volts for 100 minutes at room temperature. Proteins were

transferred at 4ºC, 100 volts for 1 hour to Hybond C Extra nitrocellulose membranes

(Amersham Biosciences) using 3-(cyclohexylamino)-1-propane sulphonic acid (CAPS)

buffer (10mM CAPS, 10% methanol, pH 11.0) and the membranes blocked (5% skim

milk powder in PBS containing 0.1% Tween 20 (SM-PBS-T)) for 1 hour at room

temperature with rocking. Patient samples from the seven patient groups were diluted

1:1000 in SM-PBS-T and incubated with the membranes at room temperature for 1 hour

with shaking. After washing with PBS-T (PBS, 0.1% Tween 20), membranes were

incubated with the secondary antibody, conjugated rabbit anti-human horseradish

peroxidase (HRP) IgG (Roche) diluted 1:4000 in SM-PBS-T at room temperature for 1

hour with shaking. Membranes were washed 4X for 15 minutes in PBS-T then detected

via chemiluminescence (Amersham Biosciences ECL Plus Detection Kit). The

presence of unique bands in the C. trachomatis L2-infected protein extract (I) not

present in the protein control (UI) were identified and subsequently categorised as

differential.

3.1.5 Immunoprecipitation of Chlamydia-Specific Antibodies

HEp-2 cell monolayers infected with C. trachomatis L2 for 24 hours were washed in

cold PBS, trypsinised and lysed with 5mL 1% TritonX100. A 1mL aliquot of the cell

lysate was centrifuged at 12000g for 10 minutes at 4°C. The supernatant was mixed

with 20μL of a single patient sample and incubated at 4°C for 4 hours with rotation.

During the lysate/antibody incubation, protein G attached to micro beads (Amersham

Biosciences) was prepared as per manufacturer’s instructions. The coupled

lysate/antibody mix was directly added to 200μL buffered protein G suspension and

incubated with rotation for 2 hours at 4°C. The mix was pelleted by centrifugation

(6000g for 40 seconds), resuspended in 1mL of wash buffer (50mM Tris pH 7.6,

150mM NaCl, 0.1% NP40, 0.03% SDS) and then centrifuged at 12000g for 30 seconds.

The supernatant was discarded and the pellet washed twice in wash buffer before adding


- 70 -

50μL of 2X sample buffer giving a final volume of ~100μL. The sample was then

heated at 95°C for 10 minutes and stored at -20°C until required.

3.1.6 SDS-PAGE/ Western Blot of Immunoprecipitated Chlamydial Proteins

Proteins precipitated by the protein G beads were separated using a 12.5% SDS-PAGE

gel and transferred at 4°C, in CAPS buffer, to immobilon-P Polyvinylidene Fluoride

(PVDF) membrane prepared as per manufacturer’s instructions (Amersham

Biosciences). A small segment of the membrane containing one marker lane and a

section of the immunoprecipitated proteins was reserved for Western blotting whilst the

remaining membrane was stained with 0.1% Coomassie blue stain (1g Coomassie blue,

40% methanol and 10% glacial acetic acid) for 30 minutes. The Coomassie stained

membrane was washed in 90% methanol until all background staining was removed.

Western blotting was performed on the small membrane segment as described in section

3.1.4. The Coomassie stained membrane and Western blotted section were aligned and

compared to identify the antigenic targets. Once identified on the Coomassie stained

PVDF membrane, these differential targets (labelled as 1A, 1B and 1C) were excised

from the membrane and sent for N-terminal sequencing. Protein identification was

undertaken by the Australian Proteome Analysis Facility (APAF) at Macquarie

University in Sydney. Protein samples attached to PVDF membrane were subjected to

7 cycles of N-terminal sequencing via automated Edman degradation (Applied

Biosystems 494 Procise Protein Sequencing System). A 10pmol β-lactoglobulin

standard was used to verify sequencer performance.

3.1.6.1 N-Terminal Sequence Analysis

The protein sequences were analysed using the National Center for Biotechnology

Information (NCBI) Basic Local Alignment Search Tool (BLAST), the Expert Protein

Analysis System (ExPASy) and The Institute for Genomic Research (TIGR) databases.


- 71 -

3.1.7 Antigenic Target Identification and Verification via Mass Spectrometry

NU-PAGE 4%-12% Bis-Tris 1 well SDS-PAGE gels (Invitrogen) were used to separate

100μL of extracted chlamydial protein in 1X MOPS buffer (50mM 3 N-morpholino

propane sulfonic acid, 3.5mM SDS, 50mM EDTA, pH 8.0) at 4°C, 120 volts for 4

hours. A small segment of gel containing a section of protein was transferred in CAPS

buffer to Hybond C Extra nitrocellulose membrane (Amersham Biosciences) and

reserved for standard Western blotting (section 3.1.4) using a single, strong-reacting

patient sample. The remaining gel was Coomassie stained for 30 minutes, destained

(40% methanol and 10% glacial acetic acid) until background was removed and

rehydrated in water to restore its size to that of the blotted gel section. The Coomassie

stained gel and Western blotted membrane were aligned and compared to identify the

antigenic targets. Each target (designated samples A and B) was excised from the

Coomassie gel. In preparation for MS identification, each protein band was dried and

in-gel digested, reduced and alkylated. Briefly, bands were cut into small pieces and

placed into pre-washed (50% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA))

eppendorf tubes. To each gel piece, 200μL of 200mM NH4HCO3/50% ACN was added

then incubated at 37°C for 45 minutes and supernatant discarded (X2). Destained gel

pieces were dried in Speedivac (Savant) on low heat for 1 hour. For reduction, 100μL

of 20mM dithiotreitol (DTT) in 25mM NH4HCO3 was added. Subsequent to incubation

(1 hour at 65°C) the supernatant was discarded. To alkylate protein samples, 100μL of

50mM iodoacetamide (IAA) in 25mM NH4HCO3 was added and incubated in the dark

for 40 minutes at 37°C. Gel pieces were then washed twice in 200μL 25mM NH4HCO3

for 15 minutes at 37°C, with supernatant discarded each time. Samples were reduced

and gel fragments placed in Speedvac (Savant) for 1 hour. Gel pieces were rehydrated

by the addition of 20μL 25mM NH4HCO3, pH 8, containing 0.02μg/μL trypsin and

incubated at room temperature for 1 hour. An additional 50μL 25mM NH4HCO3 in

50% ACN was added to each gel slice and incubated at 37°C for 18 hours. To recover

peptides, the tryptic supernatant was collected and 50μL of 0.1% TFA was added to

each gel slice and incubated for 45 minutes at 37°C. The supernatant was again

collected and pooled with the original decanted supernatant and washed twice with


- 72 -

TFA. Peptides were concentrated and spotted onto a MS plate and analysed. Fragment

patterns were matched to potential proteins using Mascot software.

3.1.8 Antibody Reactivity to Recombinant Major Outer Membrane Protein

(MOMP)

Recombinant MOMP (serovar D with a 6-histidine tag) was kindly provided by Chris

Barker (QUT, Brisbane, Australia) to verify that the antibody reactivity to band C, a

44.6kDa protein (potential candidates CT157 or CT423) was not MOMP, a highly

immunoreactive 42.4kDa protein. Briefly, 10μg of host cell proteins from non-infected

HEp-2 cells (UI), HEp-2 cells infected with C. trachomatis serovar L2 (I), and 10μL of

recombinant MOMP (0.5mg/mL) were loaded onto a 12.5% SDS-PAGE gel. The

remaining Western blot protocol and chemiluminescence detection were performed as

described in section 3.1.4, using a single, strong-reacting patient sample.

3.1.9 Species and Serovar Specificity of the Identified Novel Markers

To determine the species and serovar specificity of our identified antigenic targets,

samples from patient groups I, II and IV were screened via SDS-PAGE/Western blot

against 10μg of C. trachomatis serovars L2, D, K, and C. pneumoniae (AO3). All C.

trachomatis and C. pneumoniae infections, protein extractions and Western blots were

performed as described in sections 3.1.3 and 3.1.4. SDS-PAGE gels (12.5%) were used

for this comparative study. Protein alignments of the four target antigens

(CT157,CT423, CT727 and CT3996) were performed using National Center for

Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), the

Expert Protein Analysis System (ExPASy), TIGR (www.tigr.org) and Sanger Institute

C. trachomatis L2 proteomic databases.

3.1.10 Diagnostic Potential of Antigenic Targets


- 73 -

The results obtained from the four identified target antigens were combined into a panel

format to evaluate their predictive potential for diagnosing the different stages of

chlamydial infection. Groups I and II were combined to assess the prospect of

diagnosing an acute/recovered acute infection, whilst group IV was used to determine

the potential of an assay for the detection of chronic infection/disease. Sensitivity,

specificity, positive predictive value (PPV) and negative predictive value (NPV) were

calculated for each test format as follows: Sensitivity = True Positive/(True positive +

False Negative) x 100%. Specificity = True Negative/(True Negative + False Positive)

x 100%. PPV = True Positive/(True Positive + False Positive) x 100%. NPV = True

Negative/(True Negative + False Negative) x 100%. Statistical calculation of 95%

confidence intervals (CI 95%) and standard error for PPV and NPV and the Chi-squared

value for each individual test format was performed using the 2002 Diagnostic

Effectiveness Program (http://home.clara.net/sisa/diagnos.htm ).


- 74 -

3.2 RESULTS

3.2.1 Immunoreactivity Patterns of Females with C. trachomatis Genital

Infections

Figure 3.1 shows typical Western blot profiles when samples from each female group

were used to probe uninfected (UI) and infected (I) cell preparations and exposed for 30

seconds after chemiluminescence detection. Four bands, A (>113kDa), B (72.4kDa), C

(44.6kDa) and D (13.5kDa) were differential between patient groups when probed by

samples with the majority of C. trachomatis-infected patients responsive to not less than

two of the four antigens (Tables 3.1 and 3.2). Of the four bands, the most commonly

reactive were C (65%) and D (76%) (Table 3.3). Band A was present in 10% of patient

samples in group IV (n = 10), compared to 56%, 45% and 75% of samples in groups I

(n = 9), II (n = 11) and III (n =4) respectively. Of the 40 negative control patients

tested, only one (in group V) reacted with this antigen. Band B was shown to be

reactive in 75% of group III and 80% of group IV samples. In contrast, groups I and II

demonstrated a reactivity of 56% and 27% respectively. Furthermore, 3/40 negative

controls also reacted to band B. Interestingly, group IV samples that were reactive to

band B also recognised band C. Moreover, antibody reactivities of samples in groups I,

II and III to band C were 56%, 45% and 100% respectively. In addition, 17% of

negative control group V samples reacted to band C. All group IV samples were

reactive to band D. In addition, 89%, 45% and 100% of samples in groups I, II and III

respectively showed antibody reactivity to band D. Of the three control groups, only

group VII showed zero reactivity to any of the four bands. Antibody levels to C.

pneumoniae and C. trachomatis for all groups were determined by MIF and EIA

respectively prior to Western blot analysis. C. trachomatis EIA antibody responses

generally reflected the infection status of each group. The overall percentages of

antibodies to C. pneumoniae were similar levels (25% - 55%) in adults. MIF testing

showed 25% of patient samples from the control group comprising children (group VII)

were positive for C. pneumoniae antibodies.


- 75 -

113

98

52

35

28

21

113

98

52

35

28

21

113

98

52

35

28

21

113

98

52

35

28

21

(a) (b) (c) (d)

(e) (f) (g)

Figure 3.1. Western blots of uninfected (UI) and infected (I) whole cell extracts probed

with samples from seven patient groups. Circled are the four identified differential

chlamydial antigenic bands designated A, B, C and D. Exposure time after detection

was 45 seconds.

UI I UI I UI I UI I

A A

BB

C

D

C

D D

C

A

B

C

D

kDa kDa kDa kDa

UI IUI I kDa kDa

113

98

52

35

28

21

113

98

52

35

28

21

113

98

52

35

28

21

UI I kDa

C. trachomatis- infected samples

Uninfected samples – Negative Controls

Group I Group II

Group VII Group VI Group V

Group IV Group III


- 76 -

Table 3.1. Individual patient profiles of differential bands A, B, C and D after a single

sample was probed against uninfected and C. trachomatis-infected cells for groups I -

IV.

PATIENT GROUP

PATIENT I.D. A B C D

13578 - - - + 4081 - - - - 13817 + + + + 8015 + - - + 13785 + - + + 2361176 + + + + 2248900 - + - + 2180122 + + + +

I

1996360 - + + + 1580 - - - - 10622 + + + - 339 - - - - 14697 - - + + 10306 + - - - 18456 + + + + 13619 + - + + 10553 + + - + 11036 - - + + 17096 - - - -

II

11796 - - - - 4020 + + + - 13108 - + + + 9908 + + + +

III

1132 + - + + 11011 - + + + 13114 - + + + 113 + + + + 12581 - + + + IVF012 - + + + IVF013 - - - + IVF017 - + + + IVF029 - + + + IVF031 - - - +

IV

IVF032 - + + + + = band present in Western blot - = band absent in Western blot

Differential Bands


- 77 -

Table 3.2. Individual patient profiles of differential bands A, B, C and D subsequent to

a single patient sample probed against uninfected and C. trachomatis-infected cells for

groups V – VII.

PATIENT GROUP

PATIENT I.D. A B C D

IVF008 + + + + IVF019 - - - - IVF018 - - - - IVF022 - - + + IVF034 - - - - IVF010 - - - - IVF040 - - - - IVF009 - - - - IVF016 - - - - IVF021 - - + + IVF027 - - - - IVF034 - - - - IVF018 - - - - IVF026 - + - + IVF035 - - - - IVF041 - - - + IVF020 - - - -

V

IVF011 - - - - 4120081 - - - - 4120335 - + - + Mary - - - - 4120647 - - - - 4131367 - - - + 4131329 - - - - 4374044 - - - + 4373968 - - - - 4120057 - - - + 4374317 - - - + 4131135 - - - - 4373969 - - - +

VI

4374766 - - - + CF1 - - - - CF2 - - - - CF3 - - - - CF4 - - - - CF5 - - - - CF6 - - - - CF7 - - - -

VII

CF8 - - - - + = band present in Western blot - = band absent in Western blot

Differential Bands


- 78 -

Table 3.3. The percentage prevalence and estimated molecular weights of differential

bands A, B, C and D including C. trachomatis and C. pneumoniae serology for each

patient group.

DIFFERENTIAL BANDS (%)

EIA MIF (%) PATIENT GROUPS

A B C D Estimated Band Size (kDa) >113 72.4 44.6 13.5 C. tr C. pn

I (n = 9) 56 56 56 89 56 44 II (n = 11) 45 27 45 45 45 55 III (n = 4) 75 75 100 75 100 25 IV (n = 10) 10 80 80 100 90 50 V (n = 18) 6 11 17 28 0 44 VI (n = 13) 0 8 0 54 0 31 VII (n = 8) 0 0 0 0 0 25

3.2.2 Identification and Verification of Chlamydial Antigenic Targets

All three band samples (1A, 1B and 1C) excised from the PVDF membrane were

analysed via N-terminal sequencing and yielded 7 amino acids in the major sequence,

and varying numbers of amino acids in the minor sequence (Table 3.4). The BLASTP

algorithm was used to search for any sequence similarities in the NCBI Genbank

database. Amino acid sequences EVQLVES (samples 1A and 1B) and STAALGC

(sample 1C) showed 100% homology to Homo sapiens immunoglobulin heavy chain.

Although the major sequence of sample 1B proved to be IgG, of the 24 potential minor

protein sequences that were searched for in the NCBI database, two potential C.

trachomatis candidates, CT157 – Phospholipase D Endonuclease which matched 7/7

amino acids and CT423 – Cystathionine Beta Synthase (CBS) domain returned a

positive match to 5/7 amino acids. The major sequences of sample 1A ie. EVQLVES

and DILMTQS revealed an exact match to the N-terminus of IgG heavy chain and IgG

light chains respectively, however the first 5/7 residues of the second major signal

(DILMT) of sample 1A also corresponded to C. trachomatis protein CT727 – Metal


- 79 -

transport P-type ATPase. Whilst the EVQLVES and DILMTQS sequences were an

exact match across the N-terminal region, the other identified sequences were located

53 amino acids or more from the N-terminus.

Table 3.4. Major and minor amino acid sequences obtained by N-terminal sequencing

SAMPLE CYCLE # MAJOR SIGNAL MINOR SIGNAL 1 E, D Y, R, G, S 2 V, I L 3 Q, L V 4 L, M T 5 V, T Q 6 E, Q P

1A

7 S 1 E D, G, S 2 V P, A 3 Q, L H 4 L, M K, T 5 V S, T 6 E Q

1B

7 S N 1 G S, E 2 T 3 A 4 A 5 L 6 G

1C

7 C

Band A (>113kDa identified in the original Western blot screen) could not be identified

by N-terminal sequencing as the protein was not successfully precipitated from the C.

trachomatis host-cell lysate. MS analysis using Mascot software of the band A sample

however, identified two possible candidate proteins: CT147 (Conserved Hypothetical

Protein - 162.1kDa) and CT314 (DNA-directed RNA polymerase beta chain -

154.9kDa) (Table 3.5). MS analysis was also undertaken to verify the identity of band

B and the results suggested a match to CT396 (HSP70 – 70.8kDa). Band D could not


- 80 -

be identified by either N-terminal sequencing or MS due to the limited amount of

protein isolated from the SDS-PAGE gels.

Table 3.5. Identification of bands A, B and C by N-terminal sequencing (1) or Mass

Spectrometry (2). Molecular weights, predicted gene function and N-terminal sequence

residues where obtained, are indicated for each of the two potential candidates detected

for each band.

BAND GENE FUNCTION MW (kDa)

N-terminal Sequence

CT147 Hypothetical Protein (2) 162.1 None Band A

CT314 DNA-directed RNA polymerase (2) 154.9 None

CT727 Metal transport P-type ATPase (1) 70.5 DILMT Band B

CT396 Heat Shock Protein 70 (2) 71.1 None

CT157 Phospholipase D Endonuclease (1) 45.3 SPHTSQN Band C

CT423 Hypothetical Protein (1) 41.6 DPHTSQ

The amino acid sequences produced by N-terminal sequencing for CT157, CT423 and

CT727 were found to be internal to the protein. Original culture preparations of cells

infected with C. trachomatis used trypsin to detach infected host cells, thus the trypsin

treatment may have cleaved the N-terminal region from each of the three target

antigens. To confirm this notion, all three protein sequences were analysed using the

Peptide Cutter P (ExPASy) to determine potential trypsin cleavage sites (Figure 3.2).

Potential cleavage sites predicted by Peptide Cutter for CT423 matched the results

obtained by N-terminal sequencing. In contrast, the potential cleavage sites predicted

by the ExPASy program for CT157 and CT727 were located several amino acids N-

terminal of the first residue detected by the N-terminal sequencing analysis.


- 81 -

(a). CT157 Tryps_(100%) Tryps_(84.4%) | Tryps_(30.6%)| | Tryps_(86.1%)|| | Tryps_(100%) ||| | Tryps_(100%) | ||| | Tryps_(90.7%) | | ||| | Tryps_(84.6%) | | | ||| | Tryps_(40.6%) | | | | ||| | Tryps_(100%) | | | | | ||| | | | | | | | ||| | MSVQGSSSLKYSDLFKPPEPTSSTDSSKEPPKESAWKVVSHSRGRRRARSNPSPHTSQNT 1 ---------+---------+---------+---------+---------+---------+ 60 (b). CT423 Tryps_(87%) Tryps_(100%) | Tryps_(100%) Tryps_(100%) | | Tryps_(84.4%) | Tryps_(100%) | | | Tryps_(82.6%)| | | | | | || | SRIPLFTKSIDDITGMVLVKDLSPVYYKDPHTSQPLSSIAYPPLYTPEIRRASLLLQEFR 181 ---------+---------+---------+---------+---------+---------+ 240 (c). CT727 Tryps_(100%) Tryps_(100%) | | | HWIGYQALSSLLLILTFFLAGTPALIKSFEDILDRTVNIDILMTSAAFGSIFIGGALEGA 61 ---------+---------+---------+---------+---------+---------+ 120

Figure 3.2. Prediction of trypsin cleavage sites in proteins CT157, CT423 and CT727.

The Peptide Cutter Program was used to determine possible trypsin cleavage sites

within each of the three proteins (a) CT157, (b) CT423 and (c) CT727 identified by N-

terminal sequencing. Tryptic cleavage of a protein occurs at lysine (K) and arginine (R)

residues. The potential cut sites are indicated for each target protein including the

associated probability of the sequence being cleaved.


- 82 -


(MOMP)

To verify that antibody reactivity to the 44.6kDa protein (band C) in females was not

due to the highly immunoreactive 42.4kDa MOMP protein, a single patient sample was

probed against uninfected HEp-2 cells (UI), C. trachomatis-infected HEp-2 cells (I) and

recombinant MOMP (Figure 3.3). Evident in the infected (I) lane were bands C

(44.6kDa) and D (13.5kDa), with band C showing the strongest antibody reactivity. In

addition to the two differential bands demonstrated in the Western blot (excluding the

uninfected lane), recombinant MOMP was also detected by the sample and showed

strong antibody reactivity but was clearly at a different position compared to bands C

and D.

Figure 3.3. Western blot of uninfected cells (UI), infected cells (I) and recombinant

MOMP probed with a single patient sample. Differential bands C (44.6kDa) and D

(13.5kDa) are indicated in the infected lane, whilst antibody reactivity to recombinant

MOMP is designated by an arrow. Exposure time after detection was 30 seconds.

kDa 107

21

27

31.8

48.7

81

UI I MOMP

C

D


- 83 -

3.2.4 Species and Serovar Specificity of Target Antigens

Species and serovar specificity of the four antigenic targets was determined by probing

C. trachomatis L2, D and K, and C. pneumoniae cell extracts with samples from groups

I, II and IV (Figure 3.4). Group III was not included in this comparative study as all the

samples from each patient were expended during the initial Western blot screen

(Chapter 3, Section 3.1.4). In general, none of the four target antigens were found to be

serovar or species specific (Table 3.6). Moreover, patient samples serologically

identified as positive for both C. trachomatis and C. pneumoniae appeared to be more

highly reactive across the chlamydial serovars and species tested regardless of which

group the samples were assigned. Interestingly, host-cell proteins contained within all

five protein extracts (UI, C. trachomatis L2, D and K, and C. pneumoniae) were also

more reactive when probed by samples positive for both C. trachomatis and C.

pneumoniae.

In each patient group, samples positive for C. trachomatis and negative for C.

pneumoniae demonstrated similar antibody reactivity to the antigenic targets in

comparison to the original C. trachomatis L2 extract and the D, K and C. pneumoniae

protein preparations. Bands A and B were the least reactive when samples from each of

the three groups were probed against C. pneumoniae. Interestingly, acute (group I) and

recovering acute (group II) C. trachomatis infections appear to more commonly elicit an

antibody response to bands A and B. In contrast, a chronic (group IV) C. trachomatis

infection seems to produce antibodies more frequently against bands B and C.


- 84 -

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

IgG: Ct +ve; Cpn -ve IgG: Ct -ve; Cpn -ve IgG: Ct +ve; Cpn +ve

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

IgG: Ct +ve; Cpn +ve IgG: Ct +ve; Cpn -ve IgG: Ct +ve; Cpn +ve IgG: Ct +ve; Cpn -ve

(a) (b) (c)

(d) (e) (f) (g)

Figure 3.4. Western blots showing species and serovar specificity of the four antigenic

bands in patient groups I, II, and IV. The position of the individual bands are

designated by arrows in patient group I (a – c), II (d, e) and IV (f, g). Individual results

of serology for C. trachomatis and C. pneumoniae are shown for each patient along

with I.D. numbers. (Lanes: 1 = uninfected HEp-2 cells, 2 = HEp-2 cells infected with

C. trachomatis L2, 3 = HEp-2 cells infected with C. trachomatis D, 4 = HEp-2 cells

infected with C. trachomatis K, and 5 = HEp-2 cells infected with C. pneumoniae.

Exposure time after detection was 45 seconds).

107

21

27

31.8

48.7

81 107

21

27

31.8

48.7

81

107

21

27

31.8

48.7

81 A

C

B

A

C

D

A

D

C

GROUP I

107

21

27

31.8

48.7

81 107

21

27

31.8

48.7

81

107

21

27

31.8

48.7

81 107

21

27

31.8

48.7

81

D

A

C

B

D

C C

B B

C

GROUP IVGROUP II

13785

113 1258114697 10622

224890013817


- 85 -

Table 3.6. Serovar and species specificity and serology for patient samples from

groups I, II and IV probed against C. trachomatis serovars L2, D and K, and C.

pneumoniae.

PATIENT GROUP

PATIENT I.D.

SEROLOGY (IgG) L2 D K C. pn

8015 Ct –ve; Cpn +ve A/D None None D

13817 Ct +ve; Cpn +ve A/B/C/D A/B A/B A/B/C

13785 Ct +ve; Cpn -ve A/C/D A A A/C

13578 Ct +ve; Cpn -ve D None None None

I

2248900 Ct -ve; Cpn -ve B/D B B B/D

10622 Ct +ve; Cpn -ve A/B/C B B A/B/C

14697 Ct +ve; Cpn +ve C/D None None C/D

10306 Ct +ve; Cpn -ve A None None None

18456 Ct +ve; Cpn +ve A/B/C/D B/D B/D D

II

10553 Ct –ve; Cpn -ve A/B/D None None D

11011 Ct +ve; Cpn -ve B/C/D C C B/C/D

12581 Ct +ve; Cpn -ve B/C/D C B/C C/D

113 Ct +ve; Cpn +ve A/B/C/D A/B A/B/C/D A/B

IVF017 Ct +ve; Cpn -ve B/C/D B B/D None

IV

IVF032 Ct +ve; Cpn -ve B/C/D B/C B/C/D B/C/D

To determine the level of sequence similarity between homologous proteins of C.

trachomatis serovars L2 and D, and C. pneumoniae, protein alignments were

undertaken for each of the target antigens (Figures 3.5 – 3.7). All four antigens showed

>95% sequence conservation across the serovars of C. trachomatis. In contrast, the


- 86 -

level of sequence similarity amongst the C. pneumoniae proteins and their homologues

in C. trachomatis varied from 9% to 87%. CT157 showed 94% homology between C.

trachomatis serovars L2 and D, whilst the C. pneumoniae protein shared only 9%

sequence identity to the C. trachomatis proteins. The C. pneumoniae CT423

homologue showed 44% sequence identity to the C. trachomatis proteins, which as a

group, shared 95% sequence homology between serovars. A high level of sequence

conservation is evident for CT727 and CT396 (99% and 100% respectively), in

comparison to the C. trachomatis L2 and D homologues. The level of conservation is

also reflected in the comparisons of the C. trachomatis and C. pneumoniae homologues

which show 72% and 87% sequence identity for CT727 and CT396 respectively.


- 87 -

(a). CT157 * * **** * * * * MSVQGSSSLKYSDLFKPPEPTSSTDSSKEPPKESAWKVVSHSRGRRRARSNPSP-HTSQN MSVQGFSSLKYSDLFKPPEPASSTDS----PKESEWKVVSHSRGRRRARSTPPPPHTSQN * ** * TPSPKDSSLVARTDKAATDIFNSAKHKAIETTKRSDQQSRSLHILHLLAENPEPIVFHSA TPSPKDSSLVARTDKAATDIFNSAKHKAIETTKRSDQQSRSLPILHLLAADPEPIVFHST KSEEAIIY—-S ^ ^ * HQTNHNDPQRMLCDAILQANRIITMRIFNIGSPEIIRALIRAVRRNIPVVVSAWNFPNLS HQTNHNDPQRMLCDAILQANRIITMRIFNIGSPEIIRALIRAVRRNIPVIVSAWNFPNLS NQCNE-DMRKILCDAIEHADEEIFLRIYNLSEPKIQQSLTRQAQAKNKVTIYYQKFKIPQ ^ ^ ^ ^^^^^ ^ ^ ^^ ^ ^ ^ ^ ^ ^ ^ * * NWDRESELCVELRGNPQICLHKKTTLIDNQLTIIGTANYTKSSFFKDINLTALIQNPALY NWDRESKLYVELRGNPQICLHKKTTLIDNQLTIIGTANYTKSSFFKDINLTALIQNPALY ILKQASNVTL-VEQPPAGRKLMHQKALSIDKKDAWLGSANYTNLSLRLDNNLILGMHSSE ^ ^ * * * SLILSDTRGSVSIGSQTISYYPLPFPQSNTKILPIIQEIQKAQRTIKIAMNIFSHTEIFL SLILSGTRGSVSIGSQTISYYPLPFPQSNTKTLPIIQEIQKAQRTIKIAMNIFSHPEIFL LCDLIITNTSGDFSIKDQTGKYFVLP-QDRKIAIQAVLEKIQTAQKTIQVAMFALTHSEI ^ ^ ^ ^ ^ ^ * ALEQARLRGVTITIVINKKESAHTLDILHRISALLLLKSVTTVDSLHAKICLIDNQTLIF ALEQARLRGVTITIVINKKESAHTLDILHRISALLLLKSVTTVDSLHAKICLIDDQTLIF IQALHQAKQRGIHVDIIIDRSHSKLTFKQLRQLNINKDFVSINTAPCTLHHKFAVIDNKT ^ ^ ^ ^ ^ GSPNWTYHGMHKNLEDLLIVTPLTPKQIHSIQEIWAFLLKNSSPV GSPNWTYHGMHKNLEDLLIVTPLTPKQIHSIQEIWAFLLKNSSPV LLAGSINWSKGRFSLNDESLIILENLTKQQNQKLRMIWKDLAKHS ^ ^

(b). CT423 *************** MLYILLAIIVLFLFLGSATHRRASISAYGREGLPPFSSCPKVLPLLCLIYGMLGAPVYQY GSATHRRASISAYGGEGLPPFSSCPKVLPLLCLIYGMLGAPVYQY GRE-YPPFPSAPTILATLLCILYGALGTKLYT ^^^ ^^^ ^ ^ ^ ^ ^ * IHNFFSLSPSIFWLIFLSFALVIYKFLPLCPGYSDDSFSYKVSSSTVKTLENCLAGFKTP IHNFFSLSPSIFWLIFLSLALVIYKFLPLCPGYSDDSFSYKVSSSTVKTLENCLAGFKTP LPPKTAHKDLLFWPLYSLSALIAYGFLPPWISTKVPKETTAHLRFLASVFQ---LGLFPL ^^ ^^ ^ ^^^ ^ * SITAMQQTPPPEPPNELSTNISCLDHMIAREIMTPKADIFALQGDTPISQAFPLIIDEGY SITAMQQTPPPEPPNELSTNISCLNHMIAREIMTPKADIFALQGDTPISQAFPLIIDEGY QLLFYRRRPNQQVRSSTSFQLSAFDNLIVREVMIPKVDIFALPEETTLQEALVLVSEEGY ^ ^ ^ ^ ^^ ^ ^^ ^^^^^ ^ ^ ^^^ SRIPLFTKSIDDITGMVLVKDLSPVYYKDPHTSQPLSSIAYPPLYTPEIRRASLLLQEFR SRIPLFTKSIDDITGMVLVKDLSPVYYKDPHTSQPLSSIAYPPLYTPEIRRASLLLQEFR SRVPVYKKNLDNITGILLVKDLLLLYTSSHDLSQPISSVAKPPFYAPEIKKASSLLQEFR ^^ ^ ^ ^ ^^^ ^^^^^ ^ ^^^ ^^ ^^ ^ ^^^ ^^ ^^^^^^ * * * QKRCHLAIVVNEYGFTEGLVSMEDIIEEIFGEIADEYDNQEDVHHKKIGNAWIVDGRMNI QKRCHLAIVVNEYGFTEGLVSMEDIVEEIFGEIADEYDDQEDVHYKKIGNAWIVDGRMNI QKHRHLAIIVNEYGFTEGIATMEDIIEEIIGEIADEHDVQENTPYKKIGSSWIVDGRMNI ^^ ^^^^ ^^^^^^^^^ ^^^^ ^^^ ^^^^^^ ^ ^^ ^^^^ ^^^^^^^^^ SDAEECFGLHIEHESSYDTLGGYVFHKLGAVPEKGMKIYYEDFAIDILSCSDRSVEKMKI SDAEECFGLHIEHESSYDTLGGYVFHKLGAVPEKGMKIYYEDFAIDILSCSDRSVEKMKI SDAEEYFNLKIDHENSYDTLGGHVFHKVGAVPQKGMRIHHENFDIEIITCTERNVGKLKI ^^^^^ ^ ^ ^ ^^ ^^^^^^^ ^^^^ ^^^^ ^^^^^ ^ ^ ^ ^ ^ ^ ^ ^ ^^ TPRRRKPLS TPRRRKPLS TPRKRK ^^^^^^

D -

Cpn - L2 -

Cpn - L2 - D -

- D - L2 - Cpn

- D - L2 - Cpn

Figure 3.5. Protein alignments of C. trachomatis serovars D and L2, and C. pneumoniae for target antigens (a) CT157 and (b)

CT423. (* indicates non-conserved amino acid residues in C. trachomatis serovars D and L2. ^ indicates conserved amino acid

residues in C. trachomatis serovars D and L2, and C. pneumoniae).


- 88 -

CT727 MSPQLFSSPFSRELLSYFFESGMAEENSPLLSQKNRRLSQNLTLKSAYISLALYLGSLLS MSPQLFSSPFSRELLSDFFESGMAEENSPLLSQKNRRLSQNLTLKSAYISLALYLGSLLS MFSRLFFTSFSAEVVNTFFESGMSEDTSPLLSKQNRKLSHNLPLKSAYLSLGTYLIALLS ^ ^^ ^^ ^ ^^^^^^ ^ ^^^^^ ^^^^^ ^^ ^^^^^ ^^ ^^ ^^^ HWIGYQALSSLLLILTFFLAGTPALIKSFEDILDRTVNIDILMTSAAFGSIFIGGALEGA HWIGYQALSSLLLILTFFLAGTPALIKSFEDILDRTVNIDILMTSAAFGSIFIGGALEGA FWLHAKNLSNLFVVFTFFLAGTPALIKSLDNICQKVVNIDILMTSAAFGSIFIGGALEGA ^ ^^ ^ ^^^^^^^^^^^^^ ^ ^^^^^^^^^^^^^^^^^^^^^^^^ * LLLVLFAISESLGAMVSGKAKSTLASLKHLAPTVAWVVQQDGSLQKVLVQNVKVGEIIRV LLLVLFAISESLGAMVSGKAKSTLASLKHLAPTVAWVVQQDGSLQKVLVQNVKIGEIIRV LLLVLFAISEALGQMVSGKAKSTLVSLKQLAPTTGWLVLEDGNLQKVAINKIEVGNILRI ^^^^^^^^^^ ^^ ^^^^^^^^^^ ^^^ ^^^^ ^ ^ ^^ ^^^^ ^ ^ ^ * KSGEVVPLDGKIIQGASSINLMHLTGEKIPKSCGIGDTIPAGAHNLEGSFDLQVLRIGAE KSGEVVPLDGKIIHGASSINLMHLTGEKIPKSCGIGDTIPAGAHNLEGSFDLQVLRIGAE KSGEVVPLDGEILHGSSSINLMHLTGEKVPKSCHPGSIVPAGAHNMEGSFDLRVLRTGSD ^^^^^^^^^^ ^ ^ ^^^^^^^^^^^^ ^^^^ ^ ^^^^^^ ^^^^^^ ^^^ ^ * STIAHIINLVVQAQSSKPKLQQRLDRYSSTYALTIFAISACIAIGGSLFTTLPFLGPDSA STIAHIINLVVQAQSSKPKLQQRLDRYSSTYALTIFAISACIAIGGSLFTTLPFLGPDGA STIAHIINLVIQAQNSKPRLQQRLDKYSSVYALSIFAIACGIALLVPLFTSIPLLGPQSA ^^^^^^^^^^ ^^^ ^^^^^^^^^^ ^^^ ^^^ ^^^^ ^^ ^^^ ^ ^^^ ^ FYRALAFLIAASPCALIIAIPIAYLSAINACAKHGVLLKGGVVLDRLVSCNSVVMDKTGT FYRALAFLIAASPCALIIAIPIAYLSAINACAKHGVLLKGGVVLDRLVSCNSVVMDKTGT FYRALAFLIAASPCALIIAIPIAYLSAINACAKHGVLLKGGVILDRLVSCNSVVMDKTGT ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^ LTTGDLTCSGCEDFGPESPLFYSCVLAMEQSSSHPIAQAIVNYLTEKQVRSLPATQCTTI LTTGDLTCSGCEDFGPESPLFYSCVLAMEQSSSHPIAQAIVNYLTEKQVRSLPATQCTTI LTTGELTCIGCDYFGSKNETFFPSVLALEQSSSHPIAEAIVSYLMEQKVSSLPADRYLTV ^^^^ ^^^ ^^ ^^ ^ ^^^^^^^^^^^^^ ^^^ ^^ ^ ^ ^^^^ ^ PGEGVSGEFNGEQAFVGRVSTALRYVPEEYREQLRERAQQAQERGDTCSIACLGKRVSLF PGEGVSGEFNGEQAFVGRVSTALRYVPEEYREQLRERAQQAQERGDTCSIACLGKRVSLF PGEGVRGYFNEQEAFVGRVETGLGKVPSEYLEDIEQKIYQAKQHGEICSLAYVGNSFALF ^^^^^ ^ ^^ ^^^^^^ ^ ^ ^^ ^^ ^ ^^ ^ ^^ ^ ^ ^^ YFRDVPRHDAADIVSYLKKNGYPVCMLTGDHRISAENTARLLGIDEVFYDLTPDNKLSKI YFRDVPRHDAANIVSYLKKNGYPVCMLTGDHRISAENTARLLGIDEVFYDLTPDNKLSKI YFRDIPRPQAKEIIQDLKDLGYPVSMLTGDHKVSAENTAEILGISEVFFDLTPEDKLAKI ^^^^ ^^ ^ ^ ^^ ^^^^ ^^^^^^ ^^^^^^ ^^^ ^^^ ^^^^ ^^ ^^ QELAKSRQIMMIGDGINDAPALAQATVGIAMGEAGSATAIEAADVVLLNQGLSSLPWLID QELAKSRQIMMIGDGINDAPALAQATVGIAMGEAGSATAIEAADVVLLNQGLSSLPWLID RELATQRQIMMVGDGINDAPALAQATVGIAMGEAGSATAIEAADIVLLHDSLSSLPWIIQ ^^^ ^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^ ^^^^^^ ^ KAKKTRRIVSQNLALALAIILFISGPASMGVIPLWLAVILHEGSTVIVGLNALRLLKNT KAKKTRRIVSQNLALALAIILFISGPASMGVIPLWLAVILHEGGTVIVGLNALRLLKNT KAKQTKKVVSQNLALALAIILLVSWPASLGIIPLWLAVILHEGSTVIVGLNALRLLKS ^^^ ^ ^^^^^^^^^^^^^ ^ ^^^ ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^

Figure 3.6. Protein alignment of C. trachomatis serovars D and L2, and C. pneumoniae

for target antigen CT727. (* indicates non-conserved amino acid residues in C.

trachomatis serovars D and L2. ^ indicates conserved amino acid residues in C.

trachomatis serovars D and L2, and C. pneumoniae).

Cpn - L2 - D -

- D - L2 - Cpn


- 89 -

CT396 MSEKRKSNKIIGIDLGTTNSCVSVMEGGQPKVIASSEGTRTTPSIVAFKGGETLVGIPAK MSEKRKSNKIIGIDLGTTNSCVSVMEGGQPKVIASSEGTRTTPSIVAFKGGETLVGIPAK MSEHKKSSKIIGIDLGTTNSCVSVMEGGQAKVITSSEGTRTTPSIVAFKGNEKLVGIPAK ^^^ ^^ ^^^^^^^^^^^^^^^^^^^^^ ^^^ ^^^^^^^^^^^^^^^^ ^ ^^^^^^^ RQAVTNPEKTLASTKRFIGRKFSEVESEIKTVPYKVAPNSKGDAVFDVEQKLYTPEEIGA RQAVTNPEKTLASTKRFIGRKFSEVESEIKTVPYKVAPNSKGDAVFDVEQKLYTPEEIGA RQAVTNPEKTLGSTKRFIGRKYSEVASEIQTVPYTVTSGSKGDAVFEVDGKQYTPEEIGA ^^^^^^^^^^^ ^^^^^^^^^ ^^^ ^^^ ^^^^ ^ ^^^^^^^ ^ ^ ^^^^^^^^ QILMKMKETAEAYLGETVTEAVITVPAYFNDSQRASTKDAGRIAGLDVKRIIPEPTAAAL QILMKMKETAEAYLGETVTEAVITVPAYFNDSQRASTKDAGRIAGLDVKRIIPEPTAAAL QILMKMKETAEAYLGETVTEAVITVPAYFNDSQRASTKDAGRIAGLDVKRIIPEPTAAAL ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ AYGIDKEGDKKIAVFDLGGGTFDISILEIGDGVFEVLSTNGDTHLGGDDFDGVIINWMLD AYGIDKEGDKKIAVFDLGGGTFDISILEIGDGVFEVLSTNGDTHLGGDDFDGVIINWMLD AYGIDKVGDKKIAVFDLGGGTFDISILEIGDGVFEVLSTNGDTLLGGDDFDEVIIKWMIE ^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^ ^^ EFKKQEGIDLSKDNMALQRLKDAAEKAKIELSGVSSTEINQPFITIDANGPKHLALTLTR EFKKQEGIDLSKDNMALQRLKDAAEKAKIELSGVSSTEINQPFITIDANGPKHLALTLTR EFKKQEGIDLSKDNMALQRLKDAAEKAKIELSGVSSTEINQPFITMDAQGPKHLALTLTR ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^^^^^^^^^^ AQFEHLASSLIERTKQPCAQALKDAKLSASDIDDVLLVGGMSRMPAVQAVVKEIFGKEPN AQFEHLASSLIERTKQPCAQALKDAKLSASDIDDVLLVGGMSRMPAVQAVVKEIFGKEPN AQFEKLAASLIERTKSPCIKALSDAKLSAKDIDDVLLVGGMSRMPAVQETVKELFGKEPN ^^^^ ^^^^^^^^^^ ^^ ^^ ^^^^^^ ^^^^^^^^^^^^^^^^^^ ^^^ ^^^^^^ KGVNPDEVVAIGAAIQGGVLGGEVKDVLLLDVIPLSLGIETLGGVMTPLVERNTTIPTQK KGVNPDEVVAIGAAIQGGVLGGEVKDVLLLDVIPLSLGIETLGGVMTPLVERNTTIPTQK KGVNPDEVVAIGAAIQGGVLGGEVKDVLLLDVIPLSLGIETLGGVMTTLVERNTTIPTQK ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^ KQIFSTAADNQPAVTIVVLQGERPMAKDNKEIGRFDLTDIPPAPRGHPQIEVTFDIDANG KQIFSTAADNQPAVTIVVLQGERPMAKDNKEIGRFDLTDIPPAPRGHPQIEVTFDIDANG KQIFSTAADNQPAVTIVVLQGERPMAKDNKEIGRFDLTDIPPAPRGHPQIEVSFDIDANG ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^ ILHVSAKDAASGREQKIRIEASSGLKEDEIQQMIRDAELHKEEDKQRKEASDVKNEADGM ILHVSAKDAASGREQKIRIEASSGLKEDEIQQMIRDAELHKEEDKQRKEASDVKNEADGM IFHVSAKDVASGKEQKIRIEASSGLQEDEIQRMVRDAEINKEEDKKRREASDAKNEADSM ^^^^^^^^ ^^^ ^^^^^^^^^^^^ ^^^^^ ^ ^^^^ ^^^^^ ^ ^^^^ ^^^^^ ^ IFRAEKAVKDYHDKIPAELVKEIEEHIEKVRQAIKEDASTTAIKAASDELSTHMQKIGEA IFRAEKAVKDYHDKIPAELVKEIEEHIEKVRQAIKEDASTTAIKAASDELSTRMQKIGEA IFRAEKAIKDYKEQIPETLVKEIEERIENVRNALKDDAPIEKIKEVTEDLSKHMQKIGES ^^^^^^^ ^^^ ^^ ^^^^^^^ ^^ ^^ ^ ^ ^^ ^^ ^^ ^^^^^^ MQAQSASAAASSAANAQGGPNINSEDLKKHSFSTRPPAGGSASSTDNIEDADVEIVDKPE MQAQSASAAASSAANAQGGPNINSEDLKKHSFSTRPPAGGSASSTDNIEDADVEIVDKPE MQSQSASAAASSAANAKGGPNINTEDLKKHSFSTKPPS-NNGSSEDHIEEADVEIIDNDD ^^^^^^^^^^^^^^^^ ^^^^^^ ^^^^^^^^^^ ^^ ^^ ^ ^^^^^^^^ ^

Figure 3.7. Protein alignment of C. trachomatis serovars D and L2, and C. pneumoniae

for target antigen CT396. (* indicates non-conserved amino acid residues in C.

trachomatis serovars D and L2. ^ indicates conserved amino acid residues in C.

trachomatis serovars D and L2, and C. pneumoniae).

- D - L2 - Cpn

D -

Cpn - L2 -


- 90 -

3.2.5 Diagnostic Potential of the Four Newly Identified Antigens

For the purpose of developing a serological diagnostic test capable of discriminating

between acute and chronic C. trachomatis infection, the results obtained from the four

identified antigens were combined into various panels and their predictive potential

evaluated. Of the 28 possible combinations, only those displaying the highest overall

diagnostic potential are shown (Table 3.7). Antibody response to panel format #3

(antigens A or B or C) demonstrated the greatest sensitivity (75%) and specificity

(76%) compared to other potential formats for the prediction of acute or recovered acute

infection. As a consequence, panel 3 (A or B or C) with a PPV of 60% (CI 95% 0.423)

and a NPV of 86% (CI 95% 0.736) proved to be the preferred format for diagnosing the

acute or recovery stages of C. trachomatis infection even though 8/10 PID patients

(group IV) demonstrated antibody reactivity to either of the three bands. In contrast,

other panel formats such as panel 1 (A + B or C) and panel 2 (A + C or B) showed

significantly lower sensitivities of 33% and 42% respectively, but demonstrated higher

specificity (96%). The panel 4 format (A or B or C or D) displayed the greatest

sensitivity of 79%; however the addition of antigen D when compared to the A or B or

C format decreased specificity by 21%. The removal of antigen A from the A or B or C

or D panel format ie. to B or C or D, did not improve diagnostic capabilities; in fact,

sensitivity and positive and negative predictive values were slightly reduced. Only one

combination of antigens was selected to be assessed for their predictive value in the

diagnosis of chronic infection as the initial Western blot screen of samples from group

IV (Section 3.2.1) demonstrated 8/10 samples reacted to both B and C antigens. As a

consequence, the B + C panel format was evaluated and subsequently demonstrated

80% sensitivity and 84% specificity for the potential diagnosis of chronic C.

trachomatis infection. In addition, although 6/20 patients from groups I and II

demonstrated antibody reactivity to bands B and C, only one patient from group I

exhibited the exact same banding profile as observed in those patients with chronic

infection.


- 91 -

Table 3.7. Assessment of the diagnostic potential of different panel formats for diagnosis of acute and chronic chlamydial infection.

Panel format 3 (A or B or C) proved to be the superior format for the diagnosis of acute or recovery stages of C. trachomatis infection.

Overall, the B + C format was shown to have the greatest diagnostic capability regardless of infection stage. (Sens = Sensitivity, Spec

= Specificity, PPV = Positive Predictive value, NPV = Negative Predictive Value, + = both antigens must be positive).

% % % INFECTION STAGE

PANEL FORMAT Sens. Spec. PPV

CI 95% Std Error NPV

CI 95% Std Error

CHI Squared

1. A + B or C 33 96 80 0.552 0.126 75 0.476 0.137 11.6

2. A + C or B 42 96 83 0.622 0.107 77 0.533 0.121 16.5

3. A or B or C 75 76 60 0.423 0.089 86 0.736 0.063 16.9

4. A or B or C or D 79 55 46 0.311 0.077 84 0.733 0.056 7.6

Acute and

Recovered

Acute

5. B or C or D 75 55 45 0.296 0.078 82 0.699 0.060 5.8

PID 6. B + C 80 84 44 0.215 0.117 96 0.877 0.044 19.1


- 92 -

3.2.6 Antibody Response of Identified Antigens during Different Stages of C.

trachomatis Infection

The data presented in Section 3.2.1 was used to predict and compare the antibody

response (%) (number of patients with positive antibody responses) to bands A, B, C

and D during acute (I), recovery (II), multiply infected (III) and chronic (IV) stages of

C. trachomatis infection in females (Figure 3.8).

0

25

50

75

100

Band A Antibody Response

Onset I II III IV

Infection Stage/Time Course

Ant

ibod

y Res

pons

e (%

)

0

25

50

75

100

Band B Antibody Response

Onset I II III IVInfection Stage/Time Course

Ant

ibod

y Res

pons

e (%

)

0

25

50

75

100

Band C Antibody Response


Ant

ibod

y Res

pons

e (%

)

0

25

50

75

100

Band D Antibody Response


Ant

ibod

y Res

pons

e (%

)

Figure 3.8. Comparison of female antibody responses to (a) Band A, (b) Band B, (c)

Band C, and (d) Band D for C. trachomatis-infected patient groups (I – IV). Although

the x-axis is interpreted as a temporal scale, the even spacing of the stages is purely

arbitrary, not necessarily reflecting a “to scale” time line. (Antibody response (%) =

number of patients with +ve antibody responses to bands A, B, C and D).

(d) (c)

(a) (b)


- 93 -

In general, the generation of antibodies in response to the four antigens is greater in

patients with multiple infections (patient group III) compared with other stages of

infection. Furthermore, each antigen demonstrates that the production of antibodies

against its target is decreased during the recovery phase (patient group II) of infection.

Antigens A, B and C elicit an identical production of antibodies (56%) at the acute

phase (patient group I), whilst the generation of antibody to antigen D at the same stage

of infection is markedly increased (89%).

The antibody response profile of Band A shows a moderate (56%) early antibody

response (patient group I), a slight decrease (45%) in the recovery stage, a rise (75%) in

the multiply infected stage (patient group III) and a sharp decline in the chronic phase

(patient group IV) of C. trachomatis infection. During the acute stage of infection

(patient group I), moderate levels (56%) of antibodies to Band B are produced and the

recovery phase (patient group II) shows a decrease in antibody production. The

antibody profile of Band C in the acute (patient group I) and recovering (patient group

II) stage of infection virtually mimics that of Bands A and B, however the chronic stage

of infection (patient group IV) elicits a much stronger antibody response. Antibody

levels to Band D in the early stage of infection (patient group I) elicit an antibody

response from a greater number of patients (89%) when compared to the acute profiles

of Bands A, B and C. The recovery phase of infection (patient group II) shows a rapid

drop of antibodies produced to Band D, however in patient groups III and IV, the

generation of antibodies increases from 75% to 100%, respectively, thus surpassing the

antibody response demonstrated by patients with acute infection (group I).


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3.3 DISCUSSION

The objective of this study was to identify one or more chlamydial antigens that could

be used to discriminate between acute and chronic C. trachomatis infections. We

identified four bands that potentially differentiated between acute (group I), recovered

acute (group II), acute multiply infected (group III) and PID (group IV) patients. Two

possible candidates were identified for each of bands A (CT147 and CT314), B (CT727

and CT396) and C (CT157 and CT423). Although the cellular location and function of

several of the potential candidates suggest obvious antigenic properties, it is not clear

why the remainder elicit a humoral response in the host.

Band A, found to be reactive in 38% of C. trachomatis-infected samples was identified

via mass spectrometry and produced two possible candidate proteins: CT147

(Conserved Hypothetical Protein – 162.1kDa) and CT314 (DNA-directed RNA

polymerase beta chain – 154.9kDa). Of these two candidates, only CT147 has

previously been shown to elicit a humoral response in the host (Belland et al., 2003). A

protein search of the TIGR database showed CT147 to have a domain between residues

695 and 1447 that had 21% similarity to a cytadherence domain. Typically, cytadhesion

is a mechanism employed by non-invasive bacteria which allows attachment to the

target cell and minimises its clearance by the host cell. Moreover, CT147 is an

immediate-early gene of the C. trachomatis developmental cycle, which along with

other genes expressed at this stage of chlamydial development, activate various

bacterial metabolic processes and potentially alter the bacterial envelope to evade fusion

with lytic enzymes produced by the host (Belland et al., 2003). These authors showed

that CT147 was co-localised to the EB inclusion membrane and has a significant level

of sequence similarity to the human early endosomal antigen 1 (EEA1) protein which is

involved in endosomal trafficking and fusion within mammalian cells (Mu et al., 1995;

Simonsen et al., 1998; Christoforidis et al., 1999). EEA1 regulation of endosomal

fusion is reliant upon the interaction of Rab5, however CT147 lacks a domain that

permits the interaction with Rab5 (Belland et al., 2003). Intriguingly, CT147 has

retained the C-terminal zinc finger, which suggests an altered protein function that


- 95 -

could potentially bind endosomes but circumvent endosomal fusion. The latter would

enable Chlamydia to evade lytic destruction by the host. These findings further suggest

that CT147 could potentially be a principal humoral response target. Interestingly, of

the C. trachomatis-infected samples, groups I (first-time C. trachomatis infection

acquired less than 4 months previously), and III (a history of two C. trachomatis

infections, with the most recent estimated to have been acquired less than 4 months

previously) demonstrated the highest reactivity (56% and 75% respectively) to band A

when probed against chlamydial/host cell preparations. This suggests that CT147 may

indeed also have a fundamental role in inclusion formation as anti-CT147 antibodies

were generally only present during initial and recovery stages of C. trachomatis

infection. Of interest was the presence of antibodies to CT147 in a single PID patient

sample, which given the patient’s prior history of C. trachomatis infections, may

possibly be a residual effect from the acute stage of the repeated infections. The second

band A candidate CT314, functions as a transcriptional regulator and, as such, would

not normally be presented to the host immune system at any stage during the chlamydial

developmental cycle or infection process. Therefore, given the functional role of

CT314, the probability of a humoral response targeted against this protein is unlikely

which is in direct contrast to the likelihood of CT147 eliciting an antibody response.

The two candidate proteins for band B are CT727 (P-type ATPase) and CT396

(HSP70). P-type ATPases constitute a superfamily of cation transport enzymes which

mediate transmembrane exchange of all biologically significant cations (Smith et al.,

1993). In contrast, HSP70 is associated with outer membrane complexes of EBs and

was originally thought to play a role in either attachment or entry of the EB into host

cells (Kuo et al., 1996; Raulston et al., 1993; 2002). Although HSPs are expressed

during times of stress such as bacterial invasion, Raulston et al. (2002) showed that

HSP70 is only exposed on the surface of purified EBs to a minor degree suggesting an

antigenic role for this protein during the initial stages of acute infection may be limited.

Moreover, this suggests that the antibody response of band B demonstrated in samples

of C. trachomatis-infected patients may be a result of CT727 being presented to the host


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immune system, not CT396, as 5/9 patient samples from group I (acute) demonstrated

antibodies to band B.

One of the candidate proteins detected for band C, CT157, contains two phospholipase

D (PLD) domains and is a member of the PLD superfamily which includes enzymes

that have high catalytic activity and are involved in phospholipid metabolism. PLDs,

which are known to hydrolyse phospholipids to phosphatidic acid, may be essential for

the formation of particular types of transport vesicles or be strongly involved in signal

transduction (Leiros et al., 2000). CT423, the second protein candidate for band C

contains three functional domains (two cystathionine beta synthase domains and one

transporter-associated domain) which are implicated in intracellular targeting and

trafficking as well as protein-protein interactions (Carr et al., 2003). Neither of these

band C candidate proteins have previously been reported to evoke a humoral response

in the host, yet antibody production to one or both of these proteins was demonstrated in

all but the uninfected children controls (group VII).

N-terminal sequencing of proteins isolated from bands B and C revealed sequences

contained within CT157, CT423 and CT727 not, as expected, at the N-terminus.

However, only the N-terminus sequence DPHTSQ, which identified the protein CT423,

was predicted to be cleaved by trypsin at the lysine (K) residue directly preceding the

first amino acid of the derived sequence. CT157 and CT727 were cleaved three and

four amino acids respectively from the generated N-terminal sequences. This may due

to random breakage of the peptide chain or the polypeptides being dissolved and lost in

the organic solvents used during the extraction processes. Little is known about how

CT157, CT423 and CT727 are intracellularly processed and it is possible that the amino

acids were cleaved off in the maturation of the protein through the cell. There is also a

possibility that various host cell proteases may have cleaved CT157, CT423 and CT727

thus resulting in truncated proteins. An attempt was made to N-terminal sequence the

protein isolated from band A, however Belland et al. (2003) showed that during

immunoprecipitation, CT147 is cleaved leaving a 90kDa fragment. If the identity of the

immunoreactive Band A is indeed the CT147 protein, then this would account for the


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inability of the approach to identify this protein as the immunoreactive band present on

the PVDF membrane was excised at a molecular weight >113kDa.

The comparative study of bands A, B, C and D demonstrated that none of the potential

band candidates are entirely serovar or species specific. Interestingly however,

alignment of the C. trachomatis serovars L2 and D and C. pneumoniae protein

candidates showed CT157 (band C) and CT727 (band B) to have the lowest sequence

similarity (9% and 72% respectively) with the C. pneumoniae homologues in

comparison to the other candidate proteins predicted for bands B and C. The premise

that the identity of band C is CT727 is supported by evidence that CT396 is almost

totally conserved between C. trachomatis and C. pneumoniae and, as such, anti-CT396

antibodies would be expected to demonstrate equal reactivity when probed against C.

pneumoniae. The latter was not however the case in any of the 15 patient samples

screened. The identity of band C could not be predicted based solely on the functions

of either candidate proteins CT157 or CT423. CT157 is however, the least conserved

(9%) of the band C candidate proteins and antibody reactivity for band C was shown to

be greatly decreased against C. pneumoniae. Sensitivity and specificity showed that

bands B and C were not as reactive to C. pneumoniae by comparison with C.

trachomatis. Taken together, these findings suggest that the true identities of bands B

and C are CT727 and CT157 respectively.

The IgG serology status for each patient sample was established for the species and

serovar comparative study. Interestingly, patients that were C. trachomatis IgG-

positive but C. pneumoniae IgG-negative, generally demonstrated the same level of

cross-reactivity to C. pneumoniae bands A, B, C or D. Conversely, patients that were

C. trachomatis IgG-negative but C. pneumoniae-IgG positive showed a decrease in

antibody reactivity to C. trachomatis bands A, B, C or D. This suggests that C.

trachomatis antibodies cross-react with the C. pneumoniae bands whilst, in contrast, C.

pneumoniae antibodies do not generally cross-react with the C. trachomatis bands.


- 98 -

Sensitivity and specificity of the identified antigens in various combinations showed the

A or B or C format (panel #3) to be most efficacious for diagnosing acute infection.

The addition of antigen D to the panel (A or B or C or D) was shown to increase the

sensitivity to 79%. However, given the overall prevalence of antigen D in samples from

C. trachomatis-infected patients, the diagnostic potential of antigen D for identifying

acute infection is limited due to the high C. pneumoniae cross-reactivity demonstrated

within chronic patients. Moreover, this suggests that antigen D could possibly be more

useful as a marker of general chlamydial infection rather than of a particular stage of

infection. With a sensitivity and specificity of 80% and 84% respectively, chronic

infection could potentially be diagnosed using the B + C format. Only one other assay

for the diagnosis of chronic infection is currently available, the Medac HSP60 - an

assay for the detection of anti-cHSP60 antibodies. Higher anti-cHSP60 antibody

responses in women with tubal pathology have been demonstrated compared to women

without tubal pathology (Bax et al., 2004). However, a more recent study by Gazzard

et al. (2006) showed a reduction of cHSP60 reactivity in the PID/Tubal Damage group

(20%) compared to acute patients (28%). The presence of anti-cHSP60 antibodies were

determined for all seven female groups used in this study. Of these, anti-cHSP60

antibodies were not detected in group I (acute). Despite this, the number of patient

samples from each C. trachomatis-infected group in which anti-cHSP60 antibodies

were detected was only 18%, ie. 3/38 for group II (recovering acute) and 2/38 for

groups III (acute multiply infected) and IV (PID). Of interest was the detection of anti-

cHSP60 antibodies in a single sample from each of the negative control groups.

Therefore, if we were to use the Medac HSP60 assay to detect chronic infection in our

female cohort, the sensitivity and specificity of this method would be 18% and 87%

respectively which is in significant contrast to the sensitivity (80%) of our newly

developed B + C format. As a consequence, the low sensitivity of the Medac HSP60

assay demonstrates the assay developed in this study for the diagnosis of chronic C.

trachomatis infection could potentially be a more viable option.

Since the basis of many diagnostic assays is MOMP, a well known immunogen, a

strong reacting patient serum was probed against recombinant MOMP in an attempt to


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verify that the antibody reactivity against band C was one of the identified protein

candidates CT157 or CT423 and not MOMP. MOMP from serovar D is 393 amino

acids and therefore its molecular weight should be 42.4kDa. The retained his-tag on

recombinant MOMP would have increased its size to 43.4kDa. MOMP is seen to run at

approximately 46kDa on the western blot. Unmistakably, there is a disparity in band

sizes of band C upon comparison to recombinant MOMP. Band C is running at an

estimated 44.6kDa, and clearly it is a different size to the recombinant MOMP.

Furthermore, the predicted identity of band C as CT157 is therefore still reasonable.

The protein concentration of MOMP does appear to be higher than that loaded for the

C. trachomatis-infected lane, however this has only slightly affected the migration of

recombinant MOMP through the gel given its position on the gel in relation to its

recombinant molecular weight. It is also of interest that more anti-MOMP antibodies

were not observed in the sera that were analysed. In the serum that was used to probe

MOMP, the patient had obviously mounted an antibody response to the antigen as

recombinant MOMP is visualised on the Western blot. In addition, whilst they were

present in numerous patient sera as established by the initial serology EIA testing, the

method that was employed in this project targeted antigens that were differential

between various stages of C. trachomatis infection. Furthermore, if MOMP was the

immunogenic band C, then N-terminal sequencing would have identified it accordingly

rather than amino acids from CT157 and CT423. Thus, although MOMP is highly

antigenic, within this cohort of patients, it is not differential between groups and

therefore different states of C. trachomatis infection.

The ability to differentiate between acute and chronic C. trachomatis infections is

highly significant as treatment strategies and/or management of the infection or disease

could be specifically tailored to the individual, rather than generic basis. Hence, an

accurate diagnosis would allow the correct selection of one of the various treatment

regimes (as established by the Centers for Disease Control and Prevention of sexually

transmitted diseases (Chapter 1)), for acute C. trachomatis infection or PID thus

affording the patient the best possible chance of recovery. Importantly, this specialised

treatment strategy would also lead to a substantial decrease in associated health care


- 100 -

costs. In addition, the consequence of these findings could potentially enable the early

detection of C. trachomatis-induced infertility and chronic sequelae in women.

The validation of our proposed test format will be undertaken using recombinant

proteins of all four potential candidates screened against original (batch #1) and newly

collected (batch #2) patient samples (Chapter 5).

Chapter 4: Optimisation of Expression of Antibody-Reactive Candidate Proteins

CHAPTER 4

Optimisation of Expression of Antibody-Reactive Candidate

Proteins

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4.0 INTRODUCTION

Multiple expression systems are available for the production of heterologous proteins in

vitro with selection dependent upon several factors such as host-cell growth

characteristics, intracellular and extracellular expression, post-translational

modifications and biological activity of the target protein (Goeddel, 1990; Baneyx,

1999). Eukaryotic expression systems based on yeast or mammalian cell lines often

demand considerable attention yet frequently result in limited protein (Grisshammer et

al., 1995). In contrast, bacterial gene expression in Escherichia coli host cells is both

simple and has the potential to produce high protein yields (Gold, 1990; Olins and Lee,

1993; Jana and Deb, 2005). Protein expression in E. coli requires the presence of three

important signals (the promoter, terminator and ribosome binding site) to advertise the

presence of the target gene and provide instructions for the transcriptional and

translational apparatus of the cell. Consequently, to ensure superior expression levels, a

strong promoter which can initiate and sustain a high rate of transcription is critical.

Several E. coli promoters are commonly used for this purpose and combine the desired

requirements of strength and ease of regulation. The lac promoter controls transcription

of the lacZ gene coding for β-galactosidase and is induced subsequent to addition of

isopropyl-β-D-thiogalactoside (IPTG) (Studier and Moffat, 1986). The trp promoter,

involved in tryptophan biosynthesis, is repressed by tryptophan but is induced by 3-β-

indoleacrylic acid, whilst the tac promoter, which is a hybrid of the trp and lac

promoters, is also induced by IPTG (Jana and Deb, 2005).

The general strategy for bacterial expression is the fusion of the target protein to the N-

or C- termini of specific bacterial proteins or peptides which promote efficient

expression, detection and purification of the expressed recombinant protein. The most

common fusion peptides include maltose binding protein (MBP), thioredoxin (Trx), 6-

Histidine–tag (His-tag) and glutathionine S-transferase (GST) which aid protein

stability and prevent possible degradation by the host cell. Numerous chlamydial

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studies have employed the GST fusion system for recombinant protein production

(Debattista et al., 2002; Montigiani et al., 2002; Sharma et al., 2004; Finco et al., 2005)

as expression is driven by the strong tac promoter. Furthermore, protein function and

antigenicity are typically preserved by the system’s method of purification which

targets the GST peptide. pGEX vectors (a component of the GST fusion system) also

contain an internal lacIq gene that upon translation, acts as a repressor for the tac

promoter inhibiting expression until induced with IPTG. In this manner, explicit

control over expression of the target protein is maintained.

The level of expression and solubility of the expressed protein are not solely restricted

to the use of a specific expression system. Other parameters such as culture media,

temperature, pH and aeration can influence the final product yield. The recent advent of

autoinduction as an alternative to conventional IPTG-induced expression has afforded

the ability to further increase protein levels and enhance solubility (Studier, 2005). In

addition, host-cell strains capable of producing highly toxic proteins can proliferate and

still maintain a functional plasmid for subsequent protein expression. Autoinduction is

dependent upon bacterial mechanisms which regulate the utilisation of both carbon and

energy sources present in the enriched growth medium (Studier, 2005). The presence of

glucose effectively inhibits lactose induction in the early growth phase thus promoting

high-density cell growth. As a consequence of increased cell density and subsequent to

glucose depletion, lactose uptake by the host cell initiates target protein expression and

results in greater protein production compared to traditional IPTG induction. In

contrast, given decreased culture densities, the absence of the lactose analogue IPTG

during cell growth and shorter induction times when utilising the IPTG induction

method, protein expression and thus final yields are potentially reduced.

The formation of inclusion bodies is a significant problem in cytoplasmic protein

expression as the degree of solubility impacts on the amount of recombinant protein

obtained. The major disadvantages associated with this problem are protein

insolubility, reduced protein yields and a loss of conformation resulting in a biologically

inactive protein. Inclusion bodies do have several advantages such as isolation of

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highly pure and concentrated protein, protection from host-cell proteases and the

minimisation of host-cell death by toxic heterologous proteins. In an effort to predict

protein solubility in E. coli, a statistical analysis of the composition of 81 proteins that

do and do not develop inclusion bodies was examined (Wilkinson and Harrison, 1991).

The investigation concluded that six key parameters such as charge average, cysteine

fraction, proline fraction, turn-forming residue fraction, hydrophilicity and the total

number of residues present were correlated with inclusion body formation.

Experimentally, various procedures have been developed which decrease the possibility

of formation of inclusion bodies and hence improve protein solubility. These include

temperature reduction during growth of bacterial cultures (Shirano and Shibata, 1980;

Schein, 1989, 1993), an E. coli expression species variant (Kenealy et al., 1987) and pH

alteration of the selected culture medium (Sugimoto et al., 1991). In addition to these

alternatives, a large flask capacity and liberal oxygenation enable cultures to remain

viable at saturation and therefore, further increase the amount of total protein produced.

Rare codon clusters can also adversely affect protein synthesis and thus negatively

impact protein expression (Kane, 1995). Amino acids are encoded by more than one

single codon, and each organism has its individual usage bias towards the available 61

amino acid codons. Disparity in codon usage between the expression host and intended

heterologous protein may obstruct translation of mRNA due to the need for tRNAs that

may be rare or absent in the E. coli host (Goldman et al., 1995; Kane, 1995). Of the

reported low-usage codons, AGG, AGA, CGA (which encode arginine), CUA (leucine),

AUA (isoleucine), CCC (proline), AGG and AGA are the rarest of codons.

Interestingly, clustering or interspersion of these rare codons, in particular near the 5′

end of a transcript, can potentially influence translation efficiency and hence diminish

protein yields (Goldman et al., 1995; Nakamura et al., 2000).

The aim of this part of the project was to produce a sufficient quantity and quality of the

four target proteins previously identified in chapter 3 (Section 3.2.2) to evaluate the

diagnostic potential of the novel antigens. To accomplish this, an E. coli-GST fusion

system was chosen and a range of protein expression parameters such as induction

- 104 -


methods, temperature variation and induction times were investigated and optimised for

each candidate protein.

- 105 -



4.1.1 Pilot Study using IPTG-induction for Protein Expression

The genes encoding protein candidates CT157, CT423, CT727 and CT396 were cloned

into a pGEX GST fusion vector and subsequently transformed into competent BL21

cells (Invitrogen) for protein expression (Table 4.1). Five individual colonies were

selected for each protein and transferred to 5mL of fresh Luria broth (LB) containing

100μg/mL ampicillin (LBA). Cultures were incubated at 37°C with shaking (225rpm)

until growth reached OD600 ~0.6. Prior to induction, a 200μL pre-induction sample was

collected from each culture and added to 50μL of 2X sample buffer (0.09M Tris-HCl

pH 6.8, 20% glycerol, 2% SDS, 0.1M DTT and 0.02% bromophenol blue). The

remaining cultures were halved (2.4mL) and 0.1M IPTG (Progen, Australia) was added

to one tube (IPTG induction control), whilst the other acted as an induction-negative

control. All cultures were further incubated with shaking (225rpm) at 37°C, 200μL

samples were collected at 2 hours post-induction and added to 50μL of 2X sample

buffer. IPTG-induction controls for each candidate protein were centrifuged at

3000rpm (Beckman GS-6R) for 5 minutes at 4°C. Supernatants were discarded, whilst

pellets were frozen at -80°C and retained for later analysis.

Table 4.1. Molecular weights of target genes with and without the GST fusion tag.

GENE a PROTEIN FUNCTION b MW c

(kDa) + GST d

(kDa) CT157 Phospholipase D Endonuclease 45.3 74.3

CT423 Hypothetical Protein (CBS domain) 41.6 70.6

CT727 Metal transport P-type ATPase 70.5 99.5

CT396 Heat Shock Protein 70 71.1 100.1

a, b Annotation/code from the Chlamydial (TIGR) database. c Molecular mass was calculated from the protein sequence d GST is a bacterial protein derived from Schistosoma japonicum

- 106 -


Pre-cast 4% - 20% 15 well gradient gels (Bio Rad, Australia) were used to resolve

proteins in the 200μL samples described above. Prior to electrophoresis, all samples

were denatured at 100°C for 5 minutes. Briefly, 10μL of each sample were loaded onto

the pre-cast SDS-PAGE gels and electrophoresed in Running Buffer (25mM Tris Base,

192mM glycine, 1% SDS) at 110 volts for 100 minutes at room temperature. All gels

were Coomassie stained (0.1% Coomassie blue, 40% methanol and 10% glacial acetic

acid) for 30 minutes and destained (40% methanol and 10% glacial acetic acid) until

background stain was removed. All gels were examined for candidate gene expression

prior to proceeding with fractionation of the retained pellets.

There was strong expression of CT396 from each of the clones but the remaining three

candidates genes exhibited extremely poor expression for all samples collected (data not

shown). Experiments using various expression parameters were undertaken in an

attempt to improve protein expression. A temperature reduction to 30°C and 25°C was

used and compared to the original temperature of 37°C. A temperature reduction to

25°C marginally improved protein expression for CT157 (data not shown). However,

no improvement of expression when compared to the original cultures grown at 37°C,

was evident for the three remaining target proteins (data not shown). A decrease in the

final induction concentration of IPTG from 1mM to 0.5mM did not enhance protein

expression (data not shown).

At every stage during the expression process, samples from each target protein were

tested to ascertain whether any were in fact secreted rather than solely localised and

expressed in the E. coli host’s cytoplasm (data not shown). Upon confirming none of

the protein targets were secreted, solubility levels were investigated. Determination of

protein solubility for each target protein was evaluated by disrupting the retained pellets

into soluble and insoluble fractions. All samples were then examined for solubility

levels on Coomassie-stained SDS-PAGE gels. Of the four candidate proteins, only

CT396 appeared to be highly soluble (data not shown). The expression levels of

CT727, CT423 and CT157 were extremely poor and determination of their solubilities

could not be made (data not shown). To enhance protein solubility, solubilisation

- 107 -


buffers, as directed by the Amersham protein expression manual, were added to the

repelleted insoluble fractions of each candidate protein and incubated with rocking at

4°C for 60 minutes. Samples were centrifuged at 3000rpm (Beckman GS-6R) for 15

minutes at 4°C, with the supernatant retained and sampled (200μL) prior to

resuspension of the pellet in solubilisation buffer. Incubation, centrifugation and

sampling steps were repeated and the final pellet was resuspended in 8M urea and

sampled. All collected fractions were analysed via Coomassie-stained SDS-PAGE gels.

Examination of the treated samples revealed no improvement in solubility for any of the

four candidate proteins (data not shown). As a result and given the relatively short

solubilisation incubation times, solubilisation buffer was added to the pelleted insoluble

fraction and incubated overnight at 4°C, however solubility was not improved (data not

shown). Therefore, other methods for protein expression were examined in an attempt

to increase soluble protein expression of the four target genes.

- 108 -


4.1.2 A Comparison of the Expression of Candidate Proteins by IPTG and

Autoinduction

The autoinduction of protein expression (Studier, 2005) was trialled. To test the

efficacy of the approach a direct comparison between autoinduction and IPTG-

induction was undertaken. Clones generated in Section 4.1.1 formed the basis of the

expression experiments described hereafter. To minimise disparity between the IPTG

and autoinduction comparison, single clones for each of the four genes were propagated

on LB agar plates (10% Bactotryptone, 5% yeast extracts, 0.09M NaCl, 15% agar (pH

7.5) containing 100μg/mL ampicillin. Samples were then transferred to liquid culture

(section 4.1.2.1) and induced using the two protein expression methods.

4.1.2.1 IPTG Induction of Target Proteins

A clone of each gene was cultured overnight at 37°C in 5mL LBA (LB plus ampicillin)

as described in Section 4.1.1. To compare the effect of different temperatures on

protein expression, two 5mL cultures of fresh LBA were inoculated with 250μL of each

overnight culture. Cultures were incubated at either 37°C or 25°C, with shaking

(225rpm) until growth reached OD600 0.6. Prior to induction, a 200μL pre-induction

sample was collected and added to 50μL of 2X sample buffer. The remaining cultures

were halved (2.4mL), 0.1M IPTG (Progen, Australia) was added to one tube, whilst the

other was used as an induction-negative control. Cultures were further incubated with

shaking (225rpm) at 37°C and 25°C. 200μL samples were collected at 2 hours post-

induction for each temperature and protein, and 4 hours post-induction for the 25°C

samples only, with 50μL of 2X sample buffer added. Remaining IPTG induction

cultures for both temperatures at 2 hours and 4 hours were centrifuged at 3000rpm

(Beckman GS-6R) for 5 minutes at 4°C. Supernatants were discarded and pellets frozen

at -80°C and retained for solubility determination.

- 109 -


4.1.2.2 Autoinduction of Target Proteins

In 50mL falcon tubes, a clone of each target gene was cultured overnight in 10mL of

autoinduction media (1% N-Zamine, 0.5% yeast extract, 25mM Na2HPO4, 25mM

KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4, 0.2X essential nutrient solution

(24mM CuSO4, 18mM MnSO4, 83.5mM Na2MoO4, 0.3mM H3BO3, 2mM CoCl2,

0.15M ZnCl2, 0.23M FeSO4, 0.9M SO3, 0.8mM biotin), 0.5% glycerol, 0.05% glucose

and 0.2% lactose) at 37°C and 25°C with shaking (225rpm). To 50μL of 2X sample

buffer, 200μL samples for both temperatures and all proteins were collected at 18 hour

and 24 hour intervals. Both 18 hour and 24 hour cultures were pelleted at 3000rpm

(Beckman GS-6R) for 5 minutes at 4°C and supernatant discarded. All pellets were

frozen at -80°C until required for fractionation.

4.1.3 Fractionation of Proteins Expressed via IPTG or Autoinduction

To determine protein solubility, retained cell pellets from autoinduction and IPTG-

induction experiments were thawed on ice and resuspended in 5mL 1X PBS. Lysozyme

(Roche) was added to a final concentration of 1mg/mL and incubated on ice for 20

minutes. Complete cell lysis was achieved via sonication (ultrasonic cell disruptor,

36818-Series, Cole Parmer) at 80W in 60 second bursts for 5 cycles with each sample

placed on ice between each cycle of disruption. The resultant lysates were centrifuged

at 3000rpm (Beckman GS-6R) for 15 minutes at 4°C to separate soluble and insoluble

fractions. All supernatants (soluble fraction) were retained and the pellets (insoluble

fraction) were resuspended in 5mL 1X PBS and 200μL samples for each soluble or

insoluble fraction, protein, induction method, induction time and temperature were

added to 50μL 2X sample buffer.

- 110 -


4.1.4 SDS-PAGE Determination of Protein Solubility of Each Candidate

Protein

Pre-cast 4%-20% gradient gels (BioRad, Australia) were used to resolve the

fractionated samples of each target recombinant protein. Prior to electrophoresis, all

samples were denatured at 100°C for 5 minutes. Briefly, 10μL of each fraction was

loaded onto the pre-cast SDS-PAGE gels and electrophoresed in Running Buffer

(25mM Tris Base, 192mM glycine, 1% SDS) at 110 volts for 100 minutes at room

temperature. All gels were Coomassie stained (0.1% Coomassie blue, 40% methanol

and 10% glacial acetic acid) for 30 minutes and destained (40% methanol and 10%

glacial acetic acid) until background staining was removed. Each gel was mounted onto

hydrated blotting paper and dried overnight at 80°C.

4.1.5 Level of Protein Expression in Autoinduced Cultures

To determine the level of protein expression, 10μL of the various samples were loaded

onto two pre-cast 4%-20% gradient SDS-PAGE gels (Bio Rad, Australia) and

electrophoresed in running buffer at 110 volts for 100 minutes at room temperature.

Proteins were transferred to Hybond C Extra nitrocellulose membranes (Amersham

Biosciences) at 4ºC, 100 volts for 1 hour using 3-(cyclohexylamino)-1-propane

sulphonic acid (CAPS) buffer and washed X4 in PBS and 0.1% Tween 20 (PBST).

Membranes were blocked using (5% skim milk powder in PBS containing 0.1% Tween

20 (SM-PBST)) for 1 hour at room temperature with rocking. Anti-GST polyclonal

conjugated horse radish peroxidase (HRP) antibody (Dako) was diluted 1:10000 in SM-

PBST and incubated with the membranes at room temperature for 1 hour with shaking.

After washing X4 with PBST, membranes were detected via chemiluminescence

(Amersham Biosciences ECL Plus Detection Kit) as per manufacturer’s instructions.

- 111 -


4.1.6 Densitometric Evaluation of Autoinduced and IPTG Induced Protein

Expression and Solubility

Dried gels were analysed by densitometry (BioRad Multi-AnalystTM/PC Version 1.1)

with measurements of band intensities given as Mean OD, for evaluation of protein

expression and solubility levels of each protein under the various conditions examined.

4.1.7 Determination of Rare Codon Usage of each Candidate Protein

Rare codon usage for all candidate proteins was determined by accessing the TIGR

database (codon usage display and primary sequence pages). Molecular weights of

protein fragments containing rare codons plus the GST tag were calculated by the

compute pI/MW tool program (http://ca.expasy.org/tools/pi_tool.html). Molecular

weights of the protein fragments observed on the anti-GST Western blots were

determined by using a log scale.

4.1.8 Preliminary Determination of Antibody Reactivity of Newly Expressed

Recombinant Proteins

To assess whether the expressed recombinant proteins CT157, CT423, CT727 and

CT396 were suitable for the diagnosis of C. trachomatis infections in males and females

(chapters 5 and 6), a single, strong-reacting patient sample was used to probe 10μg of

uninfected (UI), C. trachomatis L2-infected (I) and the four recombinant proteins (gel

purified - see Chapter 5, Section 5.1.2) using Western blot techniques as previously

described in Section 3.1.4.

- 112 -

http://ca.expasy.org/tools/pi_tool.html


4.2 RESULTS

4.2.1 Induction Method and Temperature Comparison of Candidate Protein

Expression

Sonicated fractions of the four proteins CT157, CT423, CT727 and CT396 collected at

differing times and temperatures from autoinduction and IPTG induction cultures were

resolved on SDS-PAGE gels and Coomassie stained (Figure 4.1). All four proteins

demonstrated enhanced expression and greater solubility in the enriched autoinduction

media, although expression levels and solubility differed significantly (Table 4.2). In

contrast and subsequent to IPTG addition, IPTG induction did not generate higher

yields of protein for any of the candidates even with longer incubation times. The

optimal autoinduction temperature for CT423, CT727 and CT396 was found to be 37°C

whilst the expression of CT157 was highest at 25°C. The 18 hour autoinduction culture

exhibited slightly higher levels of protein expression for CT423, CT727 and CT396

compared to the 24 hour interval, whereas expression of CT157 was only marginally

increased at 24 hours. The preferred expression temperature for candidate protein also

appeared to influence protein solubility. Moreover, as there was either reduced or no

protein expression, solubility of each gene product at 25°C was not examined by IPTG

induction.

Autoinduction of CT396, a 70kDa heat shock protein, resulted in an overall higher

protein expression at 18 hours (mean OD 0.60) at 37°C, compared to the 2 hour IPTG

induction (mean OD 0.47) at the same temperature. Extending autoinduction

incubation times to 24 hours did not further increase the expression of CT396, in fact, a

slight reduction in the level of protein expression (mean OD 0.09) was observed.

Moreover, improved levels of CT396 were observed for IPTG induction at 25°C with a

two hour increase in induction time, however, expression was reduced (mean OD 0.44)

compared to the autoinduction yields. Solubility of CT396 was higher (mean OD 0.60)

at the optimal temperature of 37°C in contrast to that produced at 25°C which

demonstrated a slight reduction in protein solubility levels (mean OD 0.52).

- 113 -


- 114 -

107 81

49

107 81

49

107 81

49

107 81

49

107 81

49

107 81

49

107 81

49

107 81

49

kDa kDa

kDa kDa

kDa kDa

kDa kDa

(a) (b)

(c) (d)

(e) (f)

(g) (h)

CT

157

CT

423

CT

727

Figure 4.1. Comparison of protein expression by IPTG or autoinduction. Arrows

indicate bands of the expected size for the expressed protein, whilst experiments

presented in diagrams (b), (d) and (f) failed to produce significant amounts of protein.

CT

396

AUTOINDUCTION IPTG

BL2

1

BL2

1 B

L21

BL2

1

BL2

1

BL2

1

BL2

1

18hr

BL2

1

18hr

18hr

18

hr

18hr

24hr

24hr

24

hr

24hr

24hr

24hr

inso

l

inso

l in

sol

inso

l

inso

l

inso

l

inso

l

inso

l in

sol

inso

l

inso

l

sol

sol

sol

sol

sol

sol

sol

sol

sol

sol

-ve

-ve

-ve

-ve

-ve

-ve

-ve -ve

-ve

-ve

-ve

-ve

IPTG

IPTG

IPTG

IPTG

IPTG

IPTG

IPTG

IPTG

IPTG

IPTG

IPTG

IPTG

18hr

24hr

24hr

inso

l

sol

sol

18hr

37°C 25°C 37°C 25°C

37°C 25°C 37°C 25°C 4hr 2hr37°C

25°C

4hr 2hr37°C

25°C

37°C 25°C 37°C 25°C 4hr 2hr

18hr

37°C 25°C 37°C 25°C

37°C

25°C

2hr 4hr 37°C

25°C


- 115 -

Table 4.2. Evaluation of autoinduction and IPTG expression levels for each candidate protein. For each temperature and induction

time the level of expression and protein solubility are given as Mean OD which was calculated by densitometry measurements.

AUTOINDUCTION IPTG

EXPRESSION

(Mean OD)

SOLUBILITY

(Mean OD) EXPRESSION

(Mean OD)

37°C 25°C 37°C 25°C 37°C 25°C PROTEIN 18hr 24hr 18hr 24hr 18hr 24hr 2hrs 2hrs 4hrs

CT157 0.00 0.00 0.21 0.22 0.00 0.38 0.00 0.00 0.00

CT423 0.51 0.42 0.29 0.31 0.54 0.40 0.00 0.00 0.00

CT727 0.25 0.23 0.20 0.18 0.42 0.41 0.00 0.00 0.00

CT396 0.60 0.51 0.36 0.45 0.60 0.52 0.44 0.27 0.44


CT727, a metal ion dependent P-type ATPase, showed an average level of expression

0.22OD (mean range of 0.18-0.25) under all conditions of autoinduction. Conversely,

IPTG induction did not lead to expression of CT727 under any of the trialled

conditions. Solubility levels of CT727 at 37°C and 25°C were comparable (mean OD

0.42 and 0.41, respectively) although, the slight increase in protein production at 37°C,

suggests that 37°C is the optimal temperature for expression. CT423, a hypothetical

protein containing a cystathionine beta synthase domain, exhibited higher levels of

expression in the enriched autoinduction media at the 37°C/18 hour time point (mean

OD 0.51) than 24 hours at the same temperature (mean OD 0.42) or at 25°C for either

incubation time (mean OD 0.29 and 0.31). IPTG induction did not increase the

expression of CT727 or CT423. Solubility of CT423 was greater at 37°C compared to

the reduced temperature of 25°C by 0.14 (mean OD). Protein production of CT157 was

only observed at 25°C, 18 hours and 24 hours (mean OD 0.21 and 0.22, respectively) by

autoinduction. Moreover, autoinduction at 37°C and IPTG induction failed to induce

sufficient levels of protein expression. Consequently, determination of protein

solubility was limited to the 25°C sample which, at 24 hours, was 0.38 (mean OD).

Optimum conditions determined for the expression of each protein are listed in Table

4.3.

Table 4.3. Summary of optimised conditions of induction and the levels of expression

and solubility.

EXPRESSION CONDITIONS

CT157 CT423 CT727 CT396

Method Autoinduction Autoinduction Autoinduction Autoinduction

Temperature 25°C 37°C 37°C 37°C

Expression (Mean OD) 0.22 0.51 0.25 0.60

Solubility (Mean OD) 0.38 0.54 0.42 0.60

Time 24hrs 18hrs 18hrs 18hrs

- 116 -


4.2.2 Verification of Protein Expression using Anti-GST

The level of expression of all four candidate proteins achieved using the autoinduction

method was determined by Western blotting and anti-GST antibody staining (Figure

4.2). CT423 and CT157 fractions induced by IPTG were included to assess levels of

protein expression even though none was evident in the original Coomassie stained gels

(Figure 4.2). In addition to the expressed candidate protein and the 29kDa GST tag (for

all probed samples) there were numerous partial fragments observed in each lane.

Minimal cross-reactivity was observed between the anti-GST probe and the BL21

control lane, although significant cross-reactivity of the partially expressed gene

fragments was evident.

A standard loading volume of 10μL was used for all anti-GST treated samples which

suggested that CT396 exhibited the highest levels of expression compared to the other

candidate proteins. The level of expression of CT727 was comparable to that observed

for the CT423 fractions obtained by autoinduction. In addition, both CT727 and CT423

also exhibited an equal amount of protein in the insoluble and soluble fractions.

Interestingly, IPTG induction of CT423 (2hr/25°C/IPTG) increased protein expression,

however, protein yields were considerably less than the autoinduced samples.

Autoinduction of CT157 (18hr/25°C) resulted in limited expression compared to the

other candidate proteins, however, the majority of CT157 was present in the soluble

protein fraction. Unlike the original Coomassie stained gel result (Figure 4.1), the anti-

GST antibody probe of IPTG-induced CT157 (25°C) revealed that a small amount of

protein was expressed.

- 117 -


AUTOINDUCTION IPTG

- 118 -

BL2

1 C

ontro

l

18hr

/37°

C

18hr

/37°

C/in

sol.

18hr

/37°

C/s

ol.

18hr

/37°

C

18hr

/37°

C/in

sol.

18hr

/37°

C/s

ol.

2hrs

/25°

C/-v

e

2hrs

/25°

C/IP

TG

CT396 CT727 CT157

107 81

49

34

27

20

AUTOINDUCTION IPTG

CT423CT157

18hr

/25°

C/in

sol.

18hr

/37°

C/in

sol.

2hrs

/25°

C/IP

TG

18hr

/25°

C/s

ol.

18hr

/37°

C/s

ol.

BL2

1 C

ontro

l

2hrs

/25°

C/-v

e

18hr

/25°

C

18hr

/37°

C

107 81

49

34 27

20

Figure 4.2. Verification of target protein expression by Western blot using anti-GST.

The temperature and expression times are specified for each lane and protein. Solid

arrows indicate the expected size of the recombinant candidate protein, whilst dotted

arrows designate the 29kDa GST tag.


- 119 -

4.2.2.1 Codon Usage and Expression of Candidate Proteins

It is possible that the truncated protein fragments detected in the Western blots by anti-

GST probe are the result of termination of translation at rare codons. This phenomenon

has been reported by numerous authors (Gouy and Gautier, 1982; Sharp et al., 1988;

Gutman and Hatfield, 1989; Zhang et al., 1991; Wada et al., 1992; Chen and Inouye,

1994; Goldman et al., 1995; Kane, 1995; Nakamura et al., 2000). Sequence analysis

revealed the presence of rare amino acid codons in all four genes encoding the

candidate proteins (Table 4.4). Several codons are reported to be rare, specifically

AGG, AGA, CUA, AUA, CGA and CCC (Kane, 1995). None of the candidate genes

contained the rarest AGG codon, however the remaining five codons were present in

varying amounts throughout each protein sequence. CT157 comprises 405 amino acids

and demonstrated the highest usage of rare codons (8.4%) of the candidate genes

closely followed by CT423 (6.8%). In comparison, CT727 and CT396 exhibited a rare

codon preference of 3.9% and 3.3% respectively.

For the four candidate proteins, molecular weights of the fragments evident in the anti-

GST Western blots (Figure 4.2, Section 4.2.2) were calculated. For each candidate

gene, truncated GST-fusion protein fragment sizes were predicted assuming translation

terminated at a rare codon using the ExPASy pI/MW tool program. By this approach, it

was predicted that CT157 would have two truncated GST-fusion protein fragments

whilst CT423, CT727 and CT396 would each resolve five truncated protein fragments.

Importantly, fragments observed for each candidate protein matched the occurrence and

size of a predicted fragment (Table 4.5) suggesting that these particular fragments may

indeed by generated by translation terminating at a rare codon. Additional truncated

fragments appeared in the protein fraction of CT423 (one), CT727 (four) and CT396

(four) that did not correlate with fragments predicted by the ExPASy program.


- 120 -

Table 4.4. The number of rare codons in the genes encoding the candidate proteins. Contained within each recombinant protein are

several rare codons which have the ability to cause translational problems (Chen and Inouye, 1994) and thus affect gene expression.

The number of rare codon occurrences and total percentage of rare codons within each protein are shown for all four target genes.

RARE CODON USAGE

AGG

(R)

AGA

(R)

CUA

(L)

AUA

(I)

CGA

(R)

CCC

(P)

PROTEIN PROTEIN CODON

NO.

# % # % # % # % # % # %

TOTAL CODON

NO.

TOTAL PERCENTAGE

(%)

CT157 405 0 0.00 7 1.72 9 2.22 7 1.72 6 1.48 5 1.23 34 8.4

CT423 370 0 0.00 6 1.62 7 1.89 5 1.35 1 0.27 6 1.23 25 6.8

CT727 660 0 0.00 1 0.15 9 1.36 8 1.21 3 0.45 5 0.75 26 3.9

CT396 661 0 0.0 8 1.21 7 1.05 1 0.15 4 0.60 2 0.30 22 3.3


Table 4.5. Molecular weights of observed and predicted fragments of the candidate

proteins. The molecular weights of the predicted fragments generated by translation

termination were calculated and added to the 29kDa GST fusion tag giving a final

fragment size. Estimated molecular weights were derived for each truncated protein

fragment observed in Figure 4.2.

EXPRESSED PROTEIN

AMINO ACID

No.

AMINO ACID

RARE CODON

PREDICTED FRAGMENT

MW(kDa) +GST

ESTIMATED FRAGMENT

MW (kDa)

UNMATCHED FRAGMENTS

(kDa) 20 P CCC 31.2 30

93 R AGA 39.1 CT157

99 R AGA 39.8 39

none

2 L CUA 26.2 25

22 R AGA 28.5 28

134 P CCC 43.8 43

223 P CCC 53.7

229 I AUA 54.4

231 R AGA 54.7

54

288 I AUA 61.3

CT423

311 I AUA 63.9 62

17

12 R CGA 30.3 29

95 R AGA 39.7 40 19

204 L CUA 50.9 51

273 L CUA 58.3 25

282 I AUA 59.2

285 I AUA 59.4 26

288 L CUA 59.7

58

CT727

443 L CUA 75.9 76

68

5 P CCC 29.6 29

76 R CGA 36.9

80 R AGA 37.4 36

115 R AGA 45.6 44

269 I AUA 57.9 58

CT396

393 P CCC 70.9 71

16

19

25

40

- 121 -


Two rare codons, CCC (proline) and AGA (arginine), present in CT157 appear to cause

termination of translation at amino acid positions 20 and 93 and 99 as predicted

molecular weights of 31.2kDa, 39.1kDa and 39.8kDa for the truncated proteins

respectively, match the molecular weights of 30kDa and 39kDa estimated for the

observed fragments. Of the six CT423 truncated proteins evident in Figure 4.2, only the

17kDa fragment did not match any predicted by the ExPASy program. Moreover, the

generation of the 54kDa and 62kDa CT423 correlated with the location of several rare

codons. The 54kDa fragment observed on gels may comprise the predicted 53.7kDa

(CCC), 54.4kDa (AUA) and 54.7kDa (AGA) fragments. Whilst the 62kDa fragment

potentially comprise either the 61.3kDa (AUA) or 63.9kDa (AUA) predicted protein

fragments. The other observed CT423 fragments closely match the molecular weight of

a predicted fragment.

Nine protein fragments were evident (Figure 4.2) for CT727. Of these, four fragments

(19kDa, 25kDa, 26kDa and 68kDa) did not correspond to any fragment on the basis of

occurrence of rare codons. Interestingly, the first and second rare codons situated at

amino acid positions 12 and 95 respectively, encode arginine although they differ in

codon sequence (CGA and AGA). Four rare codons located at amino acid positions

273, 282, 285 and 288 of CT727 could give rise to the observed 58kDa fragment. Four

protein fragments (16kDa, 19kDa, 25kDa and 40kDa) did not match predicted truncated

CT396 fragments, whilst the observed fragments of 29kDa, 36kDa, 58kDa and 71kDa

were a virtual match to their corresponding predicted fragments (29.6kDa, 36.9kDa,

57.9kDa and 70.9kDa respectively).

4.2.3 Antigenicity of Recombinant Proteins for the Potential Diagnosis of

Infection by C. trachomatis

To verify that the expressed recombinant proteins CT157, CT423, CT727 and CT396

were suitable for use in determining their potential for the diagnosis C. trachomatis

infections in males and females (chapters 5 and 6), a single, strong-reacting patient

sample was used to probe uninfected HEp-2 cells (UI), C. trachomatis-infected HEp-2

- 122 -


cells (I) and the four recombinant proteins (Figure 4.3). Present in the infected (I) lane

were bands B (72.4kDa) and C (44.6kDa) with band B showing the strongest antibody

reactivity. In addition to the two differential bands demonstrated in the Western blot

(excluding the uninfected lane), were the presence of all recombinant protein candidates

for bands B (CT727 and CT396) and C (CT157 and CT423). Interestingly, the patient

sample cross-reacted to the BL21 control. This suggests that various epitopes which

produced an antibody response in the host are either highly homologous or also present

in the proteins of the BL21 control.

107 81

49

34

27

20

U I BL

21 C

ontr

ol

CT

157

CT

423

CT

727

CT

396

i B

C

iv iii

ii

kDa

Figure 4.3. Western blot of recombinant proteins probed with a single patient serum

sample. Uninfected (UI) and infected (I) cell preparations and the four recombinant

proteins CT157, CT423, CT727 and CT396 were tested to ensure their suitability for

future experimental work. Differential bands B (72.4kDa) and C (44.6kDa) are

indicated in the C. trachomatis-infected lane, whilst antibody reactivity to the

recombinant proteins is designated as follows: i = CT157, ii = CT423, iii = CT727 and

iv = CT396. (Molecular weights including the 29kDa GST fusion protein for each

protein are: CT157 – 74.3kDa, CT423 – 70.6kDa, CT727 – 99.5kDa and CT396 –

100.1kDa. Exposure time after detection was 45 seconds.

- 123 -


4.3 DISCUSSION

Autoinduction, developed by Studier (2005), proved to be the simplest and most

effective method for production of usable quantities of the four chlamydial candidate

proteins. The enriched autoinduction media provided the selected E. coli host strain the

ability to attain high-density cell growth prior to expression of the heterologous protein,

thereby producing an increase in final protein yields upon comparison to conventional

induction using IPTG.

Expression of heterologous recombinant proteins is an extremely effective method for

analysing the structural, functional, immunological and possible interactive

relationships of chlamydial proteins. An E. coli expression system was chosen for

protein production of the candidate genes due to the ease of facilitating expression,

rapid high-density cultivation, well characterised genome and potential for generating

high protein yields (Baneyx, 1999). Of the four expressed candidate proteins,

regardless of induction method, only CT396 was expressed at a high level. The low

level of protein production of CT157, CT423 and CT727 may result from influences

such as sub-optimal codon usage (Gouy and Gautier, 1982; Sharp et al., 1988; Gutman

and Hatfield, 1989; Zhang et al., 1991), toxicity of the heterologous protein (Miroux

and Walker, 1996; Dumon-Seignovert et al., 2004) and rapid proteolytic degradation

(Enfors, 1992). Cloned heterologous proteins are often varied in their codon preference

to that of the expression host. Present in each candidate protein are several rare codons

which are also reported to be under-represented in E. coli, eg. AGA, CUA, AUA, CGA

and CCC (Zhang et al., 1991; Wada et al, 1992; Kane, 1995). Under typical growth

conditions, heterologous proteins are expressed in E. coli at comparable levels to the

abundantly produced endogenous proteins. However, when the mRNA of a transgene is

overexpressed in E. coli, differences in codon bias can impede translation due to the

demand for one or more tRNAs that, in the host, may be rare or totally deficient (Dong

et al., 1996). This implies that genes containing codons rarely used by E. coli may not

be efficiently expressed, thus resulting in poor protein yields and protein truncation.

Ivanov et al. (1992) showed tandem AGG triplets significantly inhibited gene

- 124 -


expression regardless of their location in the mRNA. However, the most severe effects

on protein expression have been demonstrated when consecutive low-usage codons are

situated near the 5′ end of a transcript (Goldman et al., 1995; Nakamura et al., 2000).

Interestingly, none of the chlamydial candidate genes displayed consecutive rare codons

near the N-terminus of the protein. Although, whilst multiple rare codons appear to

severely impact mRNA translation, each gene demonstrated the presence of a single N-

terminal rare codon. Moreover, given their compromised expression, in particular,

CT157, CT423 and CT727, this suggests that even singular, rare N- terminal codons can

result in a reduction of heterologous protein expression. Furthermore, of the four

candidate proteins, CT157 contained the highest percentage of rare codons (8.4%)

interspersed throughout its coding sequence and demonstrated the lowest protein

expression. CT423, CT727 and CT396 all have a lower percentage of rare codons

(6.8%, 3.9% and 3.3% respectively) which may reflect an increase in protein production

compared to CT157 expression. This implies that rare codons can affect truncation of

the intended target protein wherever the location in the mRNA or the number of codons

in tandem repeats. However, given that not all the rare codons evident in CT423,

CT727 and CT396 were represented by a corresponding protein fragment, low-codon

usage is not the sole contributing factor of their reduced protein expression.

Numerous studies have shown that heterologous proteins may be toxic to the host cell,

either from overexpression or as a result of their innate functional characteristics, can

adversely affect expression and protein solubility (O’Connor and Timmis, 1987; Brown

et al., 1993; Doherty et al., 1993; Suter-Crazzolara and Unsicker, 1995; Yike et al.,

1996). As a defence, toxic proteins aggregate in an insoluble form and are sequestered

into either the cytoplasm or periplasm (Strandberg and Enfors, 1991; Moore et al.,

1993; Suter-Crazzolara and Unsicker, 1995) where host damage is minimised and

protein expression is therefore reduced. Interestingly, the expression levels of each

candidate protein were in direct contrast to their solubility, thus indicating that the

overexpression of CT157, CT423 and CT727 were toxic to the E. coli host as inclusion

formation is not initiated unless a toxic heterologous protein is recognised and the

- 125 -


host’s own degradative capacity has been exceeded (Frydman, 2001; Hartl and Mayer-

Hartl, 2002). An established technique for limiting in vivo recombinant protein

aggregation consists of cultivation of cells and protein expression at reduced

temperatures (Schein, 1989). This approach has proven effective in enhancing the

solubility of a number of structurally and functionally diverse proteins including human

interferon α-2 (Schein and Noteborn, 1988), subtilisin E (Takagi et al., 1988), ricin A

chain (Piatak et al., 1988), bacterial luciferase (Escher et al., 1989), Fab fragments

(Cabilly, 1989), β-lactamase (Chalmers et al., 1990), rice lipoxygenase L-2 (Schirano

and Shibata, 1990), soybean lypoxygenase L-1 (Steczko et al., 1991), kanamycin

nucleotidyltransferase (Liao, 1991) and rabbit muscle glycogen phosphorylase

(Browner et al., 1991). A reduction of temperature (25°C) did decrease protein

insolubility and enhance expression of CT157 during autoinduction. In contrast,

solubility levels of CT423, CT727 and CT396 were notably greater at a higher

temperature (37°C) and expression of each of the three genes was also substantially

increased. Moreover, since low-temperature expression has the added advantage of

reducing proteolysis of the gene product (Emerick et al., 1984; Chesshyre and Hipkiss,

1989), the correlation of CT157 protein truncations to rare codon-containing fragments

demonstrates proteolytic degradation was prevented at the lowered expression

temperature, but increased at the higher temperature indicating that the additional

protein fragments of CT423, CT727 and CT396 were not solely the result of sub-

optimal codon usage.

A selection of fusion tags (MBP, GST, and Trx) can be used to facilitate the production

and solubilisation of the target protein. As a consequence, Kapust and Waugh (1999)

contrasted the effectiveness of three fusion tags at impeding the misfolding of six

aggregate-prone polypeptides. The study concluded that MBP was far more effective as

a solubilising partner compared to either GST or Trx. However, although the six

polypeptides investigated by Kapust and Waugh (1999) were diverse in structure and

function, the largest protein was only 29kDa which is significantly less than the

molecular weights of the four candidate proteins (42kDa – 71kDa) expressed in this

- 126 -


study. Several researchers have successfully employed the GST-fusion system for

expressing large (60kDa – 177kDa), highly soluble chlamydial proteins and relied upon

IPTG induction to initiate their expression (Debattista et al., 2002; Montiagiani et al.,

2002; Sharma et al., 2004; Finco et al., 2005). Montiagiani et al. (2002) cloned and

expressed the C. pneumoniae CT396 homolog by concurrently utilising a C-terminus

six-histidine tag and GST N-terminal tag as expression with only one fusion partner

resulted in poor expression and marked degradation of the protein product.

Interestingly, although CT396 was also expressed with a GST tag and showed evidence

of proteolytic degradation and protein aggregation, protein expression in the enriched

autoinduction media produced a highly soluble product. Employing the IPTG induction

method and pQE-60 expression vector, Bas et al. (2001) incorporated a C-terminal six-

histidine tag fusion protein to CT396 for use in the comparative analysis of several

diagnostic tests. High expression and solubility of CT396 was achieved however, as an

incorrect base change was introduced into the protein during cloning. In vitro site-

directed mutagenesis was required to reverse the translational error. Despite the fact

that a portion of CT396 was found to be insoluble given the increased expression times

of autoinduction compared to IPTG induction, the overall expression and unwarranted

need for further manipulation reinforces the superiority of the GST fusion system and

autoinduction for heterologous chlamydial protein expression.

In summary, heterologous protein expression and subsequent protein solubility is

dependent upon several factors: vector and fusion partner selection, sub-optimal codon

usage, protein toxicity, proteolytic degradation, induction method and cultivation

parameters such as temperature, pH and oxygenation. Evident from comparison of

autoinduction and IPTG induction is the profound impact that rare-codon usage can

impose on successful production of candidate protein. In addition, although IPTG

induction is generally the preferred option for recombinant protein expression,

autoinduction, employed at the optimal temperature for the candidate protein, has the

ability to further enhance otherwise poor protein production and solubility. Despite the

low protein level of production of CT157 by autoinduction, the newly derived

expression protocols for CT157, CT423, CT727 and CT396, will enable the evaluation

- 127 -


of their diagnostic potential for the discrimination of acute and chronic C. trachomatis

infections. (Chapters 5 and 6).

- 128 -

Chapter 5: Diagnostic Potential of Antigens for Identification of Chronic Infection

CHAPTER 5

Diagnostic Potential of Novel Antigens for the Identification

of Chronic Infection in Females

- 129 -


5.0 INTRODUCTION

The process of acute chlamydial infection is not entirely understood. The host response

to an initial chlamydial infection at the mucosal level occurs within 1 - 2 days post-

infection (Williams et al., 1981). Intense inflammation and predominant mucosal

infiltration of neutrophils and monocytes are accompanied by attraction of T cells to the

infection site where they play a pivotal role in the control of infection (Williams et al.,

1981; Rank et al., 1985; Stephens, 2003). Host immune reaction manifests along the

Th1 pathway in response to bacterial entry in an attempt to drive the infection towards

resolution (Debattista et al., 2002; McGuirk and Mills, 2002). Conversely, the hallmark

of chronic C. trachomatis infection is an inflammatory process that is exacerbated by

reinfection and may culminate in fibrosis, extensive tissue damage, scarring and PID

(Cumming et al., 1988; Cates and Wasserheit, 1991; Cohen and Brunham, 1999; Mabey

et al., 2003). To resolve a persistent or chronic chlamydial infection the Th1 pathway

and its associated cytokine response is critical. In contrast, a Th2 immune response

which produces antigen-specific antibodies, has the potential to initiate a chronic

infection and promote associated adverse sequelae (Debattista et al., 2002; McGuirk

and Mills, 2002). Moreover, Th2 responses impede chlamydial destruction and favour

a chronic or persistent state. Alternatively, hyperstimulation resultant from excess Th1

cytokine production may evoke extensive tissue degradation and scarring via cytotoxic

T lymphocyte (CTL) action (Debattista et al., 2002). Long-term sequelae such as tubal

occlusion leading to infertility, ectopic pregnancy and chronic pelvic pain develop in

approximately 25% of women with C. trachomatis-induced PID (Svensson et al.,

1981).

The most common long-term complication of acute PID is tubal factor infertility (TFI)

(Westrom, 1980; Westrom et al., 1992). In a longitudinal study (1960 – 1984)

conducted by Westrom et al. (1992) on a large cohort of 2501 women treated for

suspected PID, laparoscopic investigation revealed that 1844 women had acute PID and

657 had no sign of the disease (control group). Confirmed TFI was established in 141

PID patients with no evidence of the disease found in the control group. The rate of

- 130 -


infertility was reported to be directly associated with the number and severity of PID

infections. Hence, every subsequent episode of PID roughly doubled the rate of TFI,

increasing from 8% with one C. trachomatis infection, to 19.5% with two exposures

resulting in infection and 40% with three or more episodes. Damage to the fallopian

tube/s as a result of PID is a well established cause of ectopic pregnancy (Cumming et

al., 1988; Westrom, 1991; Gerard et al., 1998). Interestingly, Cumming et al. (1988)

reported that ongoing, low-grade post inflammatory salpingitis was not solely localised

to the ectopic pregnancy site, thus suggesting that surgical diagnosis and microscopic

examination does not preclude inflammation and/or infection in women where a

diagnosis of no disease has previously been established. Epidemiological evidence of

ectopic pregnancy accumulated over a 28 year period (1970 – 1997) demonstrated a ≥2

fold increase of ectopic pregnancy subsequent to C. trachomatis infection with the

greatest occurrence in women ≥25 years of age (Kamwendo et al., 2000). The authors

also reported that a reduction of PID was strongly associated with a decline of ectopic

pregnancy.

Available techniques used to diagnose C. trachomatis infections comprise direct

detection methods such as enzyme immuno-assays (EIA) and nucleic acid amplification

(NAATs) eg. PCR, and indirect serological tests that include microimmunofluorescence

(MIF) and enzyme-linked immuno-assays (ELISAs) (Black, 1997; Battle et al., 2001).

Importantly, all these methods are variable in their performance with regards to

sensitivity and specificity. Moreover, PCR as a diagnostic method for the detection of

chlamydial infection is to some degree limited as clinical specimens are routinely

sampled from the lower genital tract, hence infections in the fallopian tube where

inflammatory damage is most significant, escape detection. Whilst other serological

assays aim to specifically discriminate between acute and chronic C. trachomatis

infection, only one ie. Medac HSP60 test (Hamburg, Germany) has the potential to

detect chronic infection. However at present, there are conflicting reports as to the

efficacy of the test as increased levels of anti-HSP60 antibodies have been demonstrated

in not only chronic C. trachomatis infections, but also in acute infections (Bax et al.,

2004; Gazzard et al., 2006).

- 131 -


As a consequence, this study aimed to utilise the antigens identified in Chapter 3 which

were subsequently expressed as recombinant proteins (Chapter 4) to evaluate their

diagnostic potential for the discrimination of acute and chronic C. trachomatis

infections in female samples.

- 132 -




Two samples collected from 1998 – 2002 (batch #1) (Chapter 2, Section 2.2) from all

seven patient groups and one sample collected in 2006 (batch # 2) (Chapter 2, Section

2.2) from groups I –IV were analysed for this aspect of the project.

5.1.2 Production of Recombinant Chlamydial Proteins

The four candidate antigens CT157, CT423, CT727 and CT396 were produced on a

large scale by myself and Drs. Gomez and Wan (QUT, Brisbane, Australia) utilising the

optimised protein expression protocols outlined in Chapter 4. All four recombinant

proteins were gel purified from a SDS-PAGE gel. Briefly, using a one-welled comb,

400μL of each recombinant protein was loaded onto separate SDS-PAGE gels and

resolved. Each gel was Coomassie stained and the target proteins were excised using a

horizontal cut above and below the protein. Vertical edges were scored and the gel

slices were transferred to separate 2mL eppendorf tubes. An 800μL aliquot of 8M urea

was added and tubes were vortexed for 60 seconds. Tubes were incubated at room

temperature for 20 minutes and then centrifuged at 14 000g for 30 minutes at room

temperature. Supernatants were transferred to a fresh Eppendorf tube, 200μL of 8M

urea was added and samples were again incubated at room temperature for 20 minutes

and subsequently centrifuged at 14 000g for 30 minutes at room temperature. The

extracted proteins were resolved on a SDS-PAGE gel and extraction of the correct band

was verified via normal Western blotting methods.

5.1.3 Evaluation of Recombinant Proteins Diagnostic Potential by Western

Blotting

To determine the identity of Bands B and C, individual patient samples (n = 3) from

groups I - VII were probed against 10μg of the four recombinant proteins and BL21

- 133 -


untransformed cell control. In addition, to directly compare antibody reactivity of the

recombinant proteins to the previously observed individual banding profiles, 10μg of

chlamydia/host cell proteins from non-infected HEp-2 cells (UI) and HEp-2 cells

infected with C. trachomatis serovar L2 (I) were also included in each Western blot.

Western blot detection methods were as previously described in Chapter 3, Section

3.1.4.


Western Blots

Banding intensity for each of the four recombinant proteins when probed by patient

samples from groups I – VII were graded on an arbitrary scale of 1+ - 6+, with 1+

signifying a very weak antibody response to the protein, whilst 6+ was indicative of an

extremely strong banding intensity. Moreover, given the limited amount of patient

samples available, a visual method of grading was chosen over quantitative estimation

using dilution series and densitometry.

- 134 -


5.2 RESULTS

5.2.1 Evaluation of Candidate Antigens Diagnostic Potential via Western

Blotting with Patient Samples

Figures 5.1 - 5.3 show typical Western blot profiles obtained when three patient

samples from each of groups I – VII were probed against uninfected HEp-2 cells (UI),

HEp-2 cells infected with C. trachomatis L2 (I), non-transformed BL21 E. coli cells

(negative control) and CT157 (band C candidate), CT423 (band C candidate), CT727

(band B candidate) and CT396 (band B candidate) recombinant proteins. Figure 5.4

indicates how banding profiles for each of the four recombinant proteins were

individually graded on a scale of 1+ to 6+, according to their intensity. All infected

patient groups demonstrated an antibody response, in varying banding intensities, to the

four recombinant proteins (Table 5.1). Of the four candidates for bands B and C,

CT727 (band B) and CT157 (band C) exhibited the strongest overall antibody reactivity

upon comparison to the two other (CT423 and CT396) antigenic candidates.

Furthermore, samples from acute multiply infected patients (group III) demonstrated a

relatively constant level of banding intensity against the four recombinant proteins.

Interestingly, CT423 (band C candidate) demonstrated no reactivity when probed by

samples obtained from patients diagnosed with PID. Moreover, antibody reactivity

against CT423 for all the stages of C. trachomatis infection was the lowest when

compared to the three other proteins.

Recombinant CT157 demonstrated the strongest band intensity (mean = 5.7) when

probed against samples from group III (a history of two or more C. trachomatis

infections, with the most recent estimated to have been acquired less than 4 months

previously). In comparison, groups I (first-time C. trachomatis infection estimated to

have been acquired less than 4 months previously), II (first-time infection estimated to

have been acquired more than 12 months previously) and IV (C. trachomatis-induced

PID) showed mean band intensities of 2.0, 2.7 and 5.0 respectively (Figure 5.5). All

three patient samples from the PID group (IV) were negative for an antibody response

- 135 -


when probed against recombinant CT423 (Figure 5.5). As observed for CT157,

antibody reactivity to CT423 was the strongest when probed by samples from group III.

Moreover, the mean band intensity of antibody binding to CT423 when probed by

samples was 2.0 and 2.7 for groups I and II respectively. The antibody response to

recombinant CT727 when probed by samples from groups I and II showed a mean value

of 2.7 and 2.3 respectively, whilst the tested samples from groups III and IV

demonstrated an increased response (4.7 and 4.0 respectively) (Figure 5.6). Of the four

patient groups, group II samples had the lowest banding intensity (mean = 1.7) when

probed against CT396, whilst groups I, II and IV exhibited a similar level of intensity

(Figure 5.6). Of the four recombinant proteins, only CT157 and CT423 showed an

antibody response when probed by samples from group VI (adult negative control).

- 136 -

Chapter 5: Diagnostic Potential of Antigens for Identification of Chronic Infection Chapter 5: Diagnostic Potential of Antigens for Identification of Chronic Infection

GROUP I GROUP I

BATCH #2 SAMPLES BATCH #2 SAMPLES BATCH #1 SAMPLES BATCH #1 SAMPLES

107 107

49 49

20 20

34 34

27 27

81 81

107

49

20

34

27

81

107

49

20

34

27

81

(a). (b).

(c). (d). Figure 5.1. Reactivity of recombinant candidate proteins to patient samples - part A.

Samples from groups I - (a) and (b); II - (c) and (d) were probed against recombinant

CT157 & CT423 (band C) and CT727 & CT396 (band B). MWs of each protein

including the GST tag were as follows: CT157=74kDa, CT423=71kDa, CT727=99kDa

and CT396=100kDa. The number under/beside each band indicates the respective

banding intensities as derived by the grading system. Patient I.D.’s are indicated.

Exposure time was 45 seconds.

Figure 5.1. Reactivity of recombinant candidate proteins to patient samples - part A.

Samples from groups I - (a) and (b); II - (c) and (d) were probed against recombinant

CT157 & CT423 (band C) and CT727 & CT396 (band B). MWs of each protein

including the GST tag were as follows: CT157=74kDa, CT423=71kDa, CT727=99kDa

and CT396=100kDa. The number under/beside each band indicates the respective

banding intensities as derived by the grading system. Patient I.D.’s are indicated.

Exposure time was 45 seconds.

GROUP II GROUP II

C C

107

49

20

34

27

81

UI I -ve

cont

rol

CT

157

CT

423

CT

727

CT

396

B

C C C

UI I -ve

cont

rol

-ve

cont

rol

CT

157

CT

157

CT

423

CT

423

CT

727

CT

727

CT

396

CT

396

B B

UI I UI I ve c

ontr

ol

CT

157

CT

423

CT

727

CT

396

-ve

cont

rol

-ve

cont

rol

CT

157

CT

157

CT

423

CT

423

CT

727

CT

727

CT

396

CT

396

-

BATCH #1 SAMPLES BATCH #1 SAMPLES

- 137 -

107

49

20

34

27

81

107

49

20

34

27

81

(a). (b).

(c). (d).

107

49

20

34

27

81 B

C

UI I -ve

cont

rol

CT

157

CT

423

CT

727

CT

396

UI I kDa kDa

2+ 2+ 3+ 4+ 3+ 2+ 5+

13817 QUTNP12

BATCH #2 SAMPLES

UI I UI I -ve

cont

rol

CT

157

CT

423

CT

727

CT

396 1+

kDa kDa

2+ 4+ 1+ 1+ 2+ 3+ 2+ 4+

NPQUT007 10622

- 137 -


GROUP III

(a). (b).

(c). (d).

Figure 5.2. Reactivity of recombinant candidate proteins to patient samples – part B.

Samples from groups III - (a) and (b); IV - (c) and (d) were probed against recombinant

CT157 and CT423 (band C), and CT727 and CT396 (band B). MWs of each protein

including the GST tag were as follows: CT157 = 74kDa, CT423 = 71kDa, CT727 =

99kDa and CT396 = 100kDa. The number under/beside each band represents banding

intensities as derived by the grading system. Patient ID’s are indicated. Exposure time

was 45 seconds.

107

49

20

34

27

81

107

49

20

34

27

81

UI I UI I

B

C

B

C

-ve

cont

rol

CT

157

CT

423

CT

727

CT

396

-ve

cont

rol

CT

157

CT

423

CT

727

CT

396

107

49

20

34

27

81

GROUP IV


kDa kDa

3+ 2+ 3+ 5+ 4+ 6+ 2+ 6+

4020 QUTNP11


UI I -ve

cont

rol

CT

157

CT

423

CT

727

CT

396

UI I -ve

cont

rol

CT

157

CT

423

CT

727

CT

396

kDa kDa

107 4+ 2+ 3+

49

B 5+ 81 6+ 4+

C

34

27

20

12581 QUTNP004

- 138 -


NEGATIVE CONTROL

UI I -ve

cont

rol

CT

157

CT

423

CT

727

CT

396

kDa

107

49

34

27

81

Figure 5.3. Reactivity of samples from negative control groups to the recombinant

candidate proteins. A single sample from each of the negative control groups (V – VII)

were probed against recombinant CT157 and CT423 (band C) and CT727 and CT396

(band B). Only bands in the BL21 negative control were reactive to the control sample.

Patient I.D. is indicated. Exposure time was 45 seconds.

4120081

-ve

cont

rol

kDa

CT

157

CT

423

CT

727

CT

396

UI I 107

49

34

27

81

3+ 6+ 6+ 2+

Figure 5.4. Banding intensity grading for each of the four recombinant proteins when

probed by patient samples. Exposure time was 45 seconds.

- 139 -


Table 5.1. Reactivity of individual patient samples against the recombinant candidate

proteins. Three samples from groups I - IV and one from each group V – VII were

probed against each recombinant protein and produced the following banding

intensities.

Band C candidates Band B candidates

PATIENT GROUP

PATIENT I.D. CT157 CT423 CT727 CT396

2361176 + + +++ ++++

13817 ++ +++++ +++ ++++ I

QUTNP12 +++ - ++ ++

13619 ++ +++ ++ ++

10622 ++ +++ + + II

QUTNP007 ++++ ++ ++++ ++

4020 +++++ +++ ++ +++

9908 ++++++ +++++ ++++++ ++++ III

QUTNP11 ++++++ ++ ++++++ ++++

12581 ++++ - ++ ++++

11011 +++++ - +++++ ++ IV

QUTNP004 ++++++ - +++++ +++

IVF034 - - - -

4120335 ++ ++ - - V, VI and

VII CF1 - - - -

Band intensity: + = very weak; ++ = weak; +++ = medium; ++++ = strong; +++++ = very strong; ++++++ = extremely strong; - = no band present.

- 140 -


(a)

CT157

0

1

2

3

4

5

6

I II III IV Neg

Patient Groups

Ban

d In

tens

ity

0

1

2

3

4

5

CT423

Patient Groups

I II III IV Neg

Ban

d In

tens

ity

(b)

Figure 5.5. Band intensity levels (scale of +1 - +6) for patient samples from groups I –

VII when probed against recombinant proteins (a) CT157 and (b) CT423. Each dot is

representative of an individual sample, whilst the bars indicate the mean band intensity

for each patient group.

- 141 -


0

1

2

3

4

5

6

CT727

Patient Groups

I II III IV Neg

Ban

d In

tens

ity

(a)

(b)

0

1

2

3

4

CT396

Patient Groups

I II III IV Neg

Ban

d In

tens

ity

Figure 5.6. Band intensity levels (scale of +1 - +6) for patient samples from groups I –

VII when probed against recombinant proteins (a) CT727 and (b) CT396. Each dot is



- 142 -


5.2.2 Antibody Response of Recombinant Antigens during stages of C.

trachomatis Infection

The data presented in Section 5.2.1 were used to compare the antibody response of the

four candidate antigens in patients groups showing acute, recovering acute, and chronic

of C. trachomatis in women and those that are multiply infected (Figure 5.7).

Importantly, these patient groups do not show a progression through stages but rather,

are suggestive of stages, recoveries and multiple infections. In general, fewer patients

from group I (acute) compared to those in groups II (recovering acute), III (multiple)

and IV (chronic) demonstrated an antibody response to the four recombinant antigens.

An antibody response from a greater number of multiply-infected patients (group III) to

CT157 is shown whilst a fewer number of acute patients (group I) recognise this

antigen. The antibody response from acute patients (group I) to CT423 is similar to that

of CT157, however the response by patients that have been re-infected (group III) is

greater whilst the PID group (IV) show no antibody response to CT423. Antibody

responses by patients to CT727 show the same profile as CT157 although fewer patients

recognised CT727 by comparison. Of the four candidate antigens, CT396 had an

antibody response from the greatest number of patients.

- 143 -


0

1

2

3

4

5

6

Onset I II III IV

CT157 Antibody Response


Ant

ibod

y R

espo

nse

0

1

2

3

4

5

6

Onset I II III IV


Infection Stage/Time CourseA

ntib

ody

Res

pons

e

0

1

2

3

4

5

6

Onset I II III IV



Ant

ibod

y R

espo

nse

0

1

2

3

4

5

6

Onset I II III IV



Ant

ibod

y R

espo

nse

Figure 5.7. Responses to recombinant antigens (a) CT157, (b) CT423, (c) CT727 and

(d) CT396 during C. trachomatis infection in the defined patient groups. Although the

x-axis is interpreted as a temporal scale, the even spacing of the stages is purely

arbitrary, not necessarily reflecting a “to scale” time line. (Antibody response = mean

band intensity of each candidate protein derived from the four patient groups).

(b)(a)

(c) (d)

- 144 -


5.3 DISCUSSION

The objective of this study was to assess the diagnostic potential of the four previously

identified candidate antigens (Chapter 3) for the discrimination of acute versus chronic

C. trachomatis infection. Different levels of antibody reactivity to the candidate

proteins CT157, CT423, CT727 and CT396 were shown when probed by samples from

C. trachomatis-infected females. Overall, CT157 and CT727 (band C and B candidates

respectively) demonstrated similar antibody response profiles that were much higher

than those observed for CT423 and CT396.

Surface components of the chlamydial elementary body trigger an immune response in

the host since it is this infectious particle that is first encountered. Infection by C.

trachomatis in humans instigates an immune response in the formation of antibodies

primarily directed against MOMP, the main EB component (Su and Caldwell, 1993;

Knight et al., 1995; Ortiz et al., 1996). The humoral (Th2) and cell-mediated immunity

(Th1) responses perform diverse functions and involve different effector mechanisms

for generating immunity. Humoral immunity is mediated by various antibodies which

have the ability to neutralise antigens, whilst in contrast, cell-mediated immunity exerts

its function through T-cells (Mestecky et al., 1991; Stephens et al., 2003). Given that

Chlamydia must invade host cells to replicate and initiate the dissemination of infection,

the resolution of chlamydial infection necessitates a Th1 immune response (Holland et

al., 1993; Knight et al., 1995; Magee et al., 1995; Perry et al., 1997). Interestingly, the

production of antibodies against CT157 and CT727 were lowest in patient groups I

(first-time C. trachomatis infection estimated to have been acquired less than 4 months

previously) and II (first-time C. trachomatis infection estimated to have been acquired

more than 12 months previously). This suggests that although these antigens stimulate

the host’s immune system at the initial and recovery stages of infection, they do not

elicit an immune response capable of resolution as antibody levels were higher in

patients with chronic disease (group IV). Moreover, the high antibody response to

CT157 and CT727 by group IV (even though the number of sera available for analysis

was limited tends to suggest that although antibodies are long-lived, both antigens are

- 145 -


presented to the host immune system causing continued production of antibodies.

Exposure of the immune system to a foreign antigen normally enhances the host’s

ability to respond to subsequent re-exposure of the same antigen, meaning that

subsequent infections elicit a stronger antibody response compared to that derived by

the original infection (Dutton et al., 1998). This immunologic memory is evident in

group III patients with a history of two or more C. trachomatis infections which show

extremely high levels of antibody reactivity to CT157 and CT727. This is further

supported by group III patients demonstrating the highest antibody response to antigens

CT423 and CT396 in comparison to the other patient groups.

Prior to this study, none of the four identified proteins were assessed for their antigenic

qualities as potential discriminators of acute and chronic disease. Although the cellular

location and function of CT396 (HSP70) suggests obvious antigenic properties, it is not

clear why the other three proteins studied elicit a humoral response in the host.

Previously, two studies suggested that HSP70 may play a role in either attachment or

entry of the elementary body (EB) into host cells (Schmiel et al., 1991; Raulston et al.,

1993). Further investigation by Raulston et al. (2002) using immunofluorescence and

transmission electron microscopy, demonstrated that recombinant HSP70, whilst being

associated with outer membrane complexes of EBs, is not actually a EB surface-

presented ligand. HSP70 may in fact enhance host ligand interactions subsequent to

initial endocytosis of the infectious EB particle. The research presented here appears to

support this premise as unlike the graduated immune response observed across all

infected patient groups to CT157 and CT727, the antibody reactivity of groups I and III

to CT396 was high, yet low in patients recovering from a past acute infection (group II)

and those diagnosed with chronic disease (group IV).

In an attempt to further improve current diagnostic capabilities for the discrimination of

chronic disease, studies have targeted proteins known to promote an immunogenic

response in the host (Bax et al., 2003, 2004; Dadamessi et al., 2005). Utilising the

Medac cHSP60 enzyme-linked immunosorbent assay (ELISA) test for the diagnosis of

chronic infection, Bax et al. (2004) reported that 27% of women with tubal pathology,

- 146 -


16% of women without tubal pathology and 4.8% of uninfected pregnant women

(controls) demonstrated anti-C. trachomatis HSP60 antibodies. In contrast, Gazzard et

al. (2006) who also investigated the diagnostic merit of Medac’s cHSP60 ELISA test

showed that acute patients had a higher incidence (28%) of cHSP60 antibodies in

comparison to patients with PID/Tubal damage (20%). This demonstrates that cHSP60

is not necessarily the most efficacious antigen for the detection and discrimination of

chronic C. trachomatis disease. In a case-control study of Cameroon women suffering

tubal-factor infertility, the diagnostic value of C. trachomatis heat shock protein 10

(cHSP10) and cHSP60 antibodies for the detection of secondary infertility was

evaluated (Dadamessi et al., 2005). The study demonstrated a significant association

between anti-cHSP10 or anti-cHSP60 antibodies and secondary infertility (p<0.0001).

In addition, although detection of antibodies to either cHSP10 or cHSP60 allowed

diagnosis of tubal factor infertility, the sensitivity and specificity of this combination

was only 57.4% and 75.5% respectively compared to 80% and 84% of our B + C

(CT157 + CT727) format (Chapter 3). Moreover, although the diagnostic value of an

assay capable of detecting chronic infection and disease is important, the sensitivity and

specificity of the test advocated by Dadamessi and colleagues may not be a true

representation of the general population given the ethnic background of the female

cohort that was used for this study.

Little is known about the function or antigenic potential of the hypothetical protein

CT423, apart from the fact that it contains a cystathionine beta synthase domain.

Interestingly, antibody reactivity to CT423 was evident in patient groups I – III.

However, the total absence of antibodies in group IV (PID) patient samples may

indicate that the response to CT423 is switched off during chronic infection, ie.

expression is halted in this infection phase, thereby suggesting that CT423 does not

have an immunogenic function in the pathogenesis of adverse sequelae in women.

Conversely, CT423 may in fact be highly expressed in affected tissues during chronic

infection. However, it is possible that antibodies to this antigen were not detected

because all were bound to infected tissues thereby actually mediating tissue damage.

Nevertheless, the lack of anti-CT423 antibodies in infected patient samples, high levels

- 147 -


of reactivity to CT157 and the fact that PID patients were reactive to band C (Chapter 3)

suggests that the true identity of band C therefore is most likely CT157.

Prior to testing, all patient samples were assayed for C. trachomatis and C. pneumoniae

IgG using a commercially available enzyme immunoassay (EIA) (Labsystems

Chlamydia trachomatis IgG EIA) and microimmunofluorescence (MIF) (Focus

Diagnostics (USA) Chlamydia MIF IgG) test respectively. Upon screening the control

group samples (groups V, VI and VII) against the four recombinant antigens, one

patient with positive C. pneumoniae serology demonstrated the same antibody response

level to band C candidates CT157 and CT423. This suggests that the C. pneumoniae

antibodies are able to cross-react with the C. trachomatis antigens. Although antibodies

to C. pneumoniae can cross-react with the candidate antigens, the effect on antibody

responses, ie. banding intensities, appears to be only minimal as samples from patient

groups II, III and IV who were C. pneumoniae-negative demonstrated an antibody

response to the candidate proteins. Furthermore, the banding intensity of antibody

binding to the antigens was, in general, the strongest in the C. pneumoniae-negative

samples.

In order to ensure the antibody reactivity demonstrated by the various patient groups

was valid given the age and number of freeze/thaws of the original samples, new

samples were obtained and also screened against the recombinant proteins. Of the four

C. trachomatis-infected groups, only group II demonstrated an increase in reactivity

when probed by the new patient samples. In general, groups III and IV showed an

equal antibody response to the four candidate antigens when comparing the original and

new samples. These results demonstrate both the durability of the samples’ antibodies

and reinforce the effectiveness of serological testing despite an extended delay between

sample collection and subsequent screening.

In summary, whilst the exact mechanism by which the host immune response resolves a

C. trachomatis infection or causes an inflammatory response resulting in adverse

sequelae is unknown, the increase in sexually transmitted C. trachomatis infection and

- 148 -


associated chronic disease reinforces the need for a diagnostic test that accurately

distinguishes the various infection stages. Furthermore, the increasing direct medical

costs of pelvic inflammatory disease and adverse side effects such as tubal infertility

and ectopic pregnancy caused by chronic infection are also of great concern. Previous

tests have demonstrated the ability to diagnose chlamydial infection, however, none can

adequately discriminate between acute and chronic infection. As a result, this study

focused on evaluating four candidate proteins previously identified (chapter 3) and it

was found that two chlamydial antigenic targets (CT157 and CT727) may have the

potential to discriminate between acute and chronic C. trachomatis infection. Although

these preliminary findings presented here indicate our candidate antigens do elicit an

antibody response in the host, a much larger cohort will need to be tested in order to

evaluate their true diagnostic potential.

- 149 -


- 150 -

Chapter 6: Variation in Serological Responses to C. t Infection in Males and Females

CHAPTER 6

Variation in Serological Responses to C. trachomatis Infection

in Males and Females

- 151 -

Chapter 6: Variation in Serological Responses to C.t Infection in Males & Females

6.0 INTRODUCTION

C. trachomatis infections which remain untreated can eventually result in urogenital

disease in both men and women. Urogenital infections caused by C. trachomatis have a

clinical course which varies from asymptomatic to symptomatic. In women, infections

can manifest as urethritis, cervicitis and salpingitis (Sweet, 1987; Groseclose et al.,

1996; Paavonen and Lehtinen, 1996; Dieterle et al., 1998) with long-term consequences

associated with pelvic inflammatory disease, ectopic pregnancy, (Cates and Wasserheit,

1991; Buchan et al., 1993; Barlow et al., 2001), spontaneous abortion (Witkin and

Ledger, 1992) and tubal factor infertility (Westrom et al., 1992; Barlow et al., 2001). In

males, urogenital infection causes urethritis which is the most common manifestation of

C. trachomatis infection (Lindberg, 2003). Interestingly, whilst urethritis is not gender

specific, studies have shown it to be a precursor to epididymitis and sexually-acquired

reactive arthritis (SARA) (Kousa et al., 1978; Hopkinson, 2001), the equivalent of

chronic disease in women. SARA, which appears to be an immune-mediated

inflammatory response to C. trachomatis infection, is the most frequent cause of acute

peripheral arthritis in young sexually active men (Hopkinson, 2001). Very few

individuals (1% - 3%) who present with NGU, however, develop reactive arthritis (Keat

et al., 1983; Kvien et al., 1994; Bas et al., 1999).

Humoral and cell-mediated immune responses serve different functions and involve

various effector mechanisms for generating immunity. The primary focus of humoral

immunity is the neutralisation and elimination of extracellular pathogens such as

bacteria, fungi and multicellular parasites, by the activation of naïve B lymphocytes.

Neutralisation is mediated via high-affinity IgG and IgA isotypes, opsonisation by some

classes of IgG and complement activation of IgM and subclasses of IgG. The role of

cell-mediated immunity involves the destruction of infected host cells by cytotoxic T

lymphocytes (CTLs), or the phagocytosis of intracellular pathogens such as Chlamydia

by macrophages triggered by Th1 cells (Williams et al., 1981). Chlamydiae elicit

predominantly cell-mediated immune responses (Hassell et al., 1993; Johansson et al.,

1997) in which T lymphocytes are functionally differentiated into Th1 or Th2 subtypes

- 152 -


based upon their cytokine secretion profiles (Williams et al., 1981; Rank et al., 1985;

Stephens, 2003). Type 1 cells, fuelled by IL-12, typically produce pro-inflammatory

cytokines such as IFN-γ and IL-2 and drive the infection towards resolution (Debattista

et al., 2002; McGuirk and Mills, 2002). Conversely, type 2 cells activate B cells and

secrete IL-4, IL-5, IL-9, IL-10 and IL-13 which inhibit IFN-γ production thus

suppressing a Th1 response (Constant and Bottomly, 1997; Debattista et al., 2002;

McGuirk and Mills, 2002). This type of immune response, ie. Th2, hinders chlamydial

destruction and favours a chronic or persistent state. Alternatively, hyperstimulation

resultant from excess Th1 cytokine production may evoke extensive tissue degradation

and scarring through action of CTLs (Debattista et al., 2002). Furthermore, in the

absence of IL-12, a dominant Th2 response supported by IL-18 and coupled with a

weak Th1 response (decreased IL-2 and IFN-γ levels), may impede normal adjunct

immune responses thereby inducing tissue destruction and chlamydial persistence

(Tominaga et al., 2000; McGuirk and Mills, 2002). Generally, reaction to C.

trachomatis infection by the host manifests along the Th1 pathway (Ward, 1999).

However, phagocytosed Chlamydia stimulate CD8+ T cells if bacterial antigens are

relocated from either the phagosome into the cytosol, or if the pathogen escapes and

subsequently penetrates the cytoplasm of infected host cells. In the case of the latter,

Chlamydia become impervious to phagocytosis, therefore the infection is eliminated via

the destruction of infected cells via CTLs. Moreover, the effectors of cell-mediated

immunity, specifically CD4+ T cells and CD8+ CTLs and their derived cytokines,

function synergistically in defense against chlamydial invasion and are crucial for

microbial clearance (Raupach and Kaufmann, 2001; Igietseme et al., 2003).

The diversity of humoral and cell-mediated immune responses between men and

women is not well understood. However, evidence derived from female murine models

has shown that women produce a greater number of T cells and invariably mount a

stronger Th2 response in comparison to men (Weinstein et al., 1984; Amadori et al.,

1995). This may explain to a certain degree, why females are more prone to chronic

infection and its associated adverse pathology in comparison to males. Interestingly,

Pudney and Anderson, (1995) assessed the immunobiology of the human male penile

- 153 -


urethra and showed that it possesses the full complement of necessary elements for both

humoral and cell-mediated immune responses. Moreover, to investigate the clinical

course of infection, a murine model that closely mimics the natural route of infection in

human males was established by Pal et al. (2004). Despite this, the exact impact of C.

trachomatis in male urinogenital infection and subsequent immunopathogenesis still

remains undefined.

The objective of this study was to analyse male samples against the same

Chlamydia/host cell preparations used in the female study (Chapter 3) in order to

enhance our understanding of the male immune response and directly compare to what

is currently known regarding females.

- 154 -




Samples from all four male patient groups (MI – MIV) were analysed in this part of the

project, however only those samples collected from 1998 – 2002 (batch #1) (Chapter 2,

Section 2.3) were used in sections 6.1.3 – 6.1.6. Samples from batch #1 and batch #2

(collected in 2006) were used in section 6.1.7.1.


The culture of uninfected and infected HEp-2 cells was undertaken as described in

Chapter 3, Section 3.1.3.

6.1.3 Identification of Immunoreactive C. trachomatis Proteins by Western

Blotting

Male patient samples were used to screen Western blots as described in Chapter 3,

Section 3.1.4.

6.1.4 Antigen Identification via N-terminal Sequencing

A single target band was extracted from a 7.5% SDS-PAGE gel and subjected to N-

terminal sequencing. Briefly, 10μg of the infected HEp-2 cells protein extract (I) were

separated in a 7.5%, 15 well, precast Tris-HCl gel (BioRad) and electrophoresed in

Running buffer (25mM Tris Base, 192mM glycine, 1% SDS) at 110 volts for 100

minutes. The gel was then sliced vertically and one gel section was transferred to

Hybond C Extra nitrocellulose membrane (Amersham Biosciences) at 4°C in CAPS

buffer, whilst the remaining segment was stained with 0.1% Coomassie (1g in 40%

methanol) for 30 minutes. The Coomassie treated gel was destained for 2 hours in 90%

methanol and rehydrated in water to restore its size to that of the blotted gel section.

- 155 -


The nitrocellulose membrane containing one marker lane and proteins from infected

HEp-2 cells (I) was probed with a single strong-reacting patient sample as described in

Chapter 3, Section 3.1.4. The Coomassie stained gel and Western blotted membrane

were aligned and compared to identify the antigenic target. The novel marker band was

excised as neatly and precisely as possible from the Coomassie stained gel segment and

forwarded to the Australian Proteome Analysis Facility (APAF) at Macquarie

University in Sydney for N-terminal sequencing. The gel sample was incubated

overnight at 37°C in SDS elution solution, loaded onto a Prosorb cartridge (Applied

Biosystems), then washed with 0.1% TFA to remove residual SDS and reduce

background contamination. The sample was subjected to seven cycles of N-terminal

sequencing via automated Edman degradation (Applied Biosystems 494 Procise Protein

Sequencing System). A 10pmol β-lactoglobulin standard was used to verify sequencer

performance.

6.1.4.1 N-terminal Sequence Analysis

Analysis of the N-terminal sequence and identification of proteins was undertaken as

described in Chapter 3, Section 3.1.6.1.

6.1.4.2 Isolation of Untrypsinised Proteins from Cell Culture

HEp-2 cells were cultured using DMEM, supplemented with 5% heat-inactivated foetal

calf serum (CSL, Australia), 0.002% gentamycin, 5% CO2 and infected with 2mL of C.

trachomatis L2 strain. Six hours post infection (pi), the media was discarded and 10mL

of fresh 10% FCS-DMEM was added. At 30 hours pi, Chlamydia plus host cells were

extracted as follows. In contrast to previous harvesting procedures, monolayers were

manually scraped from the flask and resuspended in 48mL PBS, centrifuged at 1000

rpm (Beckman GS-6R) for 5 minutes at 4°C and pelleted. The pellet was resuspended

in 3mL of PBS, repelleted for 15 seconds, and resuspended in 3mL of 2X sample

buffer. 10μg of host cell proteins from non-infected HEp-2 cells (UI), HEp-2 cells

infected with C. trachomatis serovar L2 (I), and non-trypsinised HEp-2 cells infected

- 156 -


with C. trachomatis serovar L2 were loaded onto a 12.5% polyacrylamide SDS-PAGE

gel. The Western blot protocol and chemiluminescence detection were performed as

described in Chapter 3, Section 3.1.3, using a single patient sample.


(MOMP)

As described in Chapter 3, Section 3.1.8.

6.1.6 Species and Serovar Specificity of the Identified Novel Marker

As described in Chapter 3, Section 3.1.9, with only samples from groups MI and MII

used as all samples from MIII had previously been depleted.

6.1.7 Production of Recombinant Chlamydial Proteins

The four target antigens CT157, CT423, CT727 and CT396 previously identified in

Chapter 3 were produced by Drs. Gomez and Wan (QUT, Brisbane, Australia) utilising

the optimised protein expression protocol outlined in Chapter 4. The purification

method used for each of the four recombinant proteins is as described in Chapter 5,

Section 5.1.3.

6.1.8 Western Blotting of Recombinant Chlamydial Proteins with Patient

Samples

Individual patient samples (n = 3) from groups MI and MII and a patient sample (n = 1)

from group MIII were probed against 10μg of the four recombinant proteins and BL21

untransformed cell control. In addition, to directly compare antibody reactivity of the

recombinant proteins to the previously observed individual banding profiles, 10μg of

chlamydia/host cell proteins from non-infected HEp-2 cells (UI) and HEp-2 cells

infected with C. trachomatis serovar L2 (I) were also included in each Western blot.

- 157 -


Western blot detection methods were as previously described in Chapter 3, Section

3.1.4.


Western Blots

As described in Chapter 5, Section 5.1.3.1. (NB: Figure 5.4 used to depict the grading

system in Chapter 5 was duplicated in chapter 6 (Figure 6.8) to maintain uniformity

across both chapters).

- 158 -


6.2 RESULTS

6.2.1 Immunoreactivity Patterns of Males with C. trachomatis Genital Infections

Figure 6.1 shows a typical differential Western blot profile when samples from each

male group were used to probe uninfected (UI) and infected (I) cell preparations, whilst

Table 6.1 demonstrates the individual results obtained for each male patient. Table 6.2

shows the percentage prevalence of reactivity of the patient groups as well as the C.

trachomatis (EIA) and C. pneumoniae (MIF) serology results.

The most striking result is the presence of a low molecular weight band (~19kDa)

resolved when Western blots were probed with patient samples from groups MI (100%

incidence) and MII (82% incidence). Also noteworthy is the absence of reactivity to

Band A (>113kDa), previously observed in samples from several infected female

patients (41%), from the infected male samples. Antibodies to Band B (72.4kDa) were

present in 100% of patient samples in group MI, but showed a decreased incidence of

55% and 50% in samples from groups MII and the adult controls (MIII) respectively.

Antibodies to Band C (44.6kDa) were detected in 36% of group MII and 50% of group

MIII samples. In contrast, 100% of group MI samples demonstrated reactivity to band

C. Antibodies to Band D were present in 75% of group MI samples and 64% of group

MII. The adult controls (group MIII) demonstrated reactivity to bands B (50%), C

(50%), D (25%) and M (50%), whilst the children controls (group MIV) showed no

reactivity to any of the five bands. MIF analysis revealed 82% of group MII and 50%

of group MI samples had antibodies to C. pneumoniae. Of the two control groups,

100% and 33% of samples from control groups MIII and MIV respectively, were

positive for antibodies to C. pneumoniae. EIA testing showed 25% and 18% of patient

samples from groups MI and MII respectively were positive for C. trachomatis

antibodies. In contrast, none of the patient samples from the adult or children controls

demonstrated antibodies to C. trachomatis.

- 159 -


GROUP MII GROUP MI

kDa 109

94

52

36

30

21

10994

52

36

30

21

10994

52

36

30

21

10994

52

36

30

21

(a) (b)

(c) (d)

Figure 6.1. Western blot of uninfected (UI) and infected (I) whole cell extracts probed

with samples from the four patient groups (a) group MI, (b) group MII, (c) group MIII

and (d) group MIV. Circled are three of the identified differential antigenic bands

(designated B, C and D) previously demonstrated in the various female patient groups.

Boxed is the novel male marker designated M. Exposure time subsequent to

chemiluminescence for all Western blots was 30 seconds.

B

C

M

B

M

I UI

UI I

kDaUI I

D D

GROUP MIII GROUP IV

UI I kDa kDa

- 160 -


Table 6.1. Individual patient profiles of differential bands A, B, C, D and M on

Western blots.

DIFFERENTIAL BANDS PATIENT GROUP

PATIENT I.D. A B C D M

7292 - + + + +

13986 - + + + +

14483 - + + + + MI

12390 - + + + +

1237 - - - + +

13664 - + - + +

8956 - + - + +

10019 - + - - +

7803 - - + + +

13651 - + - - +

2305 - + + + +

8963 - - + - +

14035 - - - + -

11917 - - + + -

MII

12367 - + - - +

Control 1 - + + + +

Control 2 - - - - +

Control J - + + - - MIII

Oras179 - - - - -

CM1 - - - - -

CM2 - - - - -

CM3 - - - - -

CM4 - - - - -

CM5 - - - - -

CM6 - - - - -

CM7 - - - - -

CM8 - - - - -

CM9 - - - - -

CM10 - - - - -

CM11 - - - - -

MIV

CM12 - - - - -

+ = band present in Western blot - = band absent in Western blot

- 161 -


Table 6.2. The percentage prevalence of all differential bands A, B, C, D and M with

positive C. trachomatis and C. pneumoniae serology for each patient group.

DIFFERENTIAL BANDS

% EIA MIF

% PATIENT GROUPS A B C D M

Estimated Band Size (kDa) >113 72.4 44.6 13.5 19 C. t C. pn

MI (n = 4) 0 100 100 75 100 25 50

MII (n = 11) 0 55 36 64 82 18 82

MIII (n = 4) 0 50 50 25 50 0 100

MIV (n = 12) 0 0 0 0 0 0 33

6.2.2 Identification of Novel Marker via N-terminal Sequencing

Figure 6.2 shows the Coomassie stained gel and accompanying Western blot used to

specifically target the novel 19kDa protein (band M). N-terminal sequencing identified

seven amino acids (ASAPAAA). Table 6.3 shows the 16 most significant BLASTP

sequence alignments with 100% homology (E value = 21.8, bits = 138) to the N-

terminal sequence of the Band M protein. The sole C. trachomatis sequence match

returned was the Probable Outer Membrane Protein B (PmpB). PmpB has a molecular

weight of 181.8kDa, approximately 9.5 times greater than the anticipated protein size.

C. trachomatis culture preparations (I) used trypsin to detach infected host cells and

may have cleaved the PmpB protein into several smaller fragments thereby producing

an ~19kDa protein. An in silico tryptic digest of the 181.8kDa pmpB gene revealed a

17.3kDa fragment which contained the identified seven amino acid sequence (Table

6.4).

- 162 -


Coomassie Gel Western Blot

I I I I kDa

109 94

B

52 C 36

30

21 19kDa

Figure 6.2. Comparison of the Western blot and Coomassie stained gel allowed

excision of the novel male marker (indicated by an arrow) for N-terminal sequencing

and protein identification. Indicated on the Western blot are differential bands B and C.

(I = C. trachomatis L2 infected HEp-2 cells).

Table 6.3. Matches of the N-terminal sequence of the Band M protein against the

NCBI Genbank database.

Score E Sequences producing significant alignments: (Bits) Value gi|2506946|sp|P16429|HYCC_ECOLI Formate hydrogenlyase subunit... 21.8 138 gi|20138462|sp|Q9WVE9|ITSN1_RAT Intersectin-1 (EH domain and ... 21.8 138 gi|5902737|sp|Q99128|AP1G1_USTMA AP-1 complex subunit gamma-1... 21.8 138 gi|124142|sp|P28925|ICP4_EHV1B Trans-acting transcriptional p... 21.8 138 gi|39931305|sp|Q9RTG5|IF2_DEIRA Translation initiation factor IF 21.8 138 gi|1710593|sp|P51408|RLA2_TRYBB 60S acidic ribosomal protein P2 21.8 138 gi|124143|sp|P17473|ICP4_EHV1K Trans-acting transcriptional p... 21.8 138 gi|267469|sp|P29941|YCB8_PSEDE Hypothetical 19.2 kDa protein in 21.8 138 gi|118353|sp|P13187|DCOA_KLEPN Oxaloacetate decarboxylase alpha 21.8 138 gi|59797779|sp|Q6S6U0|ICP4_EHV1V Trans-acting transcriptional... 21.8 138 gi|22096375|sp|Q9V7H4|RW1_DROME RW1 protein homolog 21.8 138 gi|38257667|sp|Q8PEH5|DNAA_XANCP Chromosomal replication initiat 21.8 138 gi|81779155|sp|Q98EV7|ATPG_RHILO ATP synthase gamma chain (ATP s 21.8 138 gi|14195035|sp|O84418|PMPB_CHLTR Probable outer membrane protein 21.8 138 gi|67460982|sp|O60346|PHLPP_HUMAN PH domain leucine-rich repe... 21.8 138 gi|68565584|sp|Q6T264|MAML1_MOUSE Mastermind-like protein 1 (Mam 21.8 138

- 163 -


Table 6.4. In silico tryptic digest of the entire PmpB protein by Peptide Mass Program,

ExPASy (www.expasy.org) resulted in a 17.3kDa fragment which contained the

ASAPAAA fragment. Highlighted are the identified N-terminal sequence amino acids

contained within the protein fragment.

MASS (Da)

AMINO ACID POSITION

(within pmpB gene)

PEPTIDE SEQUENCE

17257.2 799-978 ANSSSTGVATTASAPAAAAASLQAAAAAVPSSPATPTYSG VVGGAIYGEKVTFSQCSGTCQFSGNQAIDNNPSQSSLNVQ GGAIYAKTSLSIGSSDAGTSYIFSGNSVSTGKSQTTGQIAGG AIYSPTVTLNCPATFSNNTASMATPKTSSEDGSSGNSIKDTI GGAIAGTAITLSGVSR

6.2.2.1 Confirmation of Novel Protein Identity

N-terminal sequencing and a tryptic in silico digest confirmed (Section 6.2.2) PmpB as

the likely ~19kDa candidate protein. To confirm this identity, trypsinised HEp-2 cells

infected with C. trachomatis L2 and non-trypsinised HEp-2 cells infected with C.

trachomatis L2 were probed by a single patient sample (Figure 6.3). Bands B

(72.4kDa), C (44.6kDa) and M (~19kDa) were all detected in the trypsinised lane. Only

a single band, corresponding to the molecular weight (182kDa) of the entire PmpB

protein was evident in the non-trypsinised lane.

- 164 -

http://www.expasy.org/


109

94

52

36

30

21

UI +T -T

kDaPmpB

B

C

M

Figure 6.3. Size determination of the novel band M in trypsinised and non-trypsinised

cell isolates. Trypsinised HEp-2 cells infected with C. trachomatis L2 (+T) and non-

trypsinised HEp-2 cells infected with C. trachomatis L2 (-T) were probed by a single

patient sample to confirm the identity of the novel candidate protein (M). The bold

arrow indicates band M (identified as a 17.3kDa PmpB fragment) in the trypsinised

lane. Indicated by the dotted arrow in the non-trypsinised lane is a single band

corresponding to the molecular weight (182kDa) of the entire PmpB protein. UI -

uninfected HEp-2 cells; B = band B, 72.4kDa; C = band C, 44.6kDa.


(MOMP)

To verify that antibody reactivity to the 44.6kDa protein (band C) was not due to the

highly immunoreactive 42.4kDa MOMP protein, a single patient sample was probed

against uninfected HEp-2 cells (UI), Hep-2 cells infected with C. trachomatis L2 (I) and

recombinant (his-tag) MOMP (Figure 6.4). Present in the infected (I) lane were bands

B (72.4kDa), C (44.6kDa), D (13.5kDa) and M (17.3kDa), with band C demonstrating

the strongest antibody reactivity. In addition to the four differential bands demonstrated

in the Western blot (excluding the uninfected lane), recombinant MOMP was also

- 165 -


detected by the sample and showed strong antibody reactivity but was clearly at a

different position to band C.

B

C

D

M

UI I MOMPkDa 109

Figure 6.4. Comparison of size of recombinant MOMP and band C. To confirm the

identity of band C was not the immunoreactive MOMP protein, a single patient sample

was probed against recombinant MOMP and subsequently compared with banding

patterns in uninfected (UI) and infected (I) cell preparations. Differential bands B

(72.4kDa), C (44.6kDa), D (13.5kDa) and M (17.3kDa) are indicated in the infected

lane, whilst antibody reactivity to recombinant MOMP is designated by an arrow.

Exposure time after detection was 30 seconds.

6.2.4 Species and Serovar Specificity of Novel Male Marker

Species and serovar specificity of the differential marker of chlamydial infections in

males was determined by probing C. trachomatis L2, D and K, and C. pneumoniae cell

extracts with samples from groups MI and MII only (Figure 6.5) as all samples from

MIII had previously been used. Overall, the results demonstrate that C. trachomatis

infection in males leads to the production of anti-M (PmpB) antibodies which react with

the C. trachomatis L2, D and K PmpB antigen (Table 6.5). Furthermore, these

antibodies also cross-react with the C. pneumoniae PmpB antigen.

94

52

36

30

21

- 166 -


GROUP MI

1 2 3 4 5

B

C

M

10781

48

34

27

21

kDa 107

81

21

48

34

27

kDa

B

C

M

D

IgG: Ct +ve; Cpn +ve IgG: Ct -ve; Cpn -ve

10781

48

34

27

21

10781

48

34

27

21

10781

48

34

27

21

D

IgG: Ct -ve; Cpn -ve IgG: Ct -ve; Cpn +ve IgG: Ct +ve; Cpn +ve

1 2 3 4 5 1 2 3 4 5

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

(a) (b)

(c) (d) (e)

13986 12390

GROUP MII

kDa kDakDa

B B B

C

MM M

12367 2305 13664

Figure 6.5. Species and serovar comparison of the novel 19kDa marker between

patient groups MI (figures a and b) and MII (figures c, d and e). The dotted arrows

indicate the three bands (B, C or D) previously observed in female patients. The bold

arrow indicates the male-specific novel marker (M) across the various C. trachomatis

serovars and C. pneumoniae. Individual patient IDs and C. trachomatis and C.

pneumoniae serology results are shown for each Western blot. Lanes: 1 = uninfected

HEp-2 cells, 2 = HEp-2 cells infected with C. trachomatis L2, 3 = HEp-2 cells infected

with C. trachomatis D, 4 = HEp-2 cells infected with C. trachomatis K, and 5 = HEp-2

cells infected with C. pneumoniae. Exposure time was 45 seconds.

- 167 -


Table 6.5. Serovar and species specificity and serology for all 10 patient samples

probed against C. trachomatis serovars L2, D and K, and C. pneumoniae.

PATIENT GROUP

PATIENT I.D.

SEROLOGY (IgG) L2 D K C. pn

14483 Ct –ve; Cpn -ve B/C/D None None B/C

12390 Ct –ve; Cpn -ve B/C/D/M B/C/M B/C/M B/C/D/M

7292 Ct –ve; Cpn +ve B/C/D/M B/C B/C B/C/M MI

13986 Ct +ve; Cpn +ve B/C/M B B/C M

1237 Ct –ve; Cpn -ve D/M None None D/M

8956 Ct +ve; Cpn +ve B/D/M M C/M B/M

7803 Ct –ve; Cpn +ve C/D D D C/D/M

2305 Ct +ve; Cpn +ve B/C/M C C B/C/M

12367 Ct –ve; Cpn -ve C/M C C C/M

MII

13664 Ct –ve; Cpn +ve C/DM M C C/M

As observed in Western blots using female samples (Chapter 3, Section 3.2.3), male

patient samples which tested positive for C. trachomatis and C. pneumoniae infection

were also generally more reactive across the chlamydial serovars and species examined

irrespective of group assignment. In both male patient groups, C. trachomatis negative

and C. pneumoniae positive samples showed similar antibody reactivity for band M

when compared to C. trachomatis L2, D, K and C. pneumoniae protein extracts.

Furthermore, a comparable antibody response to band M was also demonstrated in C.

trachomatis and C. pneumoniae negative samples. Interestingly, both acute (group MI)

and recovering acute (group MII) C. trachomatis infections appear to elicit the same

antibody response to band M.

Results from groups MI and MII were analysed in terms of specificity for bands B, C, D

and M (Table 6.6). In group MI, band B demonstrated decreased antibody reactivity

(75%) towards extracts of cells infected with C. trachomatis serovars D, K and C.

pneumoniae when compared to those cells infected with C. trachomatis L2 (100%). In

- 168 -


contrast for band B, group MII showed reduced antibody reactivity in cell extracts from

C. trachomatis L2 (33%), D (0%), K (0%) and C. pneumoniae (33%). Band C

demonstrated 50% - 100% and 33% - 67% reactivity to cells infected with either C.

trachomatis strains or C. pneumoniae in groups MI and MII respectively. In general,

band D was shown to be poorly reactive in either patient group irrespective of

chlamydial species or serovar. Of the four bands targeted for species and serovar

analysis, band M demonstrated the highest overall reactivity. Cells infected with C.

trachomatis L2 and C. pneumoniae yielded reactivities of 75% for group MI and 83%

and 100% respectively, for group MII. In contrast, cell lines infected with C.

trachomatis serovars D and K exhibited a lower reactivity for group MI (25%) and

group MII (33% and 16%).

Table 6.6. Species and serovar specificity of band M in infected male samples.

Samples from Groups MI and MII were probed against C. trachomatis L2, C.

trachomatis D, C. trachomatis K and C. pneumoniae.

PERCENTAGE (%) PRESENCE IN

C.t PATIENT GROUP BAND

L2 D K C.pn

B 100 75 75 75

C 100 50 75 75

D 75 0 0 25

MI

(n = 4)

M 75 25 25 75

B 33 0 0 33

C 67 33 67 67

D 50 16 16 33

MII

(n = 6)

M 83 33 16 100

- 169 -


To ascertain the potential homology of band M (PmpB) between C. trachomatis

serovars L2 and D, and C. pneumoniae, protein alignments of the 19kDa fragment were

performed (Figure 6.6). Interestingly, this 178 amino acid fragment of PmpB was

found to be 97% conserved across C. trachomatis serovars D and K. The corresponding

C. pneumoniae homolog is referred to as Pmp20. There was only 65% conservation

between C. trachomatis serovars L2 and D and C. pneumoniae PmpB for these 178

amino acids. Importantly, although there is not total homology of PmpB,…

* * * ** ** ** *** ** * Cpn - VETSLT------------------------------TSTNLYGGGIYSSGAVTLTNISGTFGITGNSVINTAT D - ANSSSTGVATTASAPAAAAASLQAAAAAVPSSPATPTYSGVVGGAIYG-EKVTFSQCSGTCQFSGNQAIDNAP L2 - ANSSSTGVATTASAPAAAAASLQAAAAAVPSSPATPTYSGVVGGAIYG-EKVTFSQCSGTCQFSGNQAIDNNP ** *** *** ***** ** ** * * ** ******* * ** * * ** * SQD-ADIQGGGIYATTSLSIN--QCNTPILFSNNSAATKKTSTTKQIAGGAIFSAAVTIENNSQPIIFLNNSA SQSSLNVQGGAIYAKTSLSIGSSDAGTSYIFSGNSVSTGKSQTTGQIAGGAIYSPTVTLN---CPATFSNNTA SQSSLNVQGGAIYARTSLSIGSSDAGTSYIFSGNSVSTGKSQTTGQIAGGAIYSPTVTLN---CPATFSNNTA ** ** ***** ** KSEATTAAT----AGN—KDSCGGAIAANSVTLTNPEIT - Cpn SMATPKTSSEDGSSGNSIKDTIGGAIAGTTITLSG-VSR - D SMATPKTSSEDGSSGNSIKDTIGGAIAGTAITLSG-VSR – L2

Figure 6.6. Protein sequence alignments of C. trachomatis serovars L2 and D, and C.

pneumoniae for band M, the PmpB fragment. The asterisks indicate conserved amino

acid residues between C. trachomatis L2 and the remaining two alignments.

6.2.5 Blotting of Recombinant Proteins with Patient Samples to Confirm the

Identity of the Immunoreactive Target Antigens

Figure 6.7 shows the typical Western blot profiles obtained when patient samples from

groups MI, MII and MIII were probed against uninfected HEp-2 cells (UI), HEp-2 cells

infected with C. trachomatis L2 (I) and the different recombinant proteins derived from

bands B and C (Table 6.7). Figure 6.8 indicates how banding profiles for each of the

four recombinant proteins were individually graded on an arbitrary scale of 1+ to 6+,

- 170 -


- 171 -

according to their intensity. Importantly, quantitative methods such as dilution series

and densitometry were not employed due to the limited amount of patient samples

available.

Recombinant CT157 demonstrated the strongest relative band intensity (mean = 4.0)

when probed against samples from group MI (patients diagnosed with first-time C.

trachomatis infection estimated to have been acquired less than four months previous),

compared to a mean value of 3.0 for group MII (first time C. trachomatis infection

estimated to have been acquired more than 12 months previously) (Figure 6.9). Three

out of six samples from groups MI and MII were negative for an antibody response

when probed against recombinant CT423 (Figure 6.9). Furthermore, the mean band

intensity of antibody binding to CT423 when probed by samples was 1.7 and 1.0 for

groups MI and MII, respectively. Group MI demonstrated an identical banding

intensity (mean = 5) to recombinant CT727 amongst all 3 tested patient samples (Figure

6.10). In contrast, the highest band intensity demonstrated by group MII was 3, whilst

the other two samples showed a mean band intensity of 2. The antibody response to

recombinant CT396 when probed by samples from group MI showed a mean value of

3.7, whilst the tested samples from group MII demonstrated a decreased response (mean

= 2.3) (Figure 6.10).

Figure 6.7. Western blots of recombinant proteins probed with samples from male patients. Samples from groups (a) MI, (b) MII and

(c) MIII – negative control were probed against CT157 and CT423 (band C candidates) and CT727 and CT396 (band B candidates)

recombinant proteins to determine the correct identity of the differential banding profiles observed in C. trachomatis-infected males.

Molecular weights of each protein including the GST tag were as follows: CT157 = 74kDa, CT423 = 71kDa, CT727 = 99kDa and

CT396 = 100kDa. The number under/beside each band indicates the respective banding intensities as derived by the grading system.

Patient IDs are also shown at the bottom of each Western blot. Exposure time was 45 seconds.


- 172 -

10994

52

36

30

21

10994

52

36

30

21

10994

52

36

30

21

(a) (b) (c)

UI I -ve

cont

rol

CT

157

CT

423

CT

396

CT

727

GROUP MIII

7292 7803 Oras179

5+ 5+ 3+

3+ 2+ 4+ 5+ 3+

kDa

GROUP MII

UI I CT

396

CT

727

CT

423

CT

157

-ve

cont

rol

kDa

GROUP MI

UI I -ve

cont

rol

CT

157

CT

423

CT

727

CT

396

kDa


UI I -ve

cont

rol

CT

157

CT

423

CT

727

CT

396

kDa

10994

52

36

30

3+ 6+ 6+ 2+

21

Figure 6.8. System used to grade intensity of bands on Western blots probed with male

samples. Banding intensity for each of the four recombinant proteins when probed by

patient samples from groups MI – MIII were graded on a scale of 1+ - 6+, with 1+

signifying a very weak antibody response to the protein, whilst 6+ indicates an

extremely strong banding intensity. This Western blot (which is the same as Figure 5.4)

is an example of the various banding intensities. 2+ = weak; 3+ = medium; 6+ =

extremely strong. Exposure time was 45 seconds.

Table 6.7. Three samples from groups MI and MII, and one from group MIII were

probed against each recombinant protein and produced the following banding

intensities.

PATIENT GROUP

PATIENT I.D. CT157 CT423 CT727 CT396

7292 +++++ +++ +++++ ++++

14483 +++++ ++ +++++ ++ MI

QUTNP005 ++ - ++ ++++

7803 +++++ +++ ++ +++

12367 ++ - ++ ++ MII

QUTNP017 ++ - +++ ++

MIII Oras179 - - - - Band intensity: + = very weak; ++ = weak; +++ = medium; ++++= strong; ++++ = very strong; ++++++ = extremely strong; - = negative.

- 173 -


(a)

CT157

0

1

2

3

4

5

NegM I M IIPatient Groups

Ban

d In

tens

ity

(b)

CT423

0

1

2

3

M I M II NegPatient Groups

Ban

d In

tens

ity

Figure 6.9. Band intensity levels for patient samples from groups MI –MIII when

probed against recombinant proteins (a) CT157 and (b) CT423. Each dot is



- 174 -


(a)

CT727

0

1

2

3

4

5


Ban

d In

tens

ity

(b)

CT396

0

1

2

3

4

5


Ban

d In

tens

ity

Figure 6.10. Band intensity levels for patient samples from groups MI – MIII when

probed against recombinant proteins (a) CT727 and (b) CT396. Each dot is



- 175 -


6.2.6 A Comparison of Acute and Recovered Acute Stages of C. trachomatis

Infection in Males and Females

The data presented in Sections 6.2.5 and 5.2.1 were used to compare the acute (MI and

I) and recovered acute (MII and II) antibody responses of the four candidate antigens in

males and females (Figures 6.11 and 6.12). The antibody profiles of CT157 and CT727

show a much stronger response during the acute stage of infection in males (MI) when

compared with females (I). Each antigen demonstrates a decrease in the antibody

response in the male patient group MII, however, only CT727 and CT396 shows this

same profile in the female group (I).

The profile of CT157 for males shows a strong antibody response in group MI and then

a decline in group MII. In contrast, whilst the antibody response in the female group I

is not quite as strong as the males, antibody reactivity to CT157 increase in samples

from group II. In male samples from group MI, antibody production to CT423 is

notably lower compared to the other antigenic targets. Furthermore, a reduction in

antibody responses to this antigen is observed in group MII samples. The female

antibody profile to CT423 is in direct contrast to that of the males with a stronger

response in group I and an increase in the recovering acute female group (II). Antibody

levels to CT727 in male (MI and MII) and female (I and II) groups are similar,

however, the strength of the response to CT727 by male groups MI and MII is slightly

greater by comparison. Of the four target antigens, CT396 demonstrates the greatest

decrease of antibody response in males and females from the recovered acute groups

(MII and II respectively).

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MALES FEMALES

CT157 Antibody Response in Males

0

1

2

3

4

Onset M I M IIInfection Stage/Time Course

Ant

ibod

y R

espo

nse

CT157 Antibody Response in Females

0

1

2

3

4

Onset I IIInfection Stage/Time Course

Ant

ibod

y R

espo

nse


0

1

2

3

4


Ant

ibod

y R

espo

nse


0

1

2

3

4


Ant

ibod

y R

espo

nse

(a)

(b)

Figure 6.11. Male antibody responses to (a) CT157 and (b) CT423 were directly

compared against females in the same defined patient groups. Although the x-axis is

interpreted as a temporal scale, the even spacing of the stages is purely arbitrary, not

necessarily reflecting a “to scale” time line. (Antibody response = mean band intensity

of each candidate protein derived from the patient groups).

- 177 -


MALES FEMALES


0

1

2

3

4


Ant

ibod

y R

espo

nse


0

1

2

3

4


Ant

ibod

y R

espo

nse


0

1

2

3

4


Ant

ibod

y R

espo

nse


0

1

2

3

4


Ant

ibod

y R

espo

nse

(a)

(b)

Figure 6.12. Male antibody responses to (a) CT727 and (b) CT396 were directly

compared against females in the same defined patient groups. Although the x-axis is

interpreted as a temporal scale, the even spacing of the stages is purely arbitrary, not

necessarily reflecting a “to scale” time line. (Antibody response = mean band intensity

of each candidate protein derived from the patient groups).

- 178 -


6.3 DISCUSSION

The objective of this study was to investigate potential humoral immune response

variation between males and females following infection by C. trachomatis. A total of

five chlamydial antigens (A, B, C, D and M) were used for this analysis. Of the four

differential bands recognised by female samples, only three (B, C and D) were

recognised by infected males. Unique to C. trachomatis-infected males was band M

which was identified by N-terminal sequencing as a 19kDa fragment of the

Polymorphic membrane protein B (PmpB). Genome sequence analysis has shown

polymorphic membrane proteins to be a large superfamily of proteins comprised of nine

members in C. trachomatis and 21 members in C. pneumoniae which are all

heterogeneous in size and amino acid sequence (Kalman et al., 1999). Pmp proteins are

potentially involved in cell envelope biogenesis (Nicholson et al., 2003) and/or protein

export (Henderson and Lam, 2001). Recently, Crane et al. (2006) showed C.

trachomatis PmpD to be surface exposed and its cognate antibodies to be neutralising in

vitro. Furthermore, the authors demonstrated that antibodies against immunodominant

EB surface antigens such as MOMP and LPS have the ability to inhibit PmpD

neutralisation and therefore may act as a host immune response decoy in vivo, thus

suggesting a functional role for PmpD in the pathogenesis of infection. Longbottom et

al. (1996) demonstrated that three polymorphic membrane proteins were highly

immunogenic and localised to the outer membrane complexes of an ovine abortion

subtype of C. psittaci. Moreover, two polymorphic membrane proteins were identified

as part of the outer membrane complex in C. pneumoniae (Knudsen et al., 1999).

Combined, these results demonstrate the high level of chlamydial Pmp variation and

further support a possible putative function in antigenic diversity and/or pathogenicity.

Investigation of the C. trachomatis genome by microarray analysis demonstrated that

the PmpB gene transcript is expressed by 18 hours post infection and remains relatively

constant for the remainder of the chlamydial developmental cycle (Nicholson et al.,

2003). Of the four groups analysed for differential serological antigens in our present

study, all patient samples from group MI (first-time C. trachomatis infection acquired

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less than 4 months previously) showed the presence of anti-PmpB antibodies. This

finding indicates that during the early or acute stage of C. trachomatis infection in vivo,

PmpB is surface exposed thereby enabling a humoral immune response targeted against

PmpB. The continued presence of antibodies to PmpB in 9 out of 11 patients during the

recovery stage of infection (group MII - first-time C. trachomatis infection acquired

more than 12 months previously) suggests that the protein is still being presented to the

host’s immune system and therefore may be involved in other, as yet undetermined,

Chlamydia/host cell immune response interactions.

Interestingly, in contrast to males, none of the female patient samples tested

demonstrated antibodies to PmpB. This either suggests significantly different infection

processes in females versus males, and/or very different immune responses to mucosal

infections in the two sexes. Several studies have demonstrated the ability of steroid

hormones to greatly influence and modulate the host immune response against C.

trachomatis infection (Wira et al., 1992; Prabhala and Wira, 1995; Crowley et al., 1997;

Pal et al., 1998; Salem, 2004; Guseva et al., 2005). Investigation into a possible link

between the female’s menstrual cycle and chlamydial infection showed a significant

increase in cervical epithelial cell sensitivity to C. trachomatis in the late stages of the

cycle (Mahmoud et al., 1994; Crowley et al., 1997). In a mouse model used to examine

a C. muridarum genital tract infection, mice pretreated with progesterone showed

enhanced vaginal infection (Morrison et al., 2002). Moreover, Kaushic et al. (2000)

demonstrated in a rat persistence model, that those animals administered progesterone,

were more susceptible to chlamydial intrauterine infection compared to rats treated with

oestradiol. In contrast, a swine model used due to the similarity of epithelial cell

physiology and oestrous cycle to humans showed that oestrogen-dominant epithelial

cells were more prone to chlamydial infection than progesterone-dominant cells

(Guseva et al., 2003). Furthermore, during the menstrual cycle when progesterone was

at its peak, EBs attached to epithelial cell surfaces but were not endocytosed into the

cell thus impeding chlamydial inclusion formation. The cellular action of steroid

hormones is facilitated by binding to their cognate receptor with the maximum number

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of oestrogen receptors (ERs) occurring during the early proliferative phase of the

menstrual cycle (Mertens et al., 2001).

Clearly, studies have demonstrated the ability of oestrogen and progesterone to

modulate chlamydial infection in females with the occurrence of ERs increased during

certain phases of the menstrual cycle. Therefore, given a humoral response to PmpB

was solely observed in males whose endogenous oestrogen, progesterone and ERs are

significantly less compared to females, it is not unreasonable to speculate that PmpB

may somehow be influenced by female hormones in vivo. PmpB is potentially involved

in envelope formation; an early chlamydial developmental process (Nicholson et al.,

2003). Since anti-PmpB antibodies were absent in female samples, PmpB may have the

ability to interact with the increased number of ERs due to a conformational similarity

which would prevent a PmpB/female host interaction. If this is the case, then the

question remains as to why males generate an immune response against PmpB as they

also produce oestrogen, although at reduced levels compared to females. Interestingly,

protein disulfide isomerase, a component of the ER was shown to enhance C.

trachomatis serovar E attachment and infectivity of endometrial epithelial cells (Davis

et al., 2002). Hence, females may be more predisposed to suffer associated adverse

sequelae due to the higher levels of oestrogen necessitating greater numbers of ERs and

the effect of an increased concentration of disulphide isomerase. The ability to bind the

ER would preclude the production of antibodies against the protein (as demonstrated in

the C. trachomatis-infected female groups) as PmpB would not be directly exposed to

the host immune system. Furthermore, the reduced need for expression of ER in males

due to lower oestrogen levels and antigenic load threshold would permit the

presentation of PmpB to the male host immune system. Also, given the duration of

chlamydial infection in males is shorter compared to females, the generation of anti-

PmpB antibodies may enhance protection which would explain the lower incidence of

pathology in males.

N-terminal sequencing of band M identified a seven amino acid sequence contained

within the PmpB protein, however the first ten amino acids from the N-terminus of the

19kDa fragment were not detected. The primary limitation of Edman degradation, as

- 181 -


with all repetitive procedures, is that errors are cumulative (Edman and Begg, 1967;

Niall, 1973). For example, an unreacted N-terminal residue remaining subsequent to a

cycle, will be present in future cycles, thereby increasing the amount and diversity of

the background material making correct identification of the amino acid difficult.

Random breakage of the polypeptide chain which is subjected to harsh conditions also

contributes to the background. Furthermore, some polypeptides may be dissolved and

lost in the organic solvents used during the extraction process. This problem can be

prevented by covalently bonding the polypeptides to a solid support matrix such as

immobilon-P Polyvinylidene Fluoride (PVDF) membrane.

Passive extraction of the protein from SDS-PAGE gel by a SDS-based buffer produces

lower protein yields for N-terminal sequencing in comparison to PVDF-bound proteins.

In addition, a significant amount (>30%) of lower molecular weight proteins have been

observed to wash out of lower percentage gels ie. <15%, subsequent to extraction

methods. For the identification of bands B and C where N-terminal sequencing was

used (Chapter 3), chlamydial proteins were transferred to PVDF membrane. In

contrast, band M was excised from a 7.5% Pre-cast SDS-PAGE gel and used directly

for sequencing. Since the gel was 7.5% from which band M (a low molecular weight

19kDa protein) was subjected to a SDS-based buffer prior to sequencing and the final

protein concentration was only 2pmol, these factors may have precluded the first ten

amino acids from being identified by N-terminal sequencing. However, since there is

only minimal information about how PmpB is intracellularly processed, it is more likely

that the 10 amino acid residues were cleaved off during the maturation of the protein

through the cell. In addition, host cell proteases which are specific for threonines may

have cleaved the PmpB fragment to reveal the ASAPAAA sequence.

The study of species and serovar specificity of band M demonstrated that C.

trachomatis infection in males invariably leads to the production of anti-PmpB

antibodies. These antibodies are able to cross-react with C. trachomatis L2, D and K

strains, indicating that the reactive epitopes are sufficiently conserved across the

majority of C. trachomatis serovars. An in silico alignment of the available amino acid

- 182 -


sequences from C. trachomatis L2 and D supports this finding. In addition, C.

trachomatis-induced PmpB antibodies also cross-react with the C. pneumoniae PmpB

antigen. This suggests that PmpB antibodies are not solely diagnostic of a C.

trachomatis infection but could also indicate C. pneumoniae infection. This is further

supported by the presence of PmpB antibodies in two negative control samples that

were serologically positive for C. pneumoniae.

The male and female immune response to C. trachomatis infection differs due to either

the varying courses of infection and/or because of the immunological differences

between the sexes. The absence of band A in male patients further emphasises these

differences. Band A, observed in 38% of C. trachomatis-infected female samples

(Chapter 3), was identified via mass spectrometry and produced two possible candidate

proteins: CT147 (Conserved Hypothetical Protein – 162.1kDa) and CT314 (DNA-

directed RNA polymerase beta chain – 154.9kDa). Of the two band A candidates, only

CT147 has been shown to be a potential humoral response target (Belland et al., 2003).

Using genomic transcriptional analysis of the C. trachomatis developmental cycle,

Belland et al. (2003) found CT147 to be an immediate-early gene which initiates

bacterial metabolic processes and potentially alters the bacterial inclusion to evade

fusion with host lysosomes. The authors showed that CT147 was co-localised to the EB

inclusion membrane and homologous to the human early endosomal antigen 1 protein

which is involved in endosomal trafficking and fusion in mammalian cells (Mu et al.,

1995; Simonsen et al., 1998; Christoforidis et al., 1999). This finding suggests that

CT147 has an important functional role in vacuole formation thus making it a prime

antigenic target for the host immune response.

Interestingly, of the C. trachomatis-infected female samples, group I (first-time C.

trachomatis infection acquired less than months previously) and III (a history of two or

more C. trachomatis infections, with the most recent acquired less than 4 months

previously) demonstrated the highest incidence (55% and 75% respectively) of band A

when probed against HEp2 cells infected with C. trachomatis serovar L2 protein

extracts. This result further demonstrates that CT147’s subsequent presentation to the

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host makes it a primary humoral response target during the initial stage of chlamydial

infection. As CT147 is localised to the periphery of the inclusion and elicits a humoral

response in females, the question arises as to why the same antibody response is not

seen in males? As discussed earlier, the cyclical nature of female sex hormone levels

during the menstrual cycle have the ability to greatly influence chlamydial infection.

Furthermore, several studies have shown that the oral contraceptive pill (OCP) can also

directly impact C. trachomatis infection (Cottingham et al., 1992; Elgaali et al., 1994;

Crowley et al., 1996; Kimani et al., 1996; Ness et al., 1997). Interestingly, not all C.

trachomatis-infected female samples from groups I and III demonstrated an antibody

response to CT147. Unfortunately, this phenomenon did not correlate with clinical

histories and symptomology of patients that demonstrated the presence of anti-CT147

antibodies and those that did not. However, given the gender differences in immune

responses and the modulating effect of steroid hormones (Kaushic et al., 1998; 2000)

are higher in women, suggests that the generation of antibodies to CT147 in females

may be influenced by the production of endogenous female hormones. If this

hypothesis is correct, the humoral response to CT147 in females would therefore be

influenced by normal hormonal fluctuations experienced by women during the various

phases of the menstrual cycle. Hence, given that oestrogen-dominant epithelial cells are

more prone to chlamydial infection (Guseva et al., 2003) and oestrogen levels are

cyclic, the stage of the menstrual cycle at which a C. trachomatis infection was

contracted would subsequently impact on the production (or lack) of antibodies to

CT147. This would therefore explain the immunoreactivity patterns observed in

samples from female patient groups I and III.

In summary, the results from this study demonstrate that whilst a humoral response to

PmpB is restricted to males, its diagnostic potential is limited by both gender bias and

C. pneumoniae cross-reactivity. Furthermore, although the potential modulation of

CT147 by female hormones and their subsequent role in humoral responses has not

been resolved in this study, future directions should investigate the potential

oestrogen/progesterone and CT147 interaction. In addition, the possible conformational

relationship between PmpB and ERs could be explored. Collectively, future research

- 184 -


focusing on these areas will further broaden our understanding into host cell interactions

and hormonal modulation of chlamydial infection.

- 185 -


- 186 -

Chapter 7: Chlamydial Persistence Markers in Samples of Males & Females

CHAPTER 7

Chlamydial Persistence Markers in Samples of Infected Males

and Females

- 187 -


7.0 INTRODUCTION

The pathology of urogenital C. trachomatis infection often involves the persistent form

of the organism residing at anatomically distant locations from that of the initial

infection site (Taylor-Robinson et al., 1992; Nanagara et al., 1995; Kohler et al., 1998;

Inman et al., 2000). Moreover, reinfection by C. trachomatis is also an important and

well established component of chlamydial pathogenesis given that disease severity

increases with each subsequent infection (Grayston et al., 1985). A chronic or

persistent infection is defined as a long-term association between the pathogen and host

in which the organism remains viable in a culture negative or non-infectious form

without overt growth or replication (Beatty et al., 1994). As such, the limited metabolic

capacity of Chlamydia in the persistent phase may influence the antigenic and

biochemical qualities of the bacteria thus potentially rendering them undetectable via

conventional diagnostic means.

In vitro studies have shown RBs in persistent cultures to be morphologically atypical

and, although latent, viable Chlamydiae are maintained as recovery of infectious

progeny are possible upon removal of the inhibitory factor (Beatty et al., 1993; Coles et

al., 1993). Several factors which favour persistent infection include the action of low

doses of penicillin (Clark et al., 1982; Hammerschlag and Vuletin, 1985) and IFN-γ

(Shemer and Sarov, 1985; Beatty et al., 1993; Rasmussen et al., 1996), and the

deprivation of amino acids (excluding valine) (Coles et al., 1993) and various nutrients

such as iron and glucose (Raulston et al., 1997; Harper et al., 2000). Transcriptional

analysis of chlamydial growth during IFN-γ-induced persistence and reactivation has

shown other chlamydial mechanisms associated with tryptophan and phospholipid

utilisation and protein translation are also altered during persistent chlamydial

development (Belland et al., 2003). In addition, aberrant chlamydial inclusions have

unique patterns of gene expression which include decreased levels of

lipopolysaccharide (LPS), outer membrane proteins and genes involved in DNA

replication and cytokinesis, coupled with increased levels of heat shock proteins as a

result of chlamydial-induced host cell stress (Byrne et al., 2001).

- 188 -


Chlamydial infection invokes cytokine responses via direct infection of host cells, and

interaction with cells of the host immune system (Fitzpatrick et al., 1991; Rasmussen et

al., 1997). The use of cytokines such as IFN-γ to generate a state of persistence of

chlamydial infections in vitro provides a novel method for understanding possible in

vivo events. Electron microscopic analysis of Chlamydia-infected cells pretreated with

IFN-γ showed RBs which were enlarged, structurally aberrant and fewer in numbers

(Belland et al., 2003). In another study, atypical RBs with poorly defined outer

membranes were more prolific in infected fibroblasts and macrophages in synovial

membrane samples from patients with C. trachomatis-induced reactive arthritis or

Reiter’s syndrome regardless of previous antibiotic treatment (Nanagara et al., 1995).

Similarly, Bragina et al. (2001) using electron microscopy also demonstrated several

morphological RB variants in urethral and endocervical specimens obtained from a

male and female following unsuccessful antibiotic treatment for a C. trachomatis

infection. Taken together, this suggests that inadequate antimicrobial therapy may also

allow Chlamydia to persist in vivo.

In this study, the IFN-γ in vitro model of persistence was utilised to identify

differentially expressed proteins which may prove useful as persistent/chronic disease

markers. This was achieved by comparing uninfected (UI), infected (I) and IFN-γ

treated and infected (IFN) protein extracts probed by male and female samples from the

previously established patient groups.

- 189 -




Samples from all seven female and four male patient groups were analysed for this

aspect of the project, however only those samples collected from 1998 – 2002 (batch

#1) were screened (Chapter 2, Sections 2.2 and 2.3).


As described in Chapter 3, Section 3.1.3.

7.1.3 IFN-γ Persistence Cell Culture

C. trachomatis serovar L2 was used for this previously well-established persistence

model (Beatty et al., 1993). The IFN-γ cell culture was maintained in DMEM,

supplemented with 5% heat-inactivated FCS (CSL Australia), 0.02% gentamycin and

5% CO2 and upon 90% confluency, cells were infected with 2mL of C. trachomatis L2

strain. Twenty-four hours prior to infection, HEp-2 cells were treated with 50U IFN-

γ/μL, a level maintained for the duration of the infection. At 30 hours pi, infection

status was observed under microscopy (Leica) to ensure a >90% aberrant infectivity rate

and then total Chlamydia plus host cell proteins were extracted (IFN) and stored as per

outlined in Chapter 3, Section 3.1.4.

7.1.4 Identification of Reactive Persistence C. trachomatis Proteins in Patient

Samples by Western Blotting

As described in Chapter 3, Section 3.1.4, however samples were also probed against

10μg of the IFN (HEp-2 cells pre-treated with IFN-γ and infected with C. trachomatis

serovar L2) protein extract.

- 190 -


- 191 -

7.2 RESULTS

7.2.1 Identification of Antigens Differentially Expressed in the IFN-γ

Persistence Model in Male and Female Patient Samples

Four bands, designated A (>113kDa), B (72.4kDa), C (44.6kDa) and D (13.5kDa), were

shown to be differential between male and female patient groups (Chapters 3 and 6)

when samples were used to probe uninfected (UI) and infected (I) cell preparations.

Furthermore, band M a 17.3kDa fragment of PmpB was found in C. trachomatis-

infected male patient samples but not in samples from infected women (Chapter 6).

Although particular attention was paid to the five bands formerly identified, male and

female C. trachomatis-infected samples were used to detect differential expression in

IFN-γ-induced persistence. In addition, since differential immune responses detected in

male (Chapter 6) and female (Chapter 3) samples of C. trachomatis-infected cells (I)

have been discussed in previous chapters, this chapter focuses on the banding profiles

observed in the IFN-γ persistence culture.

7.2.1.1 Female Patient Samples

No new immunoreactive bands were evident in the protein extracts of the persistence

cell culture. Surprisingly, of the four bands A, B, C and D identified previously, only

band C demonstrated any immunoreactivity (Figure 7.1). In this case, only 33% of

patient samples reacted to the protein extracts (IFN). Of the four C. trachomatis-

infected patient groups, only samples from groups I and III showed band C reactivity,

whereas groups II and IV did not (Table 7.2). The negative control groups (V-VIII)

demonstrated no antibody reactivity to bands A, B, C or D in the IFN cell preparation.

Interestingly, no cHSP60 band was detected in any samples from the infected groups (I

–IV) in the IFN cell culture.

Figure 7.1. Western blots showing immunoreactivity of female patient samples to proteins from IFN-γ-induced persistence cell

culture. Patient samples from all seven female patient groups were probed against uninfected (UI), infected (I) and infected IFN-γ-

treated (IFN) whole cell extracts. Depicted are the two C. trachomatis-infected patient groups which demonstrated differential

banding in the IFN lane and negative control groups: (a) Group I (b) Group III, (c) Group V and (d) Group VI. The differential bands

previously detected in the infected lane (I) are designated A (>113kDa), B (72.4kDa), C (44.6kDa) and D (13.5kDa). The block

arrows indicate a differential band in the IFN-γ lane, whilst the dotted arrows indicate approximately where cHSP60 should be.


UI I IFN

GROUP III

UI I IFN

GROUP VI

UI I IFN

GROUP V

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113

92

52

35

28

21

113

92

52

35

28

21

113

92

52

35

28

21

113

92

52

35

28

21

(a) (b) (c) (e)

NEGATIVE CONTROLS

CHSP60 cHSP60 B

C C

kDakDa

D

A kDakDa A

UI I IFN

GROUP I

D

Chapter 7: Chlamydial Persistence Markers in Clinical Samples of Males & Females

Table 7.1. Individual female patient profiles of differential bands A, B, C and D

subsequent to sample probe against IFN-γ-mediated persistence culture.

DIFFERENTIAL BANDS PATIENTGROUP

PATIENT

I.D. A B C D

13578 - - - -

4081 - - - -

13817 - - + -

8015 - - - -

13785 - - + -

2361176 - - + -

2248900 - - - -

2180122 - - - -

I

1996360 - - - -

1580 - - - -

10622 - - - -

339 - - - -

14697 - - - -

10306 - - - -

18456 - - - -

13619 - - - -

II

10553 - - - -

4020 - - - -

13108 - - + -

9908 - - - - III

1132 - - - -

11011 - - - -

13114 - - - -

113 - - - -

12581 - - - -

IVF012 - - - -

IVF013 - - - -

IVF017 - - - -

IVF029 - - - -

IVF031 - - - -

IV

IVF032 - - - -


- 193 -


- 194 -

7.2.1.2 Male Patient Samples

As observed in female samples (Section 7.2.1.1), no novel immunoreactive bands were

identified in the IFN protein extract. Figure 7.2 shows a typical differential persistence

Western bot profile when samples from each male group were used to probe IFN cell

preparations. Of the five bands previously detected in male C. trachomatis-infected

samples, band M was the sole reacting band present in the persistence cell culture

(Table 7.2). Band M in patient groups MI (first-time C. trachomatis infection estimated

to have been acquired less than 4 months previously) and MII (first-time C. trachomatis

infection estimated to have been acquired more than 12 months previously) was shown

to be reactive in 50% of patient samples. Both adult and children negative control

groups showed no reactivity to any differential band in the IFN persistence cell culture.

As observed in female samples, no cHSP60 band was detected in any male samples

from the C. trachomatis-infected groups (MI and MII) in the persistence protein extract.

Figure 7.2. Western blots showing immunoreactivity of male patient samples to proteins from IFN-γ-induced persistence cell culture.

Patient samples from all four male patient groups were probed against uninfected (UI), infected (I) and IFN-γ infected (IFN) whole

cell extracts probed with samples from the four male patient groups: (a) Group MI, (b) Group MII, (c) Group MIII and (d) Group

MIV. The differential bands previously detected in the infected lane (I) are designated B (72.4kDa), D (13.5kDa) and M (19kDa).

The solid arrows indicate a differential band in the IFN-γ lane, whilst the dotted arrows indicate approximately where cHSP60 should

be.


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10994

52

36

30

21

10994

52

36

30

21

10994

52

36

30

21

109

94

52

36

30

21

(a) (b) (c) (d)

NEGATIVE CONTROLS

UI I IFN

GROUP MIV

cHSP60 CHSP60

B

M

B

kDaUI I IFN

GROUP MIII

kDaUI I IFN

GROUP MII

M

kDaUI I IFN

GROUP MI

D

kDa


Table 7.2. Individual male patient profiles of differential bands A, B, C, D and M

subsequent to sample probe against IFN-γ-mediated persistence culture.

DIFFERENTIAL PERSISTENCE BANDS PATIENT

GROUP PATIENT

I.D. A B C D M

7292 - - - - +

13986 - - - - -

14483 - - - - + MI

12390 - - - - -

13664 - - - - +

8956 - - - - +

10019 - - - - -

7803 - - - - -

13651 - - - - +

2305 - - - - -

8963 - - - - +

14035 - - - - -

11917 - - - - -

MII

12367 - - - - +

Control 1 - - - - -

Control 2 - - - - -

Control J - - - - - MIII

Oras179 - - - - -

CM1 - - - - -

CM2 - - - - -

CM3 - - - - -

CM4 - - - - -

CM5 - - - - -

CM6 - - - - -

CM7 - - - - -

CM8 - - - - -

CM9 - - - - -

CM10 - - - - -

CM11 - - - - -

MIV

CM12 - - - - -


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7.3 DISCUSSION

In this chapter, the objective was to determine whether the five differential bands (A, B,

C, D and M) detected in male and female patient samples and/or other chlamydial

proteins were up-regulated in the IFN persistent cell preparation. Of the five bands,

only antibodies to band C (44.6kDa) and band M (17.3kDa) were observed in female

and male samples respectively when probed against a C. trachomatis persistent culture

induced by IFN-γ. Furthermore, of the four C. trachomatis-infected female groups,

detection of band C was limited to groups I (first-time C. trachomatis infection

estimated to have been acquired less than 4 months previously) and III (a history of two

or more C. trachomatis infections, with the most recent estimated to have been acquired

less than 4 months previously) ie. antibodies to band C were only found in those women

with acute or recent C. trachomatis infections. Interestingly, no additional chlamydial

antigens were shown to elicit a humoral response in either males or females as only

bands C and M were found to be up-regulated in the IFN protein extract.

Several studies have demonstrated the up-regulation of a variety of genes involved in

tryptophan utilisation, DNA repair and recombination, phospholipid biosynthesis,

general stress and translation in an in vitro model of IFN-γ-mediated persistence (Jones

et al., 2001; Belland et al., 2003; Hogan et al., 2003; Gerard et al., 2004; Polkinghorne

et al., 2004). Using transcriptome analysis of C. trachomatis growth, Belland et al.

(2003) demonstrated that IFN-γ-induced persistence in Chlamydia resulted in altered

but active biosynthetic processes eg. increased phospholipid metabolism and continued

chromosomal replication which are normalised upon IFN-γ removal. Furthermore, the

authors demonstrated that only two (CT157 - band C and CT413 - band M) of the five

differential bands detected in patient samples from this study, were up-regulated during

a persistent state (Belland et al., 2003). This result is consistent with our observations

as only CT157, present in 33% of C. trachomatis-infected samples from female groups

I and III, and CT413 (PmpB), detected in 50% of male C. trachomatis-infected samples

from groups MI and MII were shown to be up-regulated. Interestingly, the up-

regulation of these two diverse genes (CT157 - phospholipase D endonuclease and

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CT413 - polymorphic membrane protein B) suggests that both are required for aberrant

chlamydial growth and metabolism. In contrast, Belland and colleagues (2003) showed

that CT147 (band A candidate) was down-regulated during a persistent C. trachomatis

infection. As discussed in Chapter 3, CT147 which was detected in acute (group I) and

acute multiply infected (group III) female samples, is an immediate-early gene and is

associated with various metabolic processes which potentially alter the bacterial

envelope to evade fusion and subsequent lytic destruction. Therefore as expected,

CT147’s role in normal envelope biogenesis would indeed be down-regulated during a

persistent infection because although the bacteria remain viable in a non-infectious

form, no overt growth or replication occurs.

As discussed in chapter 6, PmpB (CT413) is expressed midway through a normal

chlamydial developmental cycle and is thought to be involved in cell envelope

biogenesis (Nicholson et al., 2003) and/or protein export (Henderson and Lam, 2001).

The up-regulation of PmpB and its subsequent presentation to the host immune system

which in males, resulted in the production of antibodies in 50% of group MI (first-time

C. trachomatis infection estimated to have been acquired less than 4 months previously)

samples, suggests PmpB has a critical role in envelope formation during the initial

stages of normal and persistent chlamydial development.. The continued presence of

anti-PmpB antibodies in 50% of group MII (first-time C. trachomatis infection

estimated to have been acquired more than 12 months previously) samples indicates that

because the infection was cleared more than 12 months prior, these antibodies are the

residual immune response to the earlier acute infection. A possible putative role in

antigenic diversity and/or potential pathogenicity in C. trachomatis infection have been

suggested by studies that showed members of the Pmp family to be highly

immunogenic (Longbottom et al., 1996; Crane et al., 2006). However in previous

chapters (3 and 6), an anti-PmpB antibody response was detected solely in males.

Furthermore, only C. trachomatis-infected male samples in the IFN-γ persistence model

used in this study demonstrated an up-regulation of PmpB compared to females. This

result is very interesting since persistence and as a consequence, associated adverse

pathology occurs more commonly in females compared with males. Therefore, a failure

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by women to mount an immune response to PmpB may in fact increase the risk for a

persistent C. trachomatis infection. Conversely, the ability to immunologically react to

PmpB may be a reason that males tend not to develop persistence and suffer chronic

disease.

During IFN-γ-mediated persistence, Belland et al. (2003) demonstrated that genes

located in the plasticity zone which are associated with phospholipid biosynthesis (such

as CT156 and CT158) and may participate in C. trachomatis pathogenesis (Read et al.,

2000), showed increased levels of expression. In general, the C. trachomatis plasticity

zone includes several genes, eg. the phospholipase D endonuclease family, that are

suspected to be directly involved with pathogenesis (Read et al., 2000). Interestingly,

CT157 (band C), which is also a member of the phospholipase D superfamily, was only

moderately up-regulated during the persistent state. This may in part account for the

reduced incidence (33%) of anti-CT157 antibodies in female samples from groups I

(first-time C. trachomatis infection estimated to have been acquired less than 4 months

previously) and III (a history of two or more C. trachomatis infections, with the most

recent estimated to have been acquired less than 4 months previously). Furthermore,

given that female groups I and III were both diagnosed with an acute infection and anti-

CT157 antibodies were not detected in the other C. trachomatis-infected patient groups

(II and IV) who had a previous history of chlamydial infection some considerable time

before, suggests two possible causes. Firstly, in a persistent state, CT157 is up-

regulated only during the initial stages of aberrant chlamydial development and

secondly, the antibodies generated against this protein have a short half life and do not

circulate beyond a few months post infection. Proteomic analysis of C. pneumoniae

persistence versus heat shock stress demonstrated the up-regulation of HSP70, a

potential band B candidate - CT396 (Mukhopadhyay et al., 2006). Heat shock proteins

are expressed during times of stress, however no anti-band B antibodies were detected

in any male or female samples tested regardless of stage of infection. This supports the

previous proposal that CT727 is indeed the antigenic band C candidate. The absence of

antibodies to differential bands A (candidates CT147 and CT314), B (candidates CT727

and CT396) and D (protein candidate unknown) in male and female samples suggests

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that none of these proteins are crucial for the continuing survival of Chlamydia within

the host during a persistent C. trachomatis infection.

Serological cSHP60 testing by a commercial HSP60 kit (Medac, Hamburg, Germany)

was performed on all male and female samples included in this study. Antibodies to

cHSP60 were demonstrated in 18% (6/34) and 13% (2/15) of C. trachomatis-infected

male and female samples. Interestingly, anti-cHSP60 antibodies in male and female

samples were notably absent when probed against the IFN-γ cell culture as previous

transcriptional and proteomic studies have reported increased expression of cHSP60

during persistent infections (Molestina et al., 2002; Gerard et al., 2004; Mukhopadhyay

et al., 2006). Our results are in agreement with Belland et al. (2003) who did not detect

any significant changes in levels of cHSP60 expression during IFN-γ-induced

persistence.

This study has demonstrated that no novel chlamydial proteins are up-regulated during a

C. trachomatis persistent infection. Moreover, cHSP60 which is expressed at higher

levels in a persistent infection was not present, regardless of confirmed serology, in any

male or female samples. Importantly, although bands A, B, C, D and M are differential

between male and female patient groups under normal chlamydial growth conditions

(Chapters 3 and 6), only bands C (CT157) and M (CT413 - PmpB) are up-regulated

during IFN-γ-induced persistence suggesting a potential role in chronic chlamydial

infection. In conclusion, whilst proteomic analysis has been undertaken for C.

trachomatis and C. pneumoniae during normal growth conditions, there have been no

previous studies that have focused on IFN-γ-mediated persistence in C. trachomatis.

This study therefore adds significantly to the knowledge base of gene expression during

persistent chlamydial infections.

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Chapter 8: General Discussion

CHAPTER 8

General Discussion

- 201 -


8.0 GENERAL DISCUSSION

The work presented in this thesis has made important new contributions to the field of

chlamydial research with particular emphasis on the diverse immunological responses

in men and women as a result of a general or persistent C. trachomatis infection.

Currently, diagnostic methods for the detection of chlamydial infection are variable in

their performance with regards to their sensitivity and specificity. In women, specimens

are routinely sampled from the lower genital tract, thus infections in the fallopian tube

where inflammatory damage is most significant escape detection. Importantly, several

serological assays which have incorporated various chlamydial antigens for use as

predictors or discriminators of the different stages of C. trachomatis infection have not

been totally successful in achieving this outcome (Bax et al., 2004; Dadamessi et al.,

2005; Gazzard et al., 2006). The individual studies comprising this project primarily

focused on the serological analysis of male and female samples with the overall aim of

identifying antigens that could be potentially used to accurately discriminate between

acute and chronic C. trachomatis infections.

Samples from both sexes were assigned to various patient groups according to their

infection status and were probed against C. trachomatis/host and IFN-γ-induced

persistence cell cultures to identify antibody responses capable of differentiating various

states of chlamydial infection. Serological analysis revealed the presence of five

antigens (denoted bands A, B, C, D and M) which were shown to be differential

between acute and chronic C. trachomatis infections. Two potential candidates were

identified by mass spectrometry and/or N-terminal sequencing for bands A (CT147 -

Conserved Hypothetical Protein, 162.1kDa and CT314 - DNA – directed RNA

polymerase beta chain, 154.9kDa), B (CT727 – Metal transport P-type ATPase,

70.5kDa and CT396 – Heat Shock Protein 70, 71.1kDa) and C (CT157 – Phospholipase

D Endonuclease, 45.3kDa and CT423 – Hypothetical Protein containing a cystathionine

beta synthase domain, 41.6). The confirmed identity of band M which was shown to be

exclusive to males was determined to be a fragment of a probable outer membrane

protein B (PmpB). Only the four identified band B (CT727 and CT396) and C (CT157

- 202 -


and CT423) candidates were expressed as recombinant proteins and probed by male and

female samples from each patient group to evaluate their diagnostic potential. The

common approach of antigen selection for the study of possible immunogenic reactivity

is primarily founded on known function, structure and/or host interactions. Hence,

given what is presently known regarding the candidate proteins, it is not surprising that

none have previously been targeted and thus evaluated for the ability to diagnose

chronic C. trachomatis infection.

In this study, the basis of identifying discriminatory antigens capable of distinguishing

between acute and chronic C. trachomatis was undertaken using C. trachomatis serovar

L2. Importantly, whilst previously identified immunogenic proteins such as ompB and

HSP60 are homologous between serovars, not all are conserved amongst C. trachomatis

strains (Sanchez-Campillo et al., 1999). In a study by Sanchez-Campillo et al. (1999)

the authors identified several immunoreactive proteins of C. trachomatis serovar L2 by

Western blot and 2D electrophoresis. However, in some instances, identified antigenic

proteins from serovar L2 did not reveal a serovar D homolog. Of the four novel

antigens identified ie. CT157, CT423, CT727 and CT396 all were shown to have a

serovar D homolog although the level of conservation varied from 44% (CT423) to

100% (CT396).

The euo (CT446 - DNA binding protein) and hctB (CT046 – histone-like protein 2)

genes are currently considered the most reliable general markers of chlamydial

persistence (Belland et al., 2003). Interestingly, neither gene was found to be up-

regulated in our male or female cohort during normal or IFN-γ-induced persistence.

Since the incidence of chronic disease and associated sequelae as a result of C.

trachomatis infection is significant, the need for an accurate diagnostic method that can

distinguish the various stages of infection is of great importance for the treatment and

subsequent management of chlamydial disease. In this project, the four identified

antigens were combined into diagnostic panels and their predictive potential for the

discrimination of acute and chronic C. trachomatis infection were evaluated. Of these,

the B + C panel proved to be the most efficacious diagnostic test. Furthermore, as a

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format for the diagnosis of chronic or persistent C. trachomatis infections, this antigen

combination was shown to be a viable assay with a high degree of sensitivity (80%) and

specificity (84%) not matched by any commercially available diagnostic test (Bax et al.,

2004; Gazzard et al., 2006). Several studies have demonstrated a correlation between

antibody responses to cHSP60 and pathologic sequelae in women (Brunham et al.,

1992; Dieterle and Wollenhaupt, 1996; Eckert et al., 1997), and a significant association

between the presence of anti-cHSP60 antibodies and PID (Eckert et al., 1997; Peeling et

al., 1997; Witkin et al., 1998). As a result, an enzyme-linked immunosorbent assay by

Medac (Hamburg, Germany) that targets antibodies against cHSP60 was developed.

Interestingly in this study, the presence of anti-cHSP60 antibodies was not restricted to

chronically infected females (group IV) as 29% of samples from males and females

presenting with acute (groups MI and I), recovering acute (groups MII and II), and

those acute multiply infected (group III) also demonstrated antibodies against cHSP60.

In addition, antibody reactivity to cHSP60 was detected in one male and three female

negative control samples. The IFN-γ-mediated persistence model used in this study

established that during persistence, cHSP60 is down-regulated in both men and women.

This result was unexpected and in contrast to previous studies which reported the up-

regulation of cHSP60 at both transcriptional and protein levels (Molestina et al., 2002;

Gerard et al., 2004; Mukhopadhyay et al., 2006). Consequently, given the results

demonstrated in our study, the addition of cHSP60 to the B + C format for detection of

chronic C. trachomatis infection would not increase the efficacy of the test, but would

in fact decrease sensitivity by 60%.

The diagnosis of chlamydial infection is routinely performed using the Roche PCR

assay. Whilst this method is highly sensitive and specific (~99%), it does not have the

discriminatory power to distinguish between a resolved, acute or chronic C. trachomatis

infection. Importantly, in addition to our ability to reliably diagnose chronic infection

using the B + C format, the inclusion of antigen A into the test (A or B or C), where a

positive diagnosis is achieved with the detection of antibodies against any of the three

antigens, also permits the diagnosis of acute C. trachomatis infection. Furthermore,

whilst the sensitivity (75%) and specificity (76%) of the A or B or C format is reduced

- 204 -


by comparison to the Roche assay, the ability of our tests to differentiate acute and

chronic C. trachomatis infections suggests that our formats are the superior diagnostic

method.

Recombinant proteins CT157 and CT423 (band C candidate proteins), and CT727 and

CT396 (band B candidate proteins) were assessed for their diagnostic potential for the

identification of chronic infection in males and females. Prior to this study, none of the

four candidate proteins identified in this project have been examined for a potential

antigenic role in chronic C. trachomatis infection. Varied levels of antibody reactivity

to the candidate proteins were demonstrated when probed by C. trachomatis infected

samples. In general, CT157 and CT727 showed similar antibody response profiles

which were significantly more dominant upon comparison to the antibody reactivities

produced against CT423 and CT396. Furthermore, the strong antibody responses

elicited by male and female patient samples reinforced earlier indications that the true

identity of bands B and C are indeed CT727 and CT157 respectively. The question

remains as to why the candidate proteins, in particular, CT157 and CT727 generated an

immune response? Previous studies have primarily focused on antigens that promote an

immunogenic response in the host (Bax et al., 2003; 2004; Dadamessi et al., 2005) due

to known functions and/or pathogen/host interactions. Whilst the diverse functions of

CT157, CT727 and CT396 have been established, it is only through the novel approach

undertaken in this project, ie. probing samples collected from patients suffering

different stages of C. trachomatis infection against chlamydial infected cells, that the

protein candidates have been revealed to be intrinsic components of the immune

response during the course of a C. trachomatis infection.

A limitation of this aspect of the project was the small number of patient samples (n =

3) used to assess antibody responses to the four recombinant protein candidates. Hence,

whilst different levels of antibody reactivity to CT157, CT423, CT727 and CT396 by

patients were shown, the results from this part of the project should be viewed as

preliminary only given the limited sample size.

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Male and female patient samples were collected in two batches and used for various

aspects over the course of this research. Batch #1 samples were collected in heparin-

coated vials, whereas batch #2 samples were collected in serum tubes. As a result of the

different collection methods, batch #1 samples were in fact plasma, whilst batch #2 was

sera. Moreover, plasma samples retained fibrinogen and other clotting factors such as

plasminogen compared to the sera samples. However, these added components are

unlikely to have affected results during any stage of the project as similar antibody

reactivity profiles of females were demonstrated when plasma and sera from each group

were probed against the four recombinant proteins (Chapter 5).

Amongst the different antigens used to detect antibodies to C. trachomatis is the highly

immunogenic 42.4kDa MOMP. Since MOMP is the principal component of the

infectious EB particle, an ELISA that uses synthetically derived C. trachomatis-specific

epitopes in variable domain IV of MOMP (Labsystems, Helsinki, Finland) has been

developed for commercial use. A comparative study by Bas et al. (2001) assessed

several serological assays for their effectiveness in diagnosing C. trachomatis infection.

The authors demonstrated that IgG anti-C. trachomatis MOMP antibodies were

detected in 58% (n = 45) of patients with acute C. trachomatis urogenital infection and

32% (n = 31) of healthy blood donors. Furthermore, the sensitivity and specificity of

the MOMP assay used in this female cohort was determined to be 58% and 68%

respectively. A similar study also compared the diagnostic value of the Labsystems

MOMP test to other antigens such as HSP60, outer membrane protein 2 (OMP2), the

polypeptide encoded by open reading frame 3 of the plasmid (pgp3) and a fragment of

the LPS protein (Medac, Hamburg, Germany) (Bas et al., 2001b). Interestingly, OMP2

produced the highest sensitivity (89%) but the lowest specificity (57%) possibly as a

result of cross-reactivity with the C. pneumoniae homolog. Whilst the sensitivity and

specificity of the commercial MOMP assay (Labsystems, Finland) was shown to be

61% and 84% respectively, the combination of pgp3 and MOMP improved sensitivity

(79%) but decreased the overall specificity (82%). In contrast, Land et al. (2003)

investigated the performance of five serological antibody tests in subfertile women and

showed that in 53 women with chronic tubal pathology, the Labsystems’ MOMP assay

- 206 -


had a sensitivity and specificity of 37% and 87% respectively. In our study, the absence

of a differential antibody response against MOMP in any of the male and female patient

groups was demonstrated during the initial Western blot screens as only CT157 and

CT423 were identified as potential candidates for band C. This result was surprising

since infection by C. trachomatis is known to initiate an immune response targeted

against this antigen (Su and Caldwell, 1993; Knight et al., 1995; Ortiz et al., 1996).

Similarly surprising was the lack of antibody reactivity to cHSP60 in either gender

during our initial study (Chapter 3) and studies on IFN-γ-mediated persistence (Chapter

7). Moreover, whilst MOMP and cHSP60 promote a humoral response in the host, the

variable immune response in men and women as a consequence of a C. trachomatis

infection indicates that neither antigens are reliable diagnostic candidates for the

discrimination of acute or chronic infection.

The high sensitivity and specificity of our proposed assay (CT727 (B) + CT157 (C))

would have major implications for the current management and treatment of chronic C.

trachomatis infections and associated adverse pathology. Clinically, patients presenting

with suspected infertility could be routinely screened via this method without the need

for the invasiveness of surgery to confirm the presence of disease. This alone would

greatly benefit the patient as there would be no surgical recovery period and therefore,

associated health costs would be considerably reduced. The implementation of our test

would also provide rapid diagnosis. If positive, a treatment regime individually tailored

rather than a generic approach could be applied. Conversely, a negative result would

eliminate C. trachomatis as a cause of the infertility and allow the clinician to

investigate other possible causative agents thereby hastening the diagnostic process.

Another application for our assay would be the introduction into general practice.

Although standard sexually transmitted disease (STD) health checks are performed

either upon request or routinely in STD clinics, the inclusion of this test could aid in

earlier detection of chronic infection before the infection or disease has the chance to

progress thus reducing further reproductive damage.

- 207 -


The most appropriate diagnostic format of our proposed assay would be to utilise the

ELISA method. To accomplish this, epitopes from each antigen once identified would

be combined into a single well and attached to the ELISA plate. For diagnosis of acute

or chronic infection, serial dilutions of the patient’s serum would be allowed to bind and

the resulting reactivity to the various antigens would determine the stage of infection.

This method of choice would minimise cross-reactivity with C. pneumoniae as only the

epitope and not the entire antigen would be used.

The production of antibodies to CT157 in women and PmpB in males during IFN-γ-

mediated persistence and normal C. trachomatis infection suggests immunological

diversity between genders. Transcriptome analysis of C. trachomatis growth during

IFN-induced persistence and reactivation showed genes located in the plasticity zone,

which are thought to participate in chlamydial pathogenesis (Read et al., 2000), had

increased expression levels (Belland et al., 2003). In general, the plasticity zone

includes several genes of which the phospholipase D superfamily are a component.

Moreover, CT157 which is a member of the phospholipase D family was found to be

up-regulated during persistence. Interestingly, the production of antibodies against

CT157 were lowest in female patients with acute (group I) and recovering acute (group

II) infection when compared with chronic patients (group IV). This suggests that the

immune response generated against CT157 during the early and recovery phases of

infection is not capable of fully resolving the infection. In contrast, the antibody

response to CT157 in males was extremely high in patients with acute (group MI)

infection. Unlike females whose antibody response to this antigen increased in

recovering acute patients (group II), there was a significant reduction of anti-CT157

antibodies in group MII male samples. Unfortunately, the antibody response to CT157

in chronically infected males was not investigated given our inability to obtain samples

due to the low incidence of chronic disease in men. However, since PID patients

demonstrated such a vigorous antibody response to CT157, this may indeed indicate

that CT157 does have an important role in C. trachomatis pathogenesis.

- 208 -


In this study, PmpB a probable outer membrane protein B was shown to elicit an

immune response solely in males as no antibodies against this protein were detected by

female samples. This finding was surprising although it further emphasises the

disparate immune responses between men and women. As discussed in Chapter 6, this

suggests different infection progression and/or immune responses between the two

sexes. Numerous studies have shown that steroid hormones such as oestrogen and

progesterone can both influence and modulate the host immune response upon infection

by C. trachomatis (Wira et al., 1992; Prabhala and Wira, 1995; Crowley et al., 1997;

Pal et al., 1998; Salem, 2004; Guseva et al., 2005). Furthermore, differing phases of the

female menstrual cycle can also enhance infectivity as a result of C. trachomatis

infection (Mahmoud et al., 1994; Crowley et al., 1997; Morrison et al., 2002). Since an

antibody response to PmpB was exclusive to men who do not experience the same

hormonal fluctuations as females, this finding suggests that perhaps these differing

hormone levels between sexes may in fact influence the immune response to PmpB.

These findings raise many questions. Does PmpB share conformational similarity with

oestrogen, thereby enabling interaction with the cognate ER? If so, what level of

homology is present between the two? Does the endogenous secretion of hormones

truly modulate and therefore impact the infectivity and progression of C. trachomatis

infection with regards to the presentation of PmpB? If so, is this the sole reason for the

absence of anti-PmpB antibodies in women? Does the fact that women express higher

levels of oestrogen and therefore ER in comparison to men suggest that PmpB

somehow interacts with the ER through molecular mimicry thus suppressing an immune

response in females? These questions whilst valid remain unanswered. Future studies

will see the investigation of possible interactions between PmpB and ER which will

further elucidate the complex immunological differences between males and females.

A comparative study of the four target antigens initially suggested that none of our

protein candidates (CT157, CT423, CT727 and CT396) were serovar or species

specific. Homology of the four C. trachomatis protein sequences was >95% however

when compared to their C. pneumoniae homologs, the level of sequence similarity

varied greatly (9% - 87%) Importantly, CT157 (a vital component of our proposed

- 209 -


assay ie CT727 (B) + CT157 (C)) shares 94% homology with C. trachomatis serovars

L2 and D, whereas only 9% of the protein is conserved in comparison with C.

pneumoniae. Thus the likelihood of cross-reactivity between chlamydial species is low.

Overall, species and serovar specificity of the four target antigens indicates that patients

infected with C. trachomatis cross-react with the homologous C. pneumoniae proteins,

whilst in contrast; antibodies to C. pneumoniae proteins do not generally cross-react

with the C. trachomatis proteins. This result is of great importance as the chance of a

false-positive when using our diagnostic test is reduced.

The development of a vaccine against C. trachomatis has proven difficult. This is

partially due to our lack of understanding into the regulation of the immune response in

the female genital tract, the deficiency of adjuvants that can direct the vaccines to the

genital mucosa, our inadequate knowledge of which C. trachomatis antigens induce

protective immunity and the lack of tools to genetically manipulate Chlamydia

(Igietseme et al., 2002). Nevertheless, considerable progress has been made in recent

years with the characterisation of eight C. trachomatis proteins that are known to

promote a T-cell response in the host (Brunham and Rey-Ladino, 2005). These proteins

which include CrpA, Cap1, MOMP, OMP2, HSP60, YopD, Enolase and PmpD vary in

molecular weight and locality within the cell, ie. inclusion, membrane or cytoplasm.

Since selection is based upon an antigen’s ability to elicit an immune response in the

host, the four novel markers identified in this study have the potential to be ideal

vaccine candidates. Unfortunately, although PmpB was shown to be highly

immunogenic, its uniqueness to males limits its viability as a vaccine candidate.

In summary, the novel approach used in this project has successfully identified several

antigens which have the potential to accurately discriminate between acute and chronic

C. trachomatis infection. Importantly, the studies presented in this thesis have provided

new insights into immunological responses in men and women and have laid the

foundation for future research focusing on immune responses and the possible hormonal

modulation and interaction with the target antigens.

- 210 -

Chapter 9: Literature Cited

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