SBM 2044: Lecture 10

AIMS: To provide

• Brief introduction to E. coli: a versatile pathogen

• Overview of Enterotoxigenic E. coli (ETEC)

Types of pathogenic E. coli

Enterotoxigenic E. coli (ETEC)Enteroaggregative E. coli (EAEC)Diffusely adhering E. coli (DAEC)

Enteropathogenic E. coli (EPEC)Enterohaemorrhagic E. coli (EHEC)

Enteroinvasive E. coli (EIEC)

Uropathogenic E. coli (UPEC)

Septic E. coli strains

Cholera-likewatery diarrhoea

Colitis orhaemorrhagic colitis

Dysentery

Urinary tract/pyelonephritis

Septicemia/meningitis

Intestinal

Extraintestinal

Disease

First recognised in 1987 as distinct from other ETEC

EAEC

Known virulence factors:

AAF - Aggregative adherence fimbriae

EAST-1 – EAEC heat stable toxin (similar to ETEC Sta)

Pet – Plasmid-encoded enterotoxin mucus release

Pic – role in intestinal colonisation? - various activities (mucinase, serum resistance, haemagglutinin)

Characteristic adhesion pattern in vitro reflects bacteria- bacteria (in addition to bacteria-host) adhesion

EAEC

Strains displaying a distinct adhesion pattern

Enteroaggregrative E. coli (EAEC)

Diffusely adhering E. coli (DAEC)

DAEC

• Doubts about their importance as pathogens

• Like EAEC and ETEC, appear to be heterogeneous group of strains, sharing certain common factors

• Four different adhesins identified – F1845 fimbriae & 3 non-fimbrial adhesins

• No significant information on potential toxins

Enteropathogenic E. coli (EPEC)

First associated with diarrhoea in 1940s

No detectable enterotoxin

Major cause of watery diarrhoea in infants (< 6 months)• Developing countries - endemic• Developed countries – sporadic outbreaks in nurseries,

paediatric wards, day centres, etc

1970s – first seen by EM to produce unique ‘attaching & effacing’ effects on enterocytes - producing unique lesions, now called A/E lesions

1. Initial (non-intimate) attachment

2. Intimate adhesion + effacement

3. Formation of pedestals

EPEC interactions with epithelial cells

Studies on mechanisms very limited until late 1980s

1. Initial (non-intimate) attachment

2. Intimate adhesion + effacement

3. Formation of pedestals

EPEC interactions with epithelial cells

Involved: Intimin (EaeA) [OM protein]

BFP Actinrearrangements

Anti-EspA

Filaments ‘connecting’EPEC & host cells

Knutton et al (1998) EMBO Journal 17: 2166-2176

Activation of EPEC Type III secretion

Immuno-gold antibodies

Filaments composed of EspA

EM

Host cell membrane

EPEC OM

EPEC IM

EPEC Type III secretion filamentEspB/D pore

Esc R,S,T,U,V,N,DIM-associated ‘machinery’

EscC

EscJ

Deduced from studies combining: Mutants protein-protein binding Labelling with specific Ab Electron microscope

Similar ‘needles’ in EPEC, but Salmonella lack ‘filament’

Kubori et al (1998) Science 280: 602-605

Salmonella Type III ‘needles’

EPEC: Kenny et al (1997) Cell 91: 511 - 520

Receptor for intimin (adhesin) is NOT a host cell protein

Remarkable discovery:

It is an EPEC protein

Translocated into host cell, phosphorylated, & inserted into the host cell membrane

Tir: Translocated intimin receptor

EPEC: Model of A/E Lesion Formation

Initial adhesion (via Bfp )

Activation of Type III secretion

Actin polymerizationpedestal formation

Tir + ? signals translocated into host cell Tir- P

Intimate adhesion by EaeA to Tir- P

Ca++

Details of signalling still unclear

filaments EspB/D pore

Quorum sensing ?

Enterohaemorrhagic E. coli (EHEC)

1982: two cases of severe food-poisoning (hamburgers) in USA associated with rare serotype of E. coli - O157:H7

1983: E. coli O157:H7 produces a Shiga-like toxin

1986: Like EPEC, O157:H7 produce A/E lesions

‘Enterohaemorrhagic E. coli’ (EHEC) first used in 1987 to describe what seemed to be a new type of pathogenic E. coli

Main difference with EPEC is production of Shiga-toxin

Origin of E. coli O157:H7 ?

First isolated in US in 1982 - now worldwide

O157:H7 isolates throughout world are single clone that emerged relatively recently - closely related to a much less virulent EPEC clone

Quantitative population genetic studies (MLEE)

Both SLT-I & SLT-II genes encoded by bacteriophages, - could facilitate their transmission into new strains

O157:H7 probably emerged when an EPEC strain (alreadycapable of producing A/E lesions & diarrhoea) acquired slt phages, resulting in a dramatic increase in virulence

A1SSA2

Shiga-toxinsTypical A-B subunit toxins

A1 subunit - N-glycosidase

Gb3-rich cells (particularly sensitive to STx) include

Pentameric B subunit (5 x 7kDa)

• vascular endothelial cells

B

27kDa

4kDa

Receptor: glycolipid called Gb-3

• absorptive enterocytes• kidney endothelial cells

haemorrhagediarrhoearenal failure

Hydrolyses eucaryotic cell 28S rRNA

STx: Entry via RME retrograde transport via Golgi & ER

From: Groisman

E. coli Shiga-toxins (STx)

Local effects of toxin in colon

Very serious – often fatal.

Role in disease

Haemolytic uraemic syndrome (HUS)

(10% - 20% cases)

haemorrhagediarrhoea

Absorption – systemic effects Renal failure

S. dysenteriae E. coli producing highhigh levels of STx

Strongly associated:

E. coli O157:H7• Clinical reports that therapy with certain antibiotics increased severity of O157 infections

Some antibiotics (e.g. quinolones) inhibit DNA replication.

WHY ?

higher levels of STx in gut

DNA damage SOS response

Induces lysogenic phage

Urinary tract infections

(community acquired)

Uropathogenic E. coli (UPEC)

source: UPEC in colon - harmless commensals

Urethra

Bladder

Kidneys

Bloodstream

Ascending infection

UrineFlushing

Removesnon-adherent

bacteria

Ureter

septicemia

pyelonephritis

ureteritis

cystitis

urethritis

Uropathogenic E. coli - virulence mechanisms

Adhesion - essential to avoid removal by urine

Survival - studies limited to some aspects only

Toxicity inflammation

• iron-acquisition (siderophores)• capsules (evasion of host defences)

- more important in kidney than lower UT- critical if organism enters bloodstream

• LPS (endotoxin)• -haemolysin (membrane damaging toxin)• cytotoxic necrotizing factor 1(CNF-1)

E. coli -haemolysin (HlyA)

Produced by about:

Membrane-damaging toxin:

• 50% strains from upper UTI – most tissue damage

• Produces small hydrophilic pores in mammalian cells• high concentrations ion leakage osmotic lysis • lower concentrations more subtle cytotoxic effects

Suggests strong association with UTI

• 30% strains from lower UTI• 10% GI strains

E. coli -haemolysin (HlyA)

• Role in kidney damage supported by studies in mouse models:

Parent strain expressing P fimbriae + HlyA

colonized bladder + kidneys 66% mice died

Isogenic mutant expressing P fimbriae, but not HlyA

colonized as above, but no apparent damage or deaths

Cytotoxic necrotizing factor 1 (CNF-1)

Large (110,000Da) protein, produced by ca 30% UPEC

Studies in vitro:

No direct evidence for role in UTI

• epithelial cells: rearrangements of actin filaments & membrane ruffling

• bladder cells: reduced migration & proliferation – - impede repair of damage bladder ??