1. Pathogenomics Project

2. Cross-Domain Horizontal Gene Transfer Analysis

3. Horizontal Gene Transfer: Identifying Pathogenicity Islands

Pathogenomics

Goal:

Identify previously unrecognized mechanisms of microbial pathogenicity using a combination of informatics, evolutionary biology, microbiology and genetics.

Explosion of data

26 of the 36 publicly available bacterial genome sequences are for pathogens

Approximately 24,000 pathogen genes with no known function!

~177 bacterial genome projects in progress …

Data as of June, 2001

Bacterial Pathogenicity

Processes of microbial pathogenicity at the molecular level are still minimally understood

Pathogen proteins identified that manipulate host cells by interacting with, or mimicking, host proteins

Yersinia Type III secretion system

Approach

Idea: Could we identify novel virulence factors by identifying bacterial pathogen genes more similar to host genes than you would expect based on phylogeny?

Prioritize for biological study. - Previously studied in the laboratory? - Can UBC microbiologists study it? - C. elegans homolog?

Search pathogen genes against databases. Identify those with eukaryotic similarity.

Evolutionary significance. - Horizontal transfer? Similar by chance?

Modify screening method /algorithm

Approach

Genome data for…

Anthrax Necrotizing fasciitis Cat scratch disease Paratyphoid/enteric feverChancroid Peptic ulcers and gastritisChlamydia Periodontal diseaseCholera PlagueDental caries PneumoniaDiarrhea (E. coli etc.) SalmonellosisDiphtheria Scarlet feverEpidemic typhus ShigellosisMediterranean fever Strep throatGastroenteritis SyphilisGonorrhea Toxic shock syndromeLegionnaires' disease Tuberculosis Leprosy TularemiaLeptospirosis Typhoid feverListeriosis UrethritisLyme disease Urinary Tract InfectionsMeliodosis Whooping cough Meningitis +Hospital-acquired infections

Bacterial Pathogens

Chlamydophila psittaci Respiratory disease, primarily in birdsMycoplasma mycoides Contagious bovine pleuropneumoniaMycoplasma hyopneumoniae Pneumonia in pigsPasteurella haemolytica Cattle shipping feverPasteurella multicoda Cattle septicemia, pig rhinitisRalstonia solanacearum Plant bacterial wiltXanthomonas citri Citrus cankerXylella fastidiosa Pierce’s Disease - grapevines

Bacterial wilt

World Research Community

ApproachPrioritized candidates

Study function of homolog in model host (C. elegans)

Study function of gene in bacterium.

Infection of mutant in model host

C. elegansDATABASE

Collaborations with others

Informatics/Bioinformatics• BC Genome Sequence Centre• Centre for Molecular Medicine

and Therapeutics

Evolutionary Theory• Dept of Zoology

• Dept of Botany

• Canadian Institute for Advanced Research

Pathogen Functions• Dept. Microbiology

• Biotechnology Laboratory

• Dept. Medicine

• BC Centre for Disease Control

Host Functions• Dept. Medical Genetics

• C. elegans Reverse Genetics Facility

• Dept. Biological Sciences SFU

Interdisciplinary group

Coordinator

• For each complete bacterial and eukaryote genome: BLASTP (and MSP Crunch) of all deduced proteins against non-redundant SWALL database

• Overlay NCBI taxonomy information form ACEDB database

• Query database for bacterial proteins who’s top scoring hit is eukaryotic (and eukaryotic proteins who’s top hit is bacterial)

• Perform similar query, but filtering different taxonomic groups from the analysis

Development of first database: Sequence similarity-based approach

BAE-watch Database: Bacterial proteins with unusual similarity with Eukaryotic proteins

Problem: Proteins highly conserved in the three domains of life

Top hit to a protein from another domain may occur by chance.

“StepRatio” score helps detect these.

Example:Glucose-6-Phosphate Reductase

Example of a case with a high StepRatio:

Enoyl ACP reductase

BAE-watch Database: Bacterial proteins with unusual similarity with Eukaryotic proteins

Haemophilus influenzae Rd-KW20 proteins most strongly matching eukaryotic proteins

PhyloBLAST – a tool for analysisBrinkman et al. (2001) Bioinformatics. 17:385-387.

Trends in this Sequence-based Analysis

• Identifies the strongest cases of lateral gene transfer between bacteria and eukaryotes

• Most common “cross-domain” horizontal transfers:

Bacteria Unicellular Eukaryote

• Identifies nuclear genes with potential organelle origins

• A control: Method identifies all previously reported Chlamydia trachomatis “plant-like” genes.

First case: Bacterium Eukaryote Lateral Transfer

0.1

Bacillus subtilis

Escherichia coli

Salmonella typhimurium

Staphylococcua aureus

Clostridium perfringens

Clostridium difficile

Trichomonas vaginalis

Haemophilus influenzae

Acinetobacillus actinomycetemcomitans

Pasteurella multocida

N-acetylneuraminate lyase (NanA) of the protozoan Trichomonas vaginalis is 92-95% similar to NanA of Pasteurellaceae bacteria.

de Koning et al. (2000) Mol. Biol. Evol. 17:1769-1773

N-acetylneuraminate lyase – role in pathogenicity?

Pasteurellaceae

•Mucosal pathogens of the respiratory tract

T. vaginalis

•Mucosal pathogen, causative agent of the STD Trichomonas

N-acetylneuraminate lyase (sialic acid lyase, NanA)

Involved in sialic acid metabolism

Role in Bacteria: Proposed to parasitize the mucous membranes of animals for nutritional purposes

Role in Trichomonas: ?

Hydrolysis of glycosidic linkages of terminal sialic residues in glycoproteins, glycolipids SialidaseFree sialic acid

Transporter

Free sialic acid NanA

N-acetyl-D-mannosamine + pyruvate

Another case: A Sensor Histidine Kinase for a Two-component Regulation System

Signal Transduction

Histidine kinases common in bacteria

Ser/Thr/Tyr kinases common in eukaryotes

However, a histidine kinase was recently identified in fungi, including pathogens Fusarium solani and Candida albicans

How did it get there?

Candida

Neurospora crassa NIK-1

Fusarium solani FIK2 Streptomyces coelicolor SC4G10.06c

Candida albicans CaNIK1

Escherichia coli RcsC

Erwinia carotovora RpfA / ExpSEscherichia coli BarASalmonella typhimurium BarA

Pseudomonas aeruginosa GacS

Pseudomonas fluorescens GacS / ApdAPseudomonas tolaasii RtpA / PheN

Pseudomonas syringae GacS / LemA

Pseudomonas viridiflava RepAAzotobacter vinelandii GacS

0.1

Streptomyces coelicolor SC7C7.03

Xanthomonas campestris RpfCVibrio cholerae TorS

Escherichia coli TorS

Fusarium solani FIK1Fungi

Pseudomonas aeruginosa PhoQ

100

100

51100

100

100

100

100100

100

100

100

100

86

54

39

100

100

Streptomyces Histidine Kinase. The Missing Link?

virulence factor=

virulence factor ?

Brinkman et al. (2001) Infection and Immunity. In Press.

“Plant-like” genes in Chlamydia

• Chlamydiaceae: Obligate intracellular pathogens of humans

• Proteins: Unusually high number most similar to plant proteins

• Previous proposal: Obtained genes from a plant-like amoebal host? (a relative of Chlamydiaceae infects Acanthamoeba)

“Plant-like” genes in ChlamydiaNCBI GI Protein description Subcellular localization in plants

4377270 Glycyl tRNA Synthetase Chloroplast

4376626 cADP/ATP Translocase Chloroplast

4376667 cGlycogen Hydrolase Chloroplast

4377189 GTP Cyclohydratase & DHBP Synthase Chloroplast

4377237 cBeta-Ketoacyl-ACP Synthase Chloroplast

4376686 cEnoy-Acyl-Carrier Reductase Chloroplast

4376591 cThioredoxin Reductase Chloroplast

4377185 Metal Transport P-type ATPase Chloroplast

4377346 Similar to NA+/H+ Antiporter Chloroplast

4376650 cPhosphate Permease Chloroplast

4376637 GcpE protein Chloroplast

4376637 Tyrosyl tRNA Synthetase Chloroplast

4377360 cMalate Dehydrogenase Chloroplast

4376763 GTP Binding protein Chloroplast

4376911 cADP/ATP Translocase Chloroplast

3329179 Phosphoglycerate Mutase Chloroplast

4377281 cGlycerol-3-Phosphate Acyltransferase Chloroplast

4376993 ABC Transporter ATPase Chloroplast

4376509 dDeoxyoctulonosic Acid Synthetase Chloroplast

4376872 eSugar Nucleotide Phosphorylase Chloroplast

“Plant-like” genes in Chlamydia

6578112 rRNA Methytransferase Chloroplast

3329217 HSP60 Chloroplast

3328745 cPhosphoribosylanthranilate Isomerase Chloroplast

6578104 cAspartate Aminotransferase Chloroplastf

4377328 cPolyribonucleotide Nucleotidyltransferase Chloroplastf

4377362 Putative D-Amino Acid Dehydrogenase Chloroplastg

4377331 Cytosine Deaminase Chloroplast?h

4376915 Lipoate-Protein Ligase A Mitochondrial

4377272 Glycogen Synthase N/Ai

4377065 cDihydropteroate Synthase N/Ai

4377239 cInorganic Pyrophosphatase N/Ai

4376904 Uridine 5’-Monophosphate Synthase N/Ai

4377173 cUDP-Glucose Pyrophosphorylase N/Ai

4376815 GutQ/Kpsf Family Sugar-Phosphate Isomerase Mitochondrial?j

Chlamydiaceae share an ancestral

relationship with Cyanobacteria and

Chloroplast0.1

Pyrococcus furiosus (Archaea)

Thermotoga maritima

Aquifex pyrophilus

Bacillus subtilis

Chlamydophila pneumoniae

Chlamydophila psittaci

Chlamydia muridarum

Chlamydia trachomatis1000

7041000

Chlamydomonas reinhardtii

Klebsormidium flaccidum

Zea mays

Nicotiana tabacum1000

988

998

Synechococcus PCC6301

Synechocystis PCC6803

Microcystis viridis1000

1000

1000

530

Escherichia coli

Zea mays mitochondrion

Rickettsia prowazekii

Caulobacter crescentus

868986

764

349

1000

538

Chloroplasts

Cyanobacteria

Chlamydiaceae

Chlamydiaceae share an ancestral relationship with Cyanobacteria and Chloroplast

L3

L4

L23

L2

S19

L22 S3

L16 L29

S17

L14

L24

L5

S14 S8

L6

L18 S5

L30

L15S10

EscherichiaBacillusThermatogaSynechocystisChlamydia

Unique shared-derived characters unite Chlamydiaceae and Synechocystis

Chlamydiaceae “plant-like” genes reflect an ancestral relationship with Cyanobacteria and Chloroplast

•Chlamydia do not appear to be exchanging DNA with their hosts

•Existing knowledge of Cyanobacteria may stimulate ideas about the function and control of pathogenic Chlamydia?

Non-unique shared characters include a multistage developmental lifecycle, storage of glucose primarily as glycogen, and non-flagellar motility

Expanding the Cross-Domain Analysis

• Identify cross-domain lateral gene transfer between bacteria, archaea and eukaryotes

• No obvious correlation seen with protein functional classification

• Most cases: no obvious correlation seen between “organisms involved” in potential lateral transfer

Exceptions:

– Unicellular eukaryotes

– “Organelle-functioning” proteins in Rickettsia, Synechocystis, and Chlamydiaceae

Horizontal Gene Transfer and Bacterial Pathogenicity

Transposons: ST enterotoxin genes in E. coli

Prophages:Shiga-like toxins in EHECDiptheria toxin gene, Cholera toxinBotulinum toxins

Plasmids:Shigella, Salmonella, Yersinia

Pathogenicity Islands:

Uro/Entero-pathogenic E. coliSalmonella typhimuriumYersinia spp.Helicobacter pyloriVibrio cholerae

Pathogenicity Islands

Associated with

– Atypical %G+C– tRNA sequences– Transposases, Integrases and other mobility genes– Flanking repeats

IslandPath: Identifying Pathogenicity Islands

Yellow circle = high %G+C

Pink circle = low %G+C

tRNA gene lies between the two dots

rRNA gene lies between the two dots

Both tRNA and rRNA lie between the two dots

Dot is named a transposase

Dot is named an integrase

Neisseria meningitidis serogroup B strain MC58 Mean %G+C: 51.37 STD DEV: 7.57

%G+C SD Location Strand Product 39.95 -1 1834676..1835113 + virulence associated pro. homolog 51.96 1835110..1835211 - cryptic plasmid A-related 39.13 -1 1835357..1835701 + hypothetical 40.00 -1 1836009..1836203 + hypothetical 42.86 -1 1836558..1836788 + hypothetical 34.74 -2 1837037..1837249 + hypothetical 43.96 1837432..1838796 + conserved hypothetical 40.83 -1 1839157..1839663 + conserved hypothetical 42.34 -1 1839826..1841079 + conserved hypothetical 47.99 1841404..1843191 - put. hemolysin activ. HecB 45.32 1843246..1843704 - put. toxin-activating 37.14 -1 1843870..1844184 - hypothetical 31.67 -2 1844196..1844495 - hypothetical 37.57 -1 1844476..1845489 - hypothetical 20.38 -2 1845558..1845974 - hypothetical 45.69 1845978..1853522 - hemagglutinin/hemolysin-rel. 51.35 1854101..1855066 + transposase, IS30 family

Variance of the Mean %G+C for all Genes in a Genome: Correlation with bacteria’s clonal nature

non-clonal clonal

Pathogenomics Project: Future Developments

• Identify eukaryotic motifs and domains in pathogen genes

• Threader: Detect proteins with similar tertiary structure

• Identify more motifs associated with• Pathogenicity islands• Virulence determinants

• Functional tests for new predicted virulence factors

• Expand analysis to include viral genomes

• Jeff Blanchard (National Centre for Genome Resources, New Mexico)

• Olof Emanuelsson (Stockholm Bioinformatics Center)

• Genome Sequence Centre, BC Cancer Agency

Acknowledgements

Pathogenomics group Ann M. Rose, Yossef Av-Gay, David L. Baillie, Fiona S. L.

Brinkman, Robert Brunham, Artem Cherkasov, Rachel C. Fernandez, B. Brett Finlay, Hans Greberg, Robert E.W. Hancock, Steven J. Jones, Patrick Keeling, Audrey de Koning, Don G. Moerman, Sarah P. Otto, B. Francis Ouellette, Nancy Price, Ivan Wan.

www.pathogenomics.bc.ca