Cross-Domain and Within-Domain Horizontal Gene Transfer: Implications

for Bacterial Pathogenicity

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

23 of the 37 publicly available microbial genome sequences are for bacterial pathogens

Approximately 21,000 pathogen genes with no known function!

>95 bacterial pathogen genome projects in progress …

The need for new tools

Prioritize new genes for further laboratory study

Capitalize on the existing genomic data

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

Pathogenomics Database: Bacterial proteins with unusual similarity with Eukaryotic proteins

Haemophilus influenzae Rd-KW20 proteins most strongly matching eukaryotic proteins

PhyloBLAST – a tool for analysis Brinkman et al. (2001) Bioinformatics. In Press.

Trends in the Initial 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 “eukaryote-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

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Streptomyces Histidine Kinase. The Missing Link?

virulence factor=

virulence factor ?

Reduced virulence of a Pseudomonas aeruginosa transposon mutant disrupted in the

histidine kinase gene gacS

 

Groups of 7-8 neutropenic mice challenged on two separate occasions with doses ranging from 8 to 8 x 106 bacteria

Wildtype LD50 = 10 1 bacteria

gacS mutant LD50 = 7,500 100 bacteria

750-fold increase

Recent report: P. aeruginosa eukaryote-type Phospholipase plays a role in infection

Wilderman et al. 2001. Mol Microbiol 39:291-304

• Phospholipase D (PLDs) virtually ubiquitous in eukaryotes (relatively uncommon in prokaryotes)

• P. aeruginosa expresses PLD with significant (1e-38 BLAST Expect) similarity to eukaryotic PLDs

• Part of a mobile 7 kb genetic element

• Role in P. aeruginosa persistence in a chronic pulmonary infection model

Eukaryote Bacteria Horizontal Transfer?

0.1Rat

Human

Escherichia coli

Caenorhabditis elegans

Pig roundworm

Methanococcus jannaschii

Methanobacterium thermoautotrophicum

Bacillus subtilis

Streptococcus pyogenes

Aquifex aeolicus

Acinetobacter calcoaceticus

Haemophilus influenzae

Chlorobium vibrioforme

E. coli Guanosine monophosphate reductase 81% similar to corresponding enzyme in humans and rats

Role in virulence not yet investigated.

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-like” proteins in Rickettsia and Synechocystis

– “Plant-like(?)” genes in the obligate intracellular bacteria Chlamydia

“Plant-like” genes in Chlamydia

Enoyl-acyl carrier protein reductase (involved in lipid metabolism) of Chlamydia trachomatis is similar to those of Plants

Organelle relationship?

Notably more similar to plants than Synechocystis

0.1

Aquifex aeolicus

Haemophilus influenza

Escherichia coli

Anabaena

Synechocystis

Chlamydia trachomatis

Petunia x hybrida

Nicotiana tabacum

Brassica napus

Arabidopsis thaliana

Oryza sativa

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Eukaryote Top Hits in Bacterial Genomes (after excluding relatives of the same Family)

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No. of Proteins in each Bacterial Genome

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Synechocystis

Eukaryote Top Hits in Bacterial Genomes (excluding "Family" and Synechocystis )

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its

Rickettsia and Chlamydia

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Proteins Homologous to Eukaryote Proteins (according to BLAST Exp=1)

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

Horizontal Gene Transfer and Bacterial Pathogenicity

Pathogenicity Islands:

Uropathogenic and Enteropathogenic 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

Variance of the Mean %G+C for all Genes in a Genome

Is this a measure of clonality of a bacterium?

Are intracellular bacteria more clonal because they are ecologically isolated from other bacteria?

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

• Fundamental research

• Interdisciplinary

• Lack of fit with alternative funding sources

Peter Wall Major Thematic Grant

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

Brinkman, Robert Brunham, 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, Ivan Wan.

www.pathogenomics.bc.ca

Universal role of this Histidine Kinase in pathogenicity?

Pathogenic Fungi•Senses change in osmolarity of the environment•Role in hyphal formation pathogenicity

Pseudomonas species plant pathogens•Role in excretion of secondary metabolites that are virulence factors or antimicrobials

Virulence factor for human opportunistic pathogen Pseudomonas aeruginosa?

A Histidine Kinase in Streptomyces.The Missing Link?

0.1

Neurospora crassa NIK-1

Streptomyces coelicolor SC7C7

Fusarium solani FIK

Candida albicans CHIK1

Erwinia carotovora EXPS

Escherichia coli BARA

Pseudomonas aeruginosa LEMA

Pseudomonas syringae LEMA

Pseudomonas viridiflava LEMA

Pseudomonas tolaasii RTPA

Euykaryotic top hits in bacterial genomes(after excluding "tertiary" relatives)

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No. of Proteins

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Synechocystis

Rikettsia