Cross-Domain and Within-Domain Horizontal Gene Transfer: Implications for Bacterial Pathogenicity

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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

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Pathogenomics
Goal Identify previously unrecognized mechanisms
of microbial pathogenicity using a combination of
informatics, evolutionary biology, microbiology
and genetics.
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• 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!
• gt95 bacterial pathogen genome projects in
  progress

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• The need for new tools
• Prioritize new genes for further laboratory study
• Capitalize on the existing genomic data

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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
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Yersinia Type III secretion system
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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?
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Approach
Search pathogen genes against databases.
Identify those with eukaryotic similarity.
Modify screening method /algorithm
Evolutionary significance. - Horizontal
transfer? Similar by chance?

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

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Genome data for
Anthrax Necrotizing fasciitis Cat scratch
disease Paratyphoid/enteric fever Chancroid
Peptic ulcers and gastritis Chlamydia
Periodontal disease Cholera Plague Dental
caries Pneumonia Diarrhea (E. coli
etc.) Salmonellosis Diphtheria Scarlet
fever Epidemic typhus Shigellosis Mediterranean
fever Strep throat Gastroenteritis
Syphilis Gonorrhea Toxic shock
syndrome Legionnaires' disease Tuberculosis
Leprosy Tularemia Leptospirosis Typhoid
fever Listeriosis Urethritis Lyme disease
Urinary Tract Infections Meliodosis Whooping
cough Meningitis Hospital-acquired
infections
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Bacterial Pathogens
Chlamydophila psittaci Respiratory disease,
primarily in birds Mycoplasma mycoides
Contagious bovine pleuropneumonia Mycoplasma
hyopneumoniae Pneumonia in pigs Pasteurella
haemolytica Cattle shipping fever Pasteurella
multicoda Cattle septicemia, pig
rhinitis Ralstonia solanacearum Plant bacterial
wilt Xanthomonas citri Citrus canker Xylella
fastidiosa Pierces Disease - grapevines
Bacterial wilt
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Approach
Prioritized candidates
Study function of homolog in model host (C.
elegans)
Study function of gene in bacterium. Infection
of mutant in model host
Collaborations with others
C. elegans
DATABASE
World Research Community
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Interdisciplinary group

• 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

Coordinator

• 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

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Pathogenomics Database Bacterial proteins with
unusual similarity with Eukaryotic proteins
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Haemophilus influenzae Rd-KW20 proteins most
strongly matching eukaryotic proteins
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PhyloBLAST a tool for analysis
Brinkman et al. (2001) Bioinformatics. In Press.
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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.

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First case Bacterium Eukaryote Lateral
Transfer
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.
171769-1773
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N-acetylneuraminate lyase role in pathogenicity?

• Pasteurellaceae
• Mucosal pathogens of the respiratory tract
• T. vaginalis
• Mucosal pathogen, causative agent of the STD
  Trichomonas

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N-acetylneuraminate lyase (sialic acid lyase,
NanA)
Hydrolysis of glycosidic linkages of terminal
sialic residues in glycoproteins, glycolipids
Sialidase Free sialic acid
Transporter Free sialic acid
NanA N-acetyl-D-mannosamine pyruvate
Involved in sialic acid metabolism Role in
Bacteria Proposed to parasitize the mucous
membranes of animals for nutritional purposes
Role in Trichomonas ?
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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
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Streptomyces Histidine Kinase. The Missing Link?
Pseudomonas aeruginosa PhoQ
Xanthomonas campestris RpfC
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Vibrio cholerae TorS
100
Escherichia coli TorS
Escherichia coli RcsC
Candida albicans CaNIK1
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100
Neurospora crassa NIK-1
100
Fungi
Fusarium solani FIK1
100
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Fusarium solani FIK2
Streptomyces coelicolor SC4G10.06c
100
Streptomyces coelicolor SC7C7.03
virulence factor ?
Pseudomonas aeruginosa GacS
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100
Pseudomonas fluorescens GacS / ApdA
100
Pseudomonas tolaasii RtpA / PheN
100
Pseudomonas syringae GacS / LemA
100
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Pseudomonas viridiflava RepA
100
Azotobacter vinelandii GacS
Erwinia carotovora RpfA / ExpS
virulence factor
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Escherichia coli BarA
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Salmonella typhimurium BarA
0.1
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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
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Recent report P. aeruginosa eukaryote-type
Phospholipase plays a role in infection

• Wilderman et al. 2001. Mol Microbiol 39291-304
• 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

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Eukaryote Bacteria Horizontal Transfer?
E. coli Guanosine monophosphate reductase 81
similar to corresponding enzyme in humans and
rats Role in virulence not yet investigated.
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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

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Plant-like genes in Chlamydia
Aquifex aeolicus
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Haemophilus influenza
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
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Escherichia coli
Anabaena
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Synechocystis
100
Chlamydia trachomatis
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Petunia x hybrida
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Nicotiana tabacum
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Brassica napus
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Arabidopsis thaliana
0.1
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Oryza sativa
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Synechocystis
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Rickettsia and Chlamydia
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Proteins Homologous to Eukaryote Proteins
(according to BLAST Exp1)
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Horizontal Gene Transfer and Bacterial
Pathogenicity
Transposons ST enterotoxin genes in E.
coli Prophages Shiga-like toxins in
EHEC Diptheria toxin gene, Cholera
toxin Botulinum toxins Plasmids Shigella,
Salmonella, Yersinia
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Horizontal Gene Transfer and Bacterial
Pathogenicity
Pathogenicity Islands Uropathogenic and
Enteropathogenic E. coli Salmonella
typhimurium Yersinia spp. Helicobacter
pylori Vibrio cholerae
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Pathogenicity Islands

• Associated with
• Atypical GC
• tRNA sequences
• Transposases, Integrases and other mobility genes
• Flanking repeats

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IslandPath Identifying Pathogenicity Islands
Yellow circle high GC Pink circle
low GC 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
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Neisseria meningitidis serogroup B strain MC58
Mean GC 51.37 STD DEV 7.57 GC 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

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Variance of the Mean GC for all Genes in a
Genome Correlation with bacterias clonal nature
non-clonal
clonal
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Variance of the Mean GC 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?

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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

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Peter Wall Major Thematic Grant

• Fundamental research
• Interdisciplinary
• Lack of fit with alternative funding sources

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• 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

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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?

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A Histidine Kinase in Streptomyces.The Missing
Link?
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
0.1
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