Virulence Factors of Pathogenic Baceria

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Virulence properties of

pathogens

Trudy M. WassenaarMolecular Microbiology and Genomics ConsultantsZotzenheim, Germany


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What is a pathogen?A pathogenic organism causes harm to the host itcolonizes:

Parasites (worms, ticks)Other eukaryotes (Fungi, Cryptosporidium)

Bacteria

Viruses

PrionsBorder of life


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What is a pathogen?There is a continious spectrum from 'good' to 'evil'

mutualist depends depend e.g. lychen (algae/fungi)

symbiont benefits benefit e.g. plants/Agromonas)

commensal benefits/neutral neutral e.g. mammals/E.coli

pathogen suffers benefit/neutral/depend

Host Bacteria


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A bacterial pathogen requiresvirulenceproperties to cause

disease;colonizationproperties for animal and/or humanhosts;escapestrategies to leave the host andsurvivalproperties in the environment

most attention (money, status) is directed to virulence

How were virulence genes identified in the past?

What are novel approaches?

What could be future strategies?


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Example: a foodborne pathogen

A food pathogen requiresvirulence properties tocause disease;

colonization properties foranimal and human hosts;survival properties in theenvironment and in food


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Definitions vary broad: All genes with a function in virulence

narrow: genes encoding factors that interact directly with the

host, causing damage to that host

Why do bacteria have virulence genes / cause disease?

Anthropocentric:To cause disease

Bacteriocentric: Niche adaptation.To survive in the host (liberatenutrients, evade immune defence, etc.); to enable spread (inducing diarrhea etc.) By accident (N. meningitidisis mostly commensal, when causing

meningitis it has blocked it's transmission to the next host; S. aureusbiofilms on implanted devises)

What makes a pathogen?

Virulence genes


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Koch's postulates (1890)search help

nIn 1890 the German physician and bacteriologist Robert Koch set out his celebrated criteria for judgingwhether a given bacteria is the cause of a given disease. Koch's criteria brought some much-neededscientific clarity to what was then a very confused field.

Bacteria are pathogens and cause an observed disease if and only if:

The bacteria are present in every case of the disease The bacteria are isolated from the host with the disease and grown in pure culture The specific disease must be reproduced when a pure culture of the bacteria is

inoculated into a healthy susceptible host The bacteria must be recoverable from the experimentally infected host

How to identify a pathogen?

Not alwayspossible

Animal modelnot alwaysavailable

Epidemiological orimmunological

evidence was added


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Nobel Prize for Medicine 2005:Barry Marshall and Robin Warren for thediscovery of Helicobacter pylori

search help

From the press: "this bacterium as the cause for ulcers and

'other diseases"A causual relationship with gastric ulcers has been provenA causual relationship with gastric cancer is considered.Depending on age, 10% ot 80% of the population is infected(cohort effect. Add roughly 10% per decade)Only in 10% of cases does disease occur

Have Koch's postulates been fullfilled?Is Helicobacter pyloria pathogen?

There is a continious spectrum from 'good' to 'evil'

Intermezzo


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Molecular Koch's postulates (1988)search help

nIn 1988 Stanley Falkow published a commentary article translating Koch's postulates into the era ofmolecular biology. He described the then common approaches to identify virulence genes and listedthe conclusive evidence needed:

Genes are considered virulence genes if and only if:

1. The phenotype or gene is associated with pathogenic strains/species2. Specific inactivation of the gene results in a measurable loss in virulence

(attenuation)3. Reversion or allelic replacement of the mutated gene restors pathogenicity4. Or to 2,3: induction of specific antibodies neutralizes pathogenicity

How to identify a virulence gene?


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1. Association of genes to virulence:

Many so-called virulence genes are present in non-virulentspecies too. E.g. flagella, LPS, iron acquisition systems

Depends on:Means for gene identification (Southern hybridization, PCR,

nowadays: genome sequences!)Gene expression (not only presence), interpretation of role in

virulence

False positives:False negatives:Depends on the strength of identifying gene similarity, functional

similarity, definition


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2,3. Phentoypic evidence:

Inactivate a gene and measure loss of virulence.Complement to an avirulent background and virulence increases.

Depends on:Means for genetic manipulationA suitable model for virulence (animal model, in vitro models)

False positives:

Housekeeping genes have a role in nice adaptation. Whenmutated, attenuation occurs. E.g. aroA, LPS genes

False negatives:Virulence strategies may be redundant. One gene mutation may

be overcome by other systems.


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4. Epidemiological and immunological evidence:

Is a gene strongly associated with strains causing disease?Is an immune response directed against the gene productprotective

Depends on:Statistics and correct datasets (landmines!)Epitope mapping, effect of the host! (more landmines)

False positives:

False negatives:


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complementation of a gene in anavirulent background enhancesvirulence

The identification of virulence genesPhenotypic

evidence

inactivation of a gene results inloss of virulence or in attenuation

Immunologicalevidence

antibodies against agene product result inimmunity from disease

infection results in animmune responsedirected to the geneproduct

Putativevirulence

genes

antigenic variation, mutationpatterns, are indicators for a rolein pathogenesis or host-defenseavoidence

genes with significant homologyto characterized virulence genesin other organisms

ComparativeGenetics

a gene present in (more) virulent

strains and absent in avirulentstrains


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Novel approach: comparative geneticsIdentify virulence genes fromgenomes by significant homology tocharacterized virulence genes inother organismsPitfalls:

what is significant homology? do homologous genes have

homologous functions in differentorganisms?

The pressure is up to annotateas many genes as possible

Annotation based on weakhomology may not be

biologically meaningful:

Evolution is driven by re-useof genes, and selection ofnovel functionsGenes may evolve for niche-

adapted functionsThus, gene similarity may notmean function similarity

Genes are not invented: they arestolen and misused.

Organims A with well-characterizedvirulence factor Vir encoded by virA

Database entry

query gene X

query gene YDatabaseentry of virAannotated:virulencegenegene X has 65%

similarity to virA.gene X is annotated:

'putative virA gene'

gene Y has 50%similarity to gene X.gene Y is annotated:'putative virA gene'

Genetic similarity X, Y, A:Gene A

::: :::: :: : 65%

Gene X

:: :::: :::: 50%

Gene Y

Functional similarity cannot be stated unlessexperimentally proven

'putativism' is spreadingthrough databases


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What do wewhat should we

call a virulence gene

Predicted virulence genes must be tested in a model,preferably in vivo.Should all genes leading to attenuation after mutation be

called virulence genes?

Should all genes whose presence results in disease be

called virulence genes?

No: housekeeping genes are not virulence genes (AroA)even though their inactivation can result in attenuation

No: many of these are colonization factors (fim) orstructural genes (fla, LPS)


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Genes of importance to food pathogens

'true' virulence genes

(toxins, inv, vir, yop)

colonization genes

Humans

animals

ex vivo survival genes

can be tested in in vitromodelsto be tested in in vivomodels

must be tested in in vitromodelscan be targets for interventions

Structural,

house-keepinggenes

Introducing a standardized nomenclature or classification in

electronic databases would greatly enhance their use.


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How much does a genome tell?Examples from the past

E. coli: genome/plasmid sequences ofapathogenic K12 and of pathogenic O:157

Genomes compared, no striking findings about

O:157 virulence properties Confirmation of prior knowledge on virulence

Could we have predicted the rise of O:157?

combination of virulence factors (hly, eae, stx)on plasmid is striking

acid tolerance genes seem conserved in K12

properties as food pathogen would probably nothave been predicted


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Microbial Genome Sequences

All are known pathogens (emerging pathogens)but no species lurking in the dark

Available sequences accelerate ongoingresearch and confirm hypotheses but have hadlimited spin-off in new directions of food safety

Experimental approach to test potential genecandidates are essential that specifically testproperties of importance to pathogens


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Virulence Factors of Pathogenic Baceria

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