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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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How to identify a pathogen?
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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How to identify a pathogen?
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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How to identify a pathogen?
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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