Virulence properties of pathogens

Trudy M. Wassenaar Molecular Microbiology and Genomics Consultants Zotzenheim, Germany

What is a pathogen?
A pathogenic organism causes harm to the host it colonizes:
Parasites (worms, ticks) Other eukaryotes (Fungi, Cryptosporidium) Bacteria Viruses Border of life Prions

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

mutualist
symbiont

depends
benefits

depend e.g. lychen (algae/fungi)

benefit e.g. plants/Agromonas)

commensal
pathogen

benefits/neutral
suffers

neutral e.g. mammals/E.coli

benefit/neutral/depend

A bacterial pathogen requires virulence properties to cause disease; colonization properties for animal and/or human hosts; escape strategies to leave the host and survival properties 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?

Example: a foodborne pathogen

A food pathogen requires virulence properties to cause disease; colonization properties for animal and human hosts; survival properties in the environment and in food

What makes a pathogen? Virulence genes

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 (liberate
nutrients, evade immune defence, etc.); to enable spread (inducing diarrhea etc.)

By accident (N. meningitidis is mostly commensal, when causing

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

How to identify a pathogen?

Koch's postulates (1890)
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1890 the German physician and bacteriologist Robert Koch set out his celebrated criteria for judging whether a given bacteria is the cause of a given disease. Koch's criteria brought some much-needed scientific 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 Animal model not always available Not always possible Epidemiological or immunological evidence was added

Intermezzo
Nobel Prize for Medicine 2005: Barry Marshall and Robin Warren for the discovery of Helicobacter pylori
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From the press: "this bacterium as the cause for ulcers and 'other diseases" A causual relationship with gastric ulcers has been proven A 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 pylori a pathogen?

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

How to identify a virulence gene?

Molecular Koch's postulates (1988)
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1988 Stanley Falkow published a commentary article translating Koch's postulates into the era of molecular biology. He described the then common approaches to identify virulence genes and listed the conclusive evidence needed:

Genes are considered virulence genes if and only if: 1. 2. 3. 4. The phenotype or gene is associated with pathogenic strains/species Specific inactivation of the gene results in a measurable loss in virulence (attenuation) Reversion or allelic replacement of the mutated gene restors pathogenicity Or to 2,3: induction of specific antibodies neutralizes pathogenicity

How to identify a pathogen?

1. Association of genes to virulence:
Many so-called virulence genes are present in non-virulent species 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

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 manipulation A suitable model for virulence (animal model, in vitro models) False positives: Housekeeping genes have a role in nice adaptation. When mutated, attenuation occurs. E.g. aroA, LPS genes False negatives: Virulence strategies may be redundant. One gene mutation may be overcome by other systems.

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 product protective Depends on: Statistics and correct datasets (landmines!) Epitope mapping, effect of the host! (more landmines) False positives: False negatives:

The identification of virulence genes

Phenotypic evidence
inactivation of a gene results in loss of virulence or in attenuation complementation of a gene in an avirulent background enhances virulence

genes with significant homology to characterized virulence genes in other organisms

Comparative Genetics

Putative virulence genes

a gene present in (more) virulent strains and absent in avirulent strains antigenic variation, mutation patterns, are indicators for a role in pathogenesis or host-defense avoidence

Immunological evidence

antibodies against a gene product result in immunity from disease

infection results in an immune response directed to the gene product

Novel approach: comparative genetics

Identify virulence genes from genomes by significant homology to characterized virulence genes in other organisms Pitfalls: what is significant homology? do homologous genes have homologous functions in different organisms? 'putativism' is spreading through databases Functional similarity can not be stated unless experimentally proven
Organims A with well-characterized virulence factor Vir encoded to annotate The pressure is up by virA

as many genes as possible Database entry Annotation based on weak query gene X homology may not be query biologically meaningful: gene Y Database
entry of virA annotated: Evolution is driven by re-use virulence of genes, and selectionY has 50% gene of gene gene X has 65% similarity to gene X. novel functions similarity to virA. gene annotated: Genes may evolve for Y isvirA gene' nichegene X is annotated: 'putative 'putative virA gene' adapted functions Genetic similarity X, Y, A:

Thus, gene similarity may not mean function similarity Gene A

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

Genes are not invented: they are :: :::: :::: 50% stolen and misused. Gene Y

Gene X

What do we call a virulence gene what should we

Predicted virulence genes must be tested in a model, preferably in vivo. Should all genes leading to attenuation after mutation be called virulence genes?
No: housekeeping genes are not virulence genes (AroA) even though their inactivation can result in attenuation Should all genes whose presence results in disease be called virulence genes? No: many of these are colonization factors (fim) or structural genes (fla, LPS)

Genes of importance to food pathogens

can be tested in in vitro models to be tested in in vivo models

Humans animals (toxins, inv, vir, yop)

'true' virulence genes

colonization genes
ex vivo survival genes must be tested in in vitro models

Structural, housekeeping genes

can be targets for interventions

Introducing a standardized nomenclature or classification in electronic databases would greatly enhance their use.

How much does a genome tell? Examples from the past

E. coli: genome/plasmid sequences of apathogenic 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 not have been predicted

Microbial Genome Sequences

All are known pathogens (emerging pathogens) but no species lurking in the dark Available sequences accelerate ongoing research and confirm hypotheses but have had limited spin-off in new directions of food safety Experimental approach to test potential gene candidates are essential that specifically test properties of importance to pathogens