Virulence properties of pathogens

1. Virulence properties of pathogens Trudy M. Wassenaar Molecular Microbiology and Genomics Consultants Zotzenheim, Germany

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

3. 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 • pathogensuffers benefit/neutral/depend Host Bacteria

4. A bacterial pathogen requiresvirulenceproperties to cause disease;colonizationproperties for animal and/or human hosts;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?

5. Example: a foodborne pathogen Virulence properties Colonization properties Survival properties A food pathogen requires virulence properties to cause disease; colonization properties for animal and human hosts; survival properties in the environment and in food

6. 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)

7. Not always possible Animal model not always available How to identify a pathogen? • Koch's postulates (1890) • search help • nIn 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 Epidemiological or immunological evidence was added

8. Intermezzo Nobel Prize for Medicine 2005: Barry Marshall and Robin Warren for the discovery 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 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'

9. How to identify a virulence gene? • Molecular Koch's postulates (1988) • search help • nIn 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: • 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

10. How to identify a pathogen? 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

11. 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.

12. 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:

13. Comparative Genetics genes with significant homology to characterized virulence genes in other organisms 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 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 Putative virulence genes infection results in an immune response directed to the gene product Immunological evidence antibodies against a gene product result in immunity from disease

14. Organims A with well-characterized virulence factor Vir encoded by virA • The pressure is up to annotate as many genes as possible • Annotation based on weak homology may not be biologically meaningful: • Evolution is driven by re-use of genes, and selection of novel functions • Genes may evolve for niche-adapted functions • Thus, gene similarity may not mean function similarity • Genes are not invented: they are stolen and misused. Database entry query gene X query gene Y Database entry of virA annotated: virulence gene gene Y has 50% similarity to gene X. gene Y is annotated: 'putative virA gene' gene X has 65% similarity to virA. gene X is annotated: 'putative virA gene' • 'putativism' is spreading through databases Genetic similarity X, Y, A: Gene A Functional similarity can not be stated unless experimentally proven ::: :::: :: : 65% Gene X :: :::: :::: 50% Gene Y 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?

15. What do we what 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) or structural genes (fla, LPS)

16. can be tested in in vitro models to be tested in in vivo models 'true' virulence genes (toxins, inv, vir, yop) Structural, house-keeping genes Humans animals colonization genes ex vivo survival genes must be tested in in vitro models can be targets for interventions Genes of importance to food pathogens Introducing a standardized nomenclature or classification in electronic databases would greatly enhance their use.

17. 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

18. 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