Identification of Novel Virulence-Associated Genes via Genome Analysis of Hypothetical Genes

1. Identification of Novel Virulence-Associated Genes via Genome Analysis of Hypothetical Genes Sara Garbom, Åke Forsberg, Hans Wolf-Watz, and Britt-Marie Kihlberg 2004, Infection and Immunity, v. 72 pp. 1333-1340

2. Hypothesis • IF{Genomes of pathogenic bacteria are reduced to smallest set needed for growth in an animal host}

3. Hypothesis • IF{Genomes of pathogenic bacteria are reduced to smallest set needed for growth in an animal host} • THEN{Genes expressed in vivo and shared by pathogens may be “amenable” targets for antibacterial agents}

4. Virulent WT Traditional Antibiotic Dead WT Virulent Mutant Why target in vivo expressed virulence factors?

5. Virulent WT Traditional Antibiotic Dead WT Virulent Mutant Why target in vivo expressed virulence factors?

6. Virulent WT Virulent WT Traditional Antibiotic Virulence-specific Antibiotic Dead WT Virulent Mutant Avirulent Mutant Why target in vivo expressed virulence factors?

7. Method: • In silico: Find novel putative virulence genes through comparative analysis

8. Method: • In silico: Find novel putative virulence genes through comparative analysis • In vitro: Assay genes for essentiality to survival

9. Method: • In silico: Find novel putative virulence genes through comparative analysis • In vitro: Assay genes for essentiality to survival • In vivo: Assay genes for virulence in an animal model

10. Goal: • “the rapid emergence of multiply [antibiotic] resistant bacterial strains…demands the development of new antibacterial agents by engaging strategies that specifically counteract the development of resistance”

11. In silico: Finding novel putative virulence genes through comparative analysis • Gathered genes of unknown function from a pathogenic organism • “Conserved hypotheticals” or “unknown”

12. In silico: Finding novel putative virulence genes through comparative analysis • Gathered genes of unknown function from a pathogenic organism • “Conserved hypotheticals” or “unknown” • Compared these genes to those of other pathogens

13. In silico: Finding novel putative virulence genes through comparative analysis • Gathered genes of unknown function from a pathogenic organism • “Conserved hypotheticals” or “unknown” • Compared these genes to those of other pathogens • Considered all genes found in all pathogens “virulence-associated genes (vag)”

14. “With the the exception of Y. pestis, all are causitive agents of chronic disease in humans.”

17. Classified vagA – vagQ “[NCBI nr] database indicated that all of the vag genes exhibited homologous sequences in at least 35 other microorganisms… nine had products that also exhibited similarity [to human proteins].”

18. Control: In vivo analysis & in silico comparison • 99 in vivo expressed genes • STM (signature tagged mutagenesis) and “selected capture of transcribed sequences”

19. Control: In vivo analysis & in silico comparison • 99 in vivo expressed genes • STM (signature tagged mutagenesis) and “selected capture of transcribed sequences” • Compared to (same) 6 genomes

20. Control: In vivo analysis & in silico comparison • 99 in vivo expressed genes • STM (signature tagged mutagenesis) and “selected capture of transcribed sequences” • Compared to (same) 6 genomes • 5 conserved genes classified as vir genes • Also conserved among many bacteria • No human homologues

21. In vitro: Assaying genes for essentiality to survival and virulence • Mutagenized conserved genes • Insertion mutagenesis

22. In vitro: Assaying genes for essentiality to survival and virulence • Mutagenized conserved genes • Insertion mutagenesis • Analyzed cytotoxicity with HeLa cells

23. In vitro: Assaying genes for essentiality to survival and virulence • Mutagenized conserved genes • Insertion mutagenesis • Analyzed cytotoxicity with HeLa cells • Measured Yop secretion • Yersinia outer proteins • Known virulence factors • Encoded on a plasmid • Belonging to a type III secretion system

24. Hypothesized: Unchanged in vitro growth patterns • 3 mutations were lethal

25. Hypothesized: Unchanged in vitro growth patterns • 3 mutations were lethal • 14 remaining mutants • vagE - impaired growth / uncharacteristic morphology / delayed cytotoxic response* • vagH - lowered Yops secretion • vagI - lowered Yops secretion but no loss of cytotoxicity

26. Hypothesized: Unchanged in vitro growth patterns • 3 mutations were lethal • 14 remaining mutants • vagE - impaired growth / uncharacteristic morphology / delayed cytotoxic response* • vagH - lowered Yops secretion • vagI - lowered Yops secretion but no loss of cytotoxicity • 11 “indistinguishable from the wild type”

27. In vivo: Assaying genes for virulence in an animal model • Infected model organisms with mutagenized strains • Oral infection of mice

28. In vivo: Assaying genes for virulence in an animal model • Infected model organisms with mutagenized strains • Oral infection of mice • Lethal vs. non-lethal/delayed-lethal classification of virulence • WT killed 50% mice at 107 CFU/mL in 5-8 days • “Attenuated” strains were not lethal at same dose

29. Hypothesized: Viable targets would be attenuated for virulence • 5 were virulent Control: • 2 were virulent

30. Hypothesized: Viable targets would be attenuated for virulence • 5 were virulent • 9 were attenuated • All 3 non-WT like (in vitro) mutants were attenuated Control: • 2 were virulent • 3 were attenuated

31. In vivo: Assaying genes for virulence in an animal model (continued) • In-frame deletion mutagenesis • Prevent downstream effects of insertion mutagenesis

32. In vivo: Assaying genes for virulence in an animal model (continued) • In-frame deletion mutagenesis • Prevent downstream effects of insertion mutagenesis • Meant to verify results of insertion mutagenesis

33. Hypothesized: Viable targets would still be attenuated for virulence • 1 deletion mutant could not be made

34. Hypothesized: Viable targets would still be attenuated for virulence • 1 deletion mutant could not be made • 3 mutants regained virulence • Genes in virulence-associated operons

35. Hypothesized: Viable targets would still be attenuated for virulence • 1 deletion mutant could not be made • 3 mutants regained virulence • Genes in virulence-associated operons • 5 mutants remained attenuated • 1 of these having exhibited non-WT like growth (in vitro)

36. Hypothesized: Viable targets would still be attenuated for virulence • 1 deletion mutant could not be made • 3 mutants regained virulence • Genes in virulence-associated operons • 5 mutants remained attenuated • 1 of these having exhibited non-WT like growth (in vitro) • 4~5 in vivo-only virulence genes were successfully discovered Control: • 3 remain attenuated

37. 211 genes initially considered 99 genes initially considered Experimental Control

38. 211 genes initially considered 17 (8%) conserved across pathogens 99 genes initially considered 5 (5%) conserved across pathogens Experimental Control

39. 211 genes initially considered 17 (8%) conserved across pathogens 9 (4%) in or around virulence genes 99 genes initially considered 5 (5%) conserved across pathogens 3 (3%) in or around virulence genes Experimental Control

40. 211 genes initially considered 17 (8%) conserved across pathogens 9 (4%) in or around virulence genes 5 (2%) confirmed virulence genes 99 genes initially considered 5 (5%) conserved across pathogens 3 (3%) in or around virulence genes 3 (3%) confirmed virulence genes Experimental Control

41. Hypothesis • IF{Genomes of pathogenic bacteria are reduced to smallest set needed for growth in an animal host} • THEN{Genes expressed in vivo and shared by pathogens may be “amenable” targets for antibacterial agents}

42. Amenable(… • Traditional screening not possible

43. Virulent WT Traditional Antibiotic Dead WT Virulent Mutant Amenable(…

44. Virulent WT Virulent WT Virulence-specific Antibiotic Traditional Antibiotic Avirulent Mutant Dead WT Virulent Mutant Amenable(…

45. Amenable(… • Traditional screening not possible • Microarrays?

46. Amenable(… • Traditional screening not possible • Microarrays? • Targeting gene products isn’t as easy as in-frame deletion mutagenesis • …especially when human homologues exist for 4 out of 5 of the genes IDed

47. Amenable(… • Traditional screening not possible • Microarrays? • Targeting gene products isn’t as easy as in-frame deletion mutagenesis • …especially when human homologues exist for 4 out of 5 of the genes IDed • Response of normal human microflora unknown

48. Amenable(… • Traditional screening not possible • Microarrays? • Targeting gene products isn’t as easy as in-frame deletion mutagenesis • …especially when human homologues exist for 4 out of 5 of the genes IDed • Response of normal human microflora unknown …)

49. Conclusion • Genes responsible for virulence were identified • I’m “amenable” to calling the method a success

50. Why start with T. pallidium when Y. pestis was the organism of interest and Y. pseudotuberculosis was used for testing? • How would deletion mutagenesis of homologous genes in non-pathogens alter their growth? • How target-able were the products of the genes knocked out? • What’s the best way to assay target-ability of an uncharacterized gene product? • Was there any overlap between the set of vag genes and the control (vivo + silico) set?