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

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

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

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:  Gathered genes of unknown function from a pathogenic organism  “Conserved hypotheticals” or “unknown” Finding novel putative virulence genes through comparative analysis

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

13. In silico:  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)” Finding novel putative virulence genes through comparative analysis

14. OrganismDisease Treponema pallidumSyphilis Yersinia pestisBlack death Neisseria gonorrhoeaeGonorrhea Heliobacter pyloriPeptic ulcer disease Borrelia bugdoreferiLyme disease Streptococcus pneumoniae Pneumococcal meningitis Pneumonia “With the the exception of Y. pestis, all are causitive agents of chronic disease in humans.”

15. OrganismGenes remaining Treponema pallidum 211 Yersinia pestis Neisseria gonorrhoeae Heliobacter pylori Borrelia bugdoreferi Streptococcus pneumoniae

16. OrganismGenes remaining Treponema pallidum 211 Yersinia pestis 73 Neisseria gonorrhoeae Heliobacter pylori Borrelia bugdoreferi Streptococcus pneumoniae

17. OrganismGenes remaining Treponema pallidum 211 Yersinia pestis 73 Neisseria gonorrhoeae 17 Heliobacter pylori Borrelia bugdoreferi Streptococcus pneumoniae 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.  99 in vivo expressed genes  STM (signature tagged mutagenesis) and “selected capture of transcribed sequences” In vivo analysis & in silico comparison Control:

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

20.  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 In vivo analysis & in silico comparison Control:

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

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

23. In vitro:  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 Assaying genes for essentiality to survival and virulence

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

25.  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 Hypothesized: Unchanged in vitro growth patterns

26.  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” Hypothesized: Unchanged in vitro growth patterns

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

28. In vivo:  Infected model organisms with mutagenized strains  Oral infection of mice  Lethal vs. non-lethal/delayed-lethal classification of virulence  WT killed 50% mice at 10 7 CFU/mL in 5-8 days  “Attenuated” strains were not lethal at same dose Assaying genes for virulence in an animal model

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

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

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

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

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

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

35.  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) Hypothesized: Viable targets would still be attenuated for virulence

36.  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 Hypothesized: Viable targets would still be attenuated for virulence

37. ExperimentalControl  211 genes initially considered  99 genes initially considered

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

39. ExperimentalControl  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

40. ExperimentalControl  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

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

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

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?