Targeting bacterial virulence mechanisms for antibiotic development: challenges and opportunities

1. Targeting bacterial virulence mechanisms for antibiotic development: challenges and opportunities Stephen Lory Harvard Medical School, Boston

2. History of antibiotic discovery

4. Sales in Billion €

5. Antibiotic resistance follows their introduction into therapy Rafael Cantón, 2006

7. Genome derived, target based strategy for discovery of novel drugs Essential targets: Required under all conditions Conditional essential: Virulence factors Mutability and depleteability Fitness in an infection model “Genome” Broad vs. narrow spectrum Bioinformatics Interspecies complementation

8. Genome derived, target based strategy for discovery of novel drugs Essential targets: Required under all conditions Conditional essential: Virulence factors Mutability and depleteability Fitness in an infection model “Genome” Broad vs. narrow spectrum Bioinformatics Interspecies complementation

9. Molecular Koch’s postulates (Proof that a gene product is an essential virulence factor) The pathogenic trait should be associated with the pathogenic members of the genus, species or strains. Inactivation of the gene associated with the pathogenic trait should result in a measurable loss of pathogenicity or virulence. Reversion, allelic replacement (complementation) of the mutated gene should restore pathogenicity. Koch’s postulates (Proof that a specific organism caused a disease) The organism occurs in every case of the disease and under circumstances and conditions which can account for the pathological changes and clinical course of the disease. The organism occurs in no other disease as a fortuitous and non-pathogenic colonizer. The organism is isolated, grown in pure culture and it can induce disease anew.

10. New approaches towards drug discovery Model organisms: Pseudomonas aeruginosa - extracellular pathogen can be genetically manipulated Chlamydia pneumoniae - obligate intracellular parasite - no genetic system

11. Infections caused by Pseudomonas aeruginosa VAP Neutropenia (malignancy, immune suppression) Burns Ocular, ear infections Prolonged hospitalization Acute- invasive, cytotoxic Cystic Fibrosis E n v i r o n m e n t a l R e s e r v o i r Chronic - biofilms

12. Chlamydia pneumoniae Causative agent of acute and chronic respiratory tract infections Bronchitis Pneumonia Association of with diseases of unknown etiology Atherosclerosis Multiple sclerosis Reactive arthritis Lung cancer Macular degeneration Alzheimer’s disease Asthma Schizophrenia Stroke

13. Functional assays Biological relevance Drug screen Inhibition of target activity restores normal growth Screen chemical inhibitors of target Drug optimization Drug discovery based on expression of virulence factors in yeast Target selection Normal growth Dividing yeast cells Effect on viability and infectivity Growth inhibition Identification of targets ORFs encoding potential targets Bacterial target Effect on target activity

14. Two mechanism of killing of mammalian cells by Pseudomonas aeruginosa P.aeruginosa ExoS ExoS ExoA Ras inactive Ras NAD+ ExoA* NAD+ ExoS EF-2 EF-2 (Active) (Inactive) FAS (cofactor) Protein synthesis Protein synthesis Human epithelial lung cells

15. Chlamydia developmental cycle

16. The type III secretion system Chlamydia in inclusions P. aeruginosa attached to an epithelial cell

17. System for expression of bacterial genes in yeast pGAL pGAL pGAL pGAL Yeast viability gfp CP orf gfp CP orf CEN6 2µ Mechanism of yeast killing Use yeast to screen for inhibitors CP orf CP orf CEN6 2µ High copy Low copy/ Integrated (LEU2) S. cerevisiae W303 S. cerevisiae PDR1-, PDR3-

18. Lethality of P.aeruginosa protein expressed in yeast 505 essential protein and virulence factors screened in yeast- 9 were lethal

19. The two activities of ExoS GTPase activation ADP-ribosyl transferase

20. The ADP-ribosyl transferase activity of ExoS is responsible for yeast lethality

21. Over-expression of the target leads to resistance to killing

22. Ras is the target of ExoS in yeast

23. Compounds capable of rescuing yeast cells from killing by ExoS Compound library screened: 56,000 molecules

24. Exosin is a competitive inhibitor of the ExoS-catalyzed ADP-ribosylation reaction

25. Analogues of Exosin protect cells from ExoS

26. Exosin analogues

27. Inhibitors of ExoS protect CHO cells from killing by P. aeruginosa Dead Live Staining with 7-Amino- actinomycin D ExoU

29. Summary (I) Expression of a number of P. aeruginosa essential genes leads to a lethal phenotype in yeast Over-expression of candidate yeast genes can be used to identify the target of cytotoxic proteins A screen of compound libraries can be used to reverse the lethal phenotype in yeast Active compounds protect mammalian cells from the cytotoxic activity of a P.aeruginosa toxin Yeast =Model for human infection?

30. Chlamydia developmental cycle  Genome sequences of most strains available  No means of genetic manipulation  No virulence factors identified to date

31. The genetic organization of the type III secretion system in selected bacteria

32. 62 62 62 62 62 62 124 123 123 123 123 124 186 185 185 185 184 185 248 247 247 247 245 246 310 308 309 309 307 308 372 370 371 371 369 370 399 397 398 398 418 421 Amino-acid Sequence Alignment and Comparison of SctW Homologs Identity 100% 67% 64% 64% 45% 45%

33. Surface localized proteins of Chlamydia pneumoniae

34. Expression of C. pneumoniae genes in yeast GFP Vector GFP-CP0358p (prot. phosphatase) GFP-CopN GFP-CP0679p (S/T kinase) GFP-CP0833p (TTSS effector) GFP-CP1062p (TTSS effector) High copy GFP-CopN GFP-CP1062p Low copy Integrative GFP-CopN CopN High copy (non-GFP) C. trachomatis GFP-CopN Y. enterocolitica GFP-YopN P. aeruginosa GFP-PopN High copy

35. Expression of C. pneumoniae CopN protein blocks yeast cell division GFP-CopN GFP

36. gfp dapi merge % of cells * Strain Time (h) No bud Small bud Large bud GFP-CopN Vector 22 30 48 2 h 4 h 77 14 9 90 5 6 h 4 50 2 h 19 31 30 4 h 43 27 26 52 22 6 h Expression of CopN in yeast results in cells with large buds and abnormally positioned nuclei

37. Expression of CopN disrupts microtubules and causes G2/M cell cycle arrest gfp anti--tubulin merge CopN Vector No detectable spindle in CopN expressing cells 1N 2N 0 h Flow cytometry Normal spindle detected in GFP control cells (Integrated GFP-CopN or GFP vector) 2 h 4 h

38. Expression of CopN in mammalian cells GFP-CopN e g f h Vector a a c d b Anti-tubulin GFP DAPI Overlay Vector CopN 2N 4N 0 h 10 h Cell cycle arrest at G2/M 12.5 h 15 h 17.5 h

39. High throughput screen of a compound library for the rescue of the CopN-induced growth defect in yeast Screen: Monitor growth (OD at 600 nm) of S. cerevisiae (PDR1-, PDR3-)in 384 well microtiter dishes following induction of CopN expression. Library: 50,000 compounds Primary hits: 28 compounds Validated hits: 12 compounds Available compounds: 8 compounds Confirmed: 2 compounds

40. 0433YC1 0433YC2

42. Inhibitors of CopN block the formation of large inclusions in infected cells CP0433YC1 DMSO Cm CP0433YC2

43. Role of Type III secretion system in Chlamydia infection

44. Conclusions (II) Expression of certain Chlamydia proteins leads to lethality in east CopN function by disrupting microtubules and causing a cell division arrest inG2/m phase Compounds that rescue yeast from CopN lethality prevent infection of mammalian cells by Chlamydia Chemical knockouts vs. genetic knockouts Yeast as a tool for drug discovery

45. Acknowledgments Chlamydia Jin Huang Cammie Lesser ExoS Anthony Arnaldo Igor Stagljar