1. Legionella pneumophila Ubiquitous in soil and water causative agent of Legionnaire’s disease Only recognized within last 20 years

2. L. pneumophila Only affects immune-compromised Air-conditioning cooling towers: –good habitat –disseminate aerosolized bacterium

3. Parasitizes Amoebae Intracellular

4. Parasitizes Amoebae Invades Human Macrophages in Lung Intracellular

5. Parasitizes Amoebae Invades Human Macrophages in Lung Lives in Soil or Water Intracellular Extracellular

6. How does L. pneumophila adapt between extracellular and varying intracellular environments?

7. More about L. pneumophila

8. Amino Acids = sole source of Carbon and Energy

9. More about L. pneumophila Amino Acids = sole source of Carbon and Energy Cells starved of amino acids

10. More about L. pneumophila Amino Acids = sole source of Carbon and Energy Cells starved of amino acids Virulent Slower growth

11. Why become virulent when starved?

12. SICK Starvation: due to sick hosts

13. Why become virulent when starved? SICK Starvation: due to sick hosts Escape sick hosts

14. Why become virulent when starved? SICK Disperse via Soil or Water Starvation: due to sick hosts Escape sick hosts

15. Why become virulent when starved? SICK Disperse via Soil or Water Finds new host cells Starvation: due to sick hosts Escape sick hosts

16. Why become virulent when starved? SICK Disperse via Soil or Water Finds new host cells Invades healthy hosts Starvation: due to sick hosts Escape sick hosts

17. If signal to escape is low [amino acids]...

18. Low [amino acids] stringent response ?

19. If signal to escape is low [amino acids]... Low [amino acids] stringent response virulence and slower growth ? ?

20. In E. coli and M. xanthus: [amino acids] [(p)ppGpp] Response (less protein synthesis, sporulation)

21. Maybe in L. pneumophila: [amino acids] [(p)ppGpp] virulence and slower growth

22. Maybe in L. pneumophila: [amino acids] [(p)ppGpp] Question 1 virulence and slower growth

23. Maybe in L. pneumophila: [amino acids] [(p)ppGpp] Question 1 Question 2 virulence and slower growth

24. Focus of experiment [(p)ppGpp] virulence and slower growth Question 2

25. SICK Starvation: due to sick hosts Escape sick hosts

26. SICK Starvation: due to sick hosts Signal =  [amino acids]?

27. SICK Starvation: due to sick hosts Signal =  [amino acids]?  [(p)ppGpp] ?

28. SICK Starvation: due to sick hosts Signal =  [amino acids]?  [(p)ppGpp] Virulence ? ?

29. SICK Starvation: due to sick hosts Escape sick hosts Signal =  [amino acids]?  [(p)ppGpp] Virulence ? ?

30. SICK Starvation: due to sick hosts Escape sick hosts Signal =  [amino acids]?  [(p)ppGpp] Virulence ? ?

31. Experimental Concept [(p)ppGpp] Change in Growth Rate?

32. Experimental Concept [(p)ppGpp] Change in virulence? Change in Growth Rate?

33. Experimental Concept [(p)ppGpp] Change in virulence? Change in Growth Rate? Hammer et. al.(1999) “Co-ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp.” Molecular Microbiology. 33: 721-731.

34. Experimental Concept [(p)ppGpp] Change in virulence? Hypothesis: Induction of virulence Change in Growth Rate?

35. Experimental Concept Change in Growth Rate? Hypothesis: ? How are growth and virulence related?

36. Experimental Concept [(p)ppGpp] Change in virulence? Change in Growth Rate?

37. Experimental Concept Can measure growth directly (OD) Need virulence marker

38. FlaA - Virulence Marker Flagellar synthesis appears co-regulated with virulence

39. P flaA /gfp fusion flaA P flaA gfp P flaA

40. P flaA /gfp fusion When FlaA expressed by cell...

41. P flaA /gfp fusion When FlaA expressed by cell... Observe GFP

42. Experimental Concept [(p)ppGpp] Change in virulence marker expression? Change in Growth Rate?

43. How do we get (p)ppGpp?

44. P tac /relA fusion relA P tac (Using E. coli’s relA)

45. P tac /relA fusion relA P tac (Using E. coli’s relA) RelA expression inducible with IPTG

46. P tac /relA fusion relA P tac (Using E. coli’s relA) RelA expression inducible with IPTG RelA[(p)ppGpp]+IPTG

47. Strain Wt Chromosome + inducible RelA + GFP virulence reporter

48. Results: No IPTG 1 10.5 1.0 OD (log scale) Relative Fluorescence time (hrs) 0 24 0

49. Results: + IPTG 1 10.5 1.0 Relative Fluorescence time (hrs) 0 24 0 OD (log scale)

50. Results: Compare 1 10.5 1.0 Relative Fluorescence time (hrs) 0 24 0 OD (log scale)

51. Conclusions SICK Starvation: due to sick hosts Escape sick hosts Signal =  [amino acids]?  [(p)ppGpp] Virulence and Slow Growth ? YES

52. Stringent Response still candidate for virulence and growth control

53. Remaining Questions:

54. Other signals? Stringent Response = candidate for virulence and growth control

55. Remaining Questions: Other signals? Can you separate virulence and growth rate?

56. Remaining Questions: Other signals? Can you separate virulence and grwoth rate? Does L. pneumophila have the stringent response system? Stringent Response = candidate for virulence and growth control

57. Native relA?

58. BLAST unfinished genome with E. coli relA:

59. Native relA? –Sequence 1: E value = 0.0 Identities = 44%, Positives = 63%, Gaps = 0% BLAST unfinished genome with E. coli relA:

60. Native relA? –Sequence 1: E value = 0.0 Identities = 44%, Positives = 63%, Gaps = 0% –Sequence 2: E value = 3e-83 Identities = 34%, Positives = 52%, Gaps = 1% BLAST unfinished genome with E. coli relA:

61. Future Directions SICK Starvation: due to sick hosts Escape sick hosts Signal =  [amino acids]?  [(p)ppGpp] Virulence ? ?

62. Future Directions Does (p)ppGpp accumulate when low [a.a.]?

63. Future Directions relA knockout mutant Loss of virulence?Effect on growth rate?