1. As late as the 1800s, causes and cures of TB remained mysterious

2. Then along came Robert Koch December 11, 1843- May 27, 1910

3. You may not remember me but I’ll bet you’ve heard of my Postulates

4. What were Koch’s Postulates?

5. I developed tests to determine whether an infectious agent causes a given disease

6. 1.The microorganism must be found in abundance in all organisms suffering from the disease, but not in healthy organisms.

7. 1.The microorganism must be found in abundance in all organisms suffering from the disease, but not in healthy organisms. 2.The microorganism must be isolated from a diseased organism and grown in pure culture

8. 1.The microorganism must be found in abundance in all organisms suffering from the disease, but not in healthy organisms. 2.The microorganism must be isolated from a diseased organism and grown in pure culture 3.The cultured microorganism should cause disease when introduced into a healthy organism.

9. 1.The microorganism must be found in abundance in all organisms suffering from the disease, but not in healthy organisms. 2.The microorganism must be isolated from a diseased organism and grown in pure culture 3.The cultured microorganism should cause disease when introduced into a healthy organism. 4.The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

10. In 1882 while working in Berlin I discovered the tuberculosis bacteria and the means of culturing it I know it’s a hard act to follow, but I wasn’t done yet

11. Nobelprize.org That was sort of a big deal The Nobel Prize in Physiology or Medicine 1905

12. Mycobacterium Tuberculosis (scanning EM) Mycobacterium Tuberculosis on Lowenstein-Jensen medium Images from CDC Aren’t I pretty? Our star: Mycobacterium Tuberculosis

13. Mycobacterium Tuberculosis (scanning EM) Mycobacterium Tuberculosis on Lowenstein-Jensen medium Images from CDC But I am very hard to grow

14. Mycobacterium Tuberculosis (scanning EM) Mycobacterium Tuberculosis on Lowenstein-Jensen medium Images from CDC What’s the doubling time= cell cycle length Of our favorite lab bacterium E. coli ?

15. Mycobacterium Tuberculosis (scanning EM) Mycobacterium Tuberculosis on Lowenstein-Jensen medium Images from CDC I am an Obligate aerobe and grow very slowly 15-20 hour doubling time I require 6-8 weeks to grow on plates!

16. Mycobacterium Tuberculosis (scanning EM) Mycobacterium Tuberculosis on Lowenstein-Jensen medium Images from CDC I am ‘acid-fast” so you can ID me using special stains

17. Mycobacterium tuberculosis (stained red) in tissue (blue). Acid-fastness refers to resistance to decolorization by acids during staining procedures The most common staining technique is Ziehl-Neelsen stain, in which the bacteria are stained bright red. Mycobacterium tuberculosis (stained red) in sputum Red is my favorite color!

18. What are the three “domains” of living things?

19. Here’s the M. tuberculosis family tree E. coli Staph. aureus TB bug Genome Research 12, 1080-1090 (2002)

20. We’re distant cousins You E. coli Staph. aureus TB bug Genome Research 12, 1080-1090 (2002)

21. Mycobacterial relatives of the TB bug cause other diseases Mycobacterium leprae causes Hansen’s disease (leprosy) Mycobacterium africanum: causes up to one-third of TB cases in West Africa. Milder in non-immunocompromised patient

22. Mycobacterial relatives of the TB bug cause other diseases Mycobacterium bovis. Causes Bovine TB. Killed many farm animals inn first half of 20th century. Destroyed by pasteurization. Causes TB in developing world. Mycobacterium avium. Causes TB in birds. Also affects HIV patients and other immunocompromised people

23. Left untreated, a person with active TB will infect 10-15 other people per year World Lung Foundation (2008) and http://pathport.vbi.vt.edu/pathinfo/pathogens/Tuberculosis_2.html Remember how it usually starts

24. TB is super cool because it lives INSIDE human cells!

25. It takes advantage of our bodies own “first-responders” Macrophage

26. Macrophages are responsible for phagocytosis of bacteria and other foreign cells and for activation of other immune cells Macrophage Bacteria

27. Can anyone define Phagocytosis?

28. www.nyas.org/.../ images/carroll3_small.jpg Macrophages love to “eat” bacteria Yum-yum!

29. Macrophages bind to mannosyl oligosaccharide residues on the bacterial cell surface using their cell surface mannose receptors Yum-yum! Clin Microbiol Rev. 2003 July; 16(3): 463–496.

30. Normally ingested bacteria and other things get sent to the lysosome for destruction by low pH and proteolysis bacterium Uh oh! Ahhhhhhh…..

31. What is the normal function of the lysosome?

32. But the TB bacterium has a better idea-- It remodels the endosome to be its new home! Very cozy

33. Avoiding the lysosome also reduces presentation of bacterial peptides to the immune system by the MHC proteins Very cozy

34. How does it do that? Let’s look for a mutant bacteria that can’t avoid destruction! Very cozy

35. How does it do that? Let’s look for a mutant bacteria that can’t avoid destruction!

36. Here’s the bacterial protein required— mutate it and Mycobacteria end up in an acidic lysosome PknG!!

37. PknG is a Serine-threonine Kinase! But wait…. Bacteria don’t have them!

38. Even cooler, it has other Domains found in NO Other kinases One may make it re-dox regulated

39. But wait— PknG is a bacterial protein. How does it alter Events inside the macrophage?

40. Bacteria Inject proteins INTO Our cells!! PknG is One such protein

41. Now we need To identify its Cellular targets!

42. The infected macrophage calls for help, generating a granuloma www.nyas.org/.../ images/carroll3_small.jpg

43. Let’s look at that in a bit more detail

44. Infected macrophages send signals that recruit nearby lymphocytes

45. Uninfected macrophages and Lymphocytes surround the infected cells

46. They encase the infected cells in a “cage” of “extracellular matrix” proteins where they can stay for years!

47. We’re actually quite good at keeping TB at bay only one-third of exposed patients will become infected and only 3-5% develop clinical TB in the first year Clin Microbiol Rev. 2003 July; 16(3): 463–496.

48. We can harness the immune response To help fight TB Albert Calmette Camille Guérin

49. We can harness the immune response To help fight TB Bacillus Calmette-Guérin = BCG! Albert Calmette Camille Guérin

50. Bacillus Calmette-Guérin = BCG! An “attenuated” strain of M tuberculosis that does not Cause disease but can stimulate the immune response http://www.cheng.cam.ac.uk/

51. Bacillus Calmette-Guérin = BCG! Interestingly, all “attentuated” strains have a common deletion In the genome, removing at least one known “virulence” gene Clin Microbiol Rev. 2003 July; 16(3): 463–496.

52. Bacillus Calmette-Guérin = BCG! BCG is clearly effective against miliary TB and TB meningitis But its effectiveness against pulmonary TB is controversial http://www.cheng.cam.ac.uk/

53. New attenuated strain vaccines and recombinant versions of BCG are now in Phase II and Phase III clinical trials Image from University of Oxford

54. Even without vaccination, in most otherwise healthy people the immune system keeps TB at bay

55. But if the immune system is compromised by malnutrition, HIV or old age….

56. What happens next and why does TB Kill people?

57. There is still much to be learned! “What makes M. tuberculosis virulent? Unfortunately, there is no simple answer yet, despite the knowledge obtained in the last 100 or more years” Clin Microbiol Rev. 2003 July; 16(3): 463–496.

58. We do know certain things Clin Microbiol Rev. 2003 July; 16(3): 463–496. 1.Uncontrolled M. tuberculosis growth is associated with extensive lung damage that ultimately causes death by suffocation.

59. We do know certain things Clin Microbiol Rev. 2003 July; 16(3): 463–496. 1.Uncontrolled M. tuberculosis growth is associated with extensive lung damage that ultimately causes death by suffocation. 2.Inflammatory responses are important in pathogenesis in brain and bone.

63. Here’s InhA with the inhibitor bound!