What’s a Legionnaire? We Didn’t Know Either.

1. What’s a Legionnaire?We Didn’t Know Either. courtesy of http://legion.org

2. Bob Courtesy of www.vodcasonic.com/dylan

3. Bob’s story • Bob went to the Legionnaires’ Conference • in 1976 • 3 days later Bob felt sick and went to the Doctor’s • What Bob caught was Legionnaires’ disease

4. Legionnaires’ Disease • Legionnaires’ Disease is a severe form of pneumonia • 1976 convention was the first major outbreak • 221 cases of pneumonia • 34 died • Dr. Joseph McDade, from CDC, isolated bacterium from lung specimen

5. Current Trends • 8,000 – 18,000 cases reported in the U.S. per year • This number is only 5-10% of estimated occurrence

6. Legionella pneumophila • Causes Legionnaires’ disease, a severe form of pneumonia • Lives naturally in freshwater streams, rivers, lakes • Optimal temperature: 35-37 degrees Celsius • Infects and reproduces in some protozoa and mammals Courtesy of diseaseworld.com/legion.htm

7. How Do Humans Get This Bacteria? • Inhalation of water vapor containing L. pneumophila • Man-made habitats of proliferation • Cooling towers (in air conditioning units) • Water distribution systems (showerheads) • Saunas Courtesy of www.q-net.net.au/~legion

8. Bob’s Symptoms • Early symptoms (24-48 hrs) • Fever, yucky feeling, muscle ache, loss of appetite, headache • Mild cough progressing to occasional phlegm • Late symptoms • Chest pain, vomiting, diarrhea, nausea, abdominal pain, neurological symptoms • Cough with phlegm containing blood

9. High Risk Patients • Elderly • Smokers • Those with repressed immune systems Courtesy of www.vodcasonic.com/dylan

10. A day (or more) in the life of L. pneumophila water droplets Attachment to host cell L. pneumophila Human lungs Entry via pseudopod coil Nuclear apoptosis begins Evasion of lysosome Replication Cell Death and release of bacteria into the extracellular fluid

11. WHAT’S NEW in the day (or more) in the life of L. pneumophila water droplets Attachment to host cell L. pneumophila Human lungs Entry via pseudopod coil Nuclear apoptosis begins Evasion of lysosome Replication Cell Death and release of bacteria into the extracellular fluid

12. Getting in the door • Attachment of opsonin • Complement receptors fix to MOMP • Phagocytosis of L.pneumophila is SPECIAL…

13. Oooooh, pseudopod coil Courtesy of Infectious Diseases, 1990

14. Signal Transduction during Legionella pneumophila Entry into Human Monocytes Patricia Y. Coxen, James T Summersgill, Julio A. Ramirez, AND Richard D. Miller Department of Immunology and Division of Infectious Disease, Department of Medicine, University of Louisville, Louisville, Kentucky Infection and Immunity, June 1998

15. Role of Kinases in Bacterial Entry • Kinase inhibitors inhibit host cell invasion in dose dependant manner • Do not inhibit attachment to host cell – once removed, the bacteria invade normally • Do not affect cellular replication Courtesy of Infection and Immunity, 1998

16. Proteins get Phosphorylated • Specific bands showed increased phosphorylation upon infection • Baseline-level fluorescence in kinase inhibited cells • Phosphorylation is essential to bacterial entry Courtesy of Infection and Immunity, 1998

17. Actin gets Polymerized • Cellular actin levels correlate with phagocytic entry of L. p. • Kinase inhibitors also inhibit actin polymerization • Role of kinase signaling in this event Courtesy of Infection and Immunity, 1998

18. Localization of Actin Courtesy of Infection and Immunity, 1998

19. WHAT’S NEW in the day (or more) in the life of L. pneumophila water droplets Attachment to host cell L. pneumophila Human lungs Entry via pseudopod coil Nuclear apoptosis begins Evasion of lysosome Replication Cell Death and release of bacteria into the extracellular fluid

20. Activation of Caspase 3 during Legionella pneumophila- Induced Apoptosis Lian- Yong Gao and Yousef Abu Kwaik University of Kentucky Chandler Medical Center Infection and Immunity, September 1999

21. L. Pneumophila induces apoptosis • Apoptosis of macrophages after just 2-3 hours post infection • Biphasic model of cell death by L. p. • Apoptosis: low levels of L. p., early infection • Necrosis: post exponential growth, late stage infection, results from cytotoxicity

22. Caspase Cascade Leads to Apoptosis • L. p. activates caspase 3 in macrophages • Leads to nuclear apoptosis Courtesy of Infection and Immunity, 1999

23. How Does L. p. Activate Caspase 3? • Can occur outside of macrophage • Three proposals • Translocation of factor into cell through DOT/ICM channel • Factor bind to death receptor on cell surface • Bacteria binds to death receptor with factor on surface Courtesy of Pac Land, Pacman Web Ring

24. Why Apoptosis So Early In Infection? • Four Theories • Blocks endocytic fusion activity • Inhibits anti-bacterial mechanisms • Method of release after reproduction in host cell • Reduces inflammation

25. WHAT’S NEW in the day (or more) in the life of L. pneumophila water droplets Attachment to host cell L. pneumophila Human lungs Entry via pseudopod coil Nuclear apoptosis begins Evasion of lysosome Cell Death and release of bacteria into the extracellular fluid Replication

26. Early Events in Phagosome Establishment Are Required for Intracellular Survival of Legionella pneumophila L.A. Wiater, K. Dunn, F.R. Maxfield, and H.A. Shuman College of Physicians and Surgeons, Columbia University Infection and Immunity, September 1998

27. Mutations in dotA and icm Row 1 • Wild-type L.p. • after 6 hrs., no PLF Row 2 • Mutated icmX • after 0.5, PLF Row 3 • Mutated dotA • after 6 hrs., PLF Row 4 • Mutated icmE • after 6 hrs., PLF courtesy of Infection and Immunity, 1998

28. Just incase that wasn’t clear . . . Wild-Type Wild-Type dotA dotA icmB icmX icmE icmX icmE icmU icmR icmU icmB icmR 0.5 hrs. 6 hrs. Adapted from Infection and Immunity, 1998

29. What’s This All Mean? • dotA and icm genes play a role in dodging PLF • no PLF increases life-span of L.p. within host • may lead to more favorable conditions for replication phagosome lysosome

30. WHAT’S NEW in the day (or more) in the life of L. pneumophila water droplets Attachment to host cell L. pneumophila Human lungs Entry via pseudopod coil Nuclear apoptosis begins Evasion of lysosome Cell Death and release of bacteria into the extracellular fluid Replication

31. Different fates of Legionella pneumophila pmi and mil mutants within macrophages and alveolar epithelial cells L.Y. Gao, B.J. Stone, J.K. Brieland & Y.A. Kwaik Deparment of Microbiology and Immunology, University of Kentucky Chandler Medical Center Unit for Laboratory Animal Medicine, University of Michigan Microbial Pathogenesis, September 1998

32. Messages from Mutants • 121 mutant L. pneumophila could not kill macrophages • 30/121 mutant L. pneumophila could not kill alveolar epithelial cells • 91/121 mutant L. pneumophila could kill alveolar epithelial cells Macrophages Alveolar epithelial cells wild type Adapted from Microbial Pathogenesis, 1998

33. Mice experiments Mutant 1 cannot kill macrophages cannot alveolar epithelial cells Mutant 2 cannot kill macrophages can kill alveolar epithelial cells injected into trachea injected into trachea 24 hrs 24 hrs No replication, bacteria killed Bacterial replication similar to wild-type

34. Why can’t some mutant L. pneumophila replicate in host cells? • If you can’t recruit rough ER, you can’t replicate • Wild type bacteria in phagosome • Many ribosomes • Mutant bacteria in phagosome • No ribosomes Courtesy of Microbial Pathogenesis, 1998

35. What does all this mean? • Most other research has focused on macrophages • Infection of alveolar epithelial cells is different • Infection of alveolar epithelial cells leads to replication of L. pneumophila in vivo • Recruitment of rough ER is needed for replication • It is important to study alveolar epithelial cells

36. “What About Bob?” • Bob presents symptoms that suggest pneumonia

37. X-ray, Day 1 Courtesy of Lattimer, 1981

38. DNA probe Courtesy of Lattimer, 1981

39. X-ray, Post-treatment Courtesy of Lattimer, 1981

40. Future Research Receptors? Attachment to host cell water droplets L. pneumophila Human lungs Entry via pseudopod coil Nuclear apoptosis begins Evasion of lysosome Replication Cell Death and release of bacteria into the extracellular fluid

41. Future Research Attachment to host cell water droplets L. pneumophila Human lungs Which proteins? Entry via pseudopod coil Nuclear apoptosis begins Evasion of lysosome Replication Cell Death and release of bacteria into the extracellular fluid

42. Future Research Attachment to host cell water droplets L. pneumophila Human lungs Death Receptors? Entry via pseudopod coil Nuclear apoptosis begins Evasion of lysosome Replication Cell Death and release of bacteria into the extracellular fluid

43. Future Research Attachment to host cell water droplets L. pneumophila Human lungs Entry via pseudopod coil Nuclear apoptosis begins Big Fish? Evasion of lysosome Replication Cell Death and release of bacteria into the extracellular fluid Courtesy of Printmaster Online Graphics

44. Future Research Attachment to host cell water droplets L. pneumophila Human lungs Entry via pseudopod coil Nuclear apoptosis begins Why aren’t they friends? Evasion of lysosome Replication Cell Death and release of bacteria into the extracellular fluid

45. Future Research Attachment to host cell water droplets L. pneumophila Human lungs Entry via pseudopod coil Nuclear apoptosis begins Evasion of lysosome Replication Cell Death and release of bacteria into the extracellular fluid Epithelial Cells?

46. Future Research Water treatment Why us? Receptors? Attachment to host cell water droplets L. pneumophila Human lungs Death Receptors? Which proteins? Immunizations Entry via pseudopod coil Nuclear apoptosis begins Big Fish? Why aren’t they friends? Evasion of lysosome Replication Cell Death and release of bacteria into the extracellular fluid Epithelial Cells?

47. Acknowledgements • Dr. DebBurman • Lisa Herron • Shea Hogan and Annette Sieg • Bob