Mycobacterium tuberculosis evades macrophage defenses by inhibiting plasma membrane repair

1. 091012 Johanna Eberhard Mycobacterium tuberculosis evades macrophagedefenses by inhibiting plasma membrane repair Maziar Divangahi, Minjian Chen, Huixian Gan, Danielle Desjardins, Tyler T Hickman, David M Lee, Sarah Fortune, Samuel M Behar & Heinz G Remold June 2009

2. Plasma membrane repair Based on exocytosisofendomembranes • Derivedfrom Golgi vesiclesorlysosomes • Calcium dependant • Calcium sensor: • Synaptotagmin-7 (Syt-7) • Neuronal Calcium sensor-1(NCS-1) Introduction

3. Mycobacterium tuberculosis Virulent: blocks phagosome maturation, leads to macrophage death by necrosis Avirulent: induces apoptosis of macrophages Is phagocytosed by pulmonary macrophages Introduction

4. Mtb infection Prostaglandin E2 (PGE2 ) Lipoxin A4 (LXA4) Introduction

5. Aim of study Analyse mechanisms by which virulent Mtb induces necrosis or inhibits apoptosis • Does Mtb induce plasma membrane disruptions? • Which inner membranes are involved in the repair? Introduction

6. Virulent Mtb causes persistent microdisruptions-FDX influx- Flow cytometry/ human macrophages • Less FDX influx in avirulent infected Mφ than in virulent infected Mφ Results

7. Recruitment of lysosomal membranes-Translocation of LAMP-1- Flow cytometry/ human macrophages After 12 h of infection: • Translocation of LAMP-1 stronger in H37Ra infected Mφ than in virulent infected Mφ Results

8. Recruitment of lysosomal membranes-Translocation of LAMP-1 and Syt-7- Flow cytometry/ human macrophages • Higher Syt-7 expression after infection with H37Ra not with H37Rv Results

9. Recruitment of Golgi derived membranes -Translocation of mannosidase II, not of BiP- Flow cytometry/ human macrophages • Higher translocation of Mannosidase II after avirulent infection • No BiP translocation, no involvement of ER derived membranes Results

10. Summary I virulent Mtb causes persistent membrane disruptions Membranes both from the lysosomal and Golgi compartments are involved in macrophage plasma membrane repair What is the function of calcium sensors in the recruitment of these membranes? Results

11. Calcium-sensors in plasma membrane repair-Silencing of Syt-7 expression- Immunoblot/ human macrophages Flow cytometry/ human macrophages • impaired LAMP-1 translocation but enhanced Mannosidase II, Posphatidylserine and annexin-1 expression Results

12. Inhibition of Golgi membrane recruitment- blocking withBrefeldin A- Flow cytometry/ human macrophages • Inhibition of mannosidase II, phosphatidylserine and annexin-1 translocation, but not of LAMP-1 Results

13. Calcium sensors in plasma membrane repair-Silencing of NCS-1 expression- Immunoblot/ human Mφ Flow cytometry/ human Mφ • Inhibition of the translocation of Golgi membranes, Phosphatetidylserine and annexin-1 Results

14. Inhibition of plasma membrane repair-FDX influx and Necrosis (7-AminoActinomycinD)- Flow cytometry/ human Mφ • Higher FDX influx after Syt-7 or NCS-1 silencing or treatment with BFA • Promotion of Mφ necrosis via Syt-7 or NCS-1 silencing Results

15. Summary II Recruitment of lysosomal and Golgi derived vesicels is critical in the repair of plasma membrane damage after Mtb infection and is recuired to prevent necrosis Is the upregulation of cAMP sufficient to trigger membrane repair? Results

16. Effect of cAMP upregulation on membrane repair-Acivation of adenylate cyclase with forskolin- Flow cytometry/ human Mφ • Greater translocation of LAMP-1 and Syt-7 • no effect on translocation of Golgi membranes Results

17. Effect of PGE2 on membrane repair Flow cytometry/ human Mφ • Reconstitution of plasma membrane repair after addition of PGE2 • No increase of Golgi membrane recruitment Results

18. Kinases involved in PGE2 activated membrane repair- Inhibition of PKA and PI(3)K - Flow cytometry/ human Mφ PKA inhibitor PI(3)K inhibitor • PGE2 requires PI(3)K activation Results

19. Effect of PGE2 on membrane repair- Infection of Mφ from Wt and Ptges -/- mice - Flow cytometry/ Ra infected mouse Mφ • No recruitment of lysosomal membranes in Mφ deficient in Prostaglandin E synthase • No effect on Golgi membrane recruitment Results

20. Summary III How do PGE2 and LXA4 affect the outcome of Mtb infection? • recruitment of lysosomal membranes is PGE2 dependent • recruitment of Golgi-derived membranes is independent of PGE2 Results

21. Influence of LXA4 and PGE on the death modality of Mφ in Mtb infecion Celldeathdetection ELISA / mouse Mφ • More necrosis and less apoptosis in Ptges deficient Mφ • More apoptosis and less necrosis in Alox5 deficient Mφ Results

22. Influence of LXA4 and PGE2 in control of Mtb growth Colonycount / mouse Mφ • enhanced Mtb growth in Ptges deficient Mφ • Lower growth of Mtb in Alox5 deficient Mφ Results

23. Innate control of infection in vivo-virulent infection of ko and wt mice - Pulmonarylavage /celldeathdetection ELISA/ mouse APCs • More apoposis in the cells of Alox5 deficient mice Results

24. Innate control of infection in vivo- adoptive transfer of Mφ into Rag1 deficient mice- Colonycount/ transferredmouse Mφ • Bacterial burden higher in Rag-1 deficient mice with Ptges deficient Mφ • lower with Alox5 deficient Mφ Results

25. Bacterial burden in spleen and lung Colonycount/ transferredmouse Mφ • Durable effect in spleen and lung Results

26. Summary IV • Transfer of Alox5 deficient Mφ restricts virulent Mtb replication in vivo • The effect is determined by the Mφ genotype and independent of adaptive immunity • The balance of PGE2 and LXA4 production affects the outcome of infection Is PGE2 involved in Syt-7 synthesis? Results

27. Induction of Syt-7 transcriptionby exogenous PGE2 Real –time PCR /uninfectedmouse Mφ • PGE2 induces Syt-7 expression in uninfected mouse Mφ Results

28. No Induction of LAMP-1 transcription Real –time PCR /uninfectedmouse Mφ • No effect of exogeneous PGE2 on LAMP-1 transcription Results

29. Induction of Syt-7 transcription in virulent Mtb infection Real –time PCR/ uninfectedandinfected Mφ • Synergistical effect of exogenous PGE2 and virulent Mtb Results

30. Inductionof Syt-7 transcription in virulent Mtbinfection Real –time PCR/ Rvinfected Mφ • Higher Syt -7 expression in Alox 5 deficient Mφ than in wt or Ptges deficient Mφ Results

31. In vivo induction of Syt-7 transcription Real –time PCR/ lungsofwtmice7d after aerosolinfection • Greater abundance of Syt-7 transcripts in avirulent infected mice than in wt or virulent infected mice Results

32. Summary V • Virulent Mtb evades innate immunity by suppressing the production of PGE2 What is the direct link among Syt-7 function , the death modality of Mtb infected Mφ and the outcome of infection? Results

33. Translocation of LAMP-1 Flow cytometry/ uninfectedandRvinfectedmouse Mφ • Translocation of Lamp-1 in Alox-5 deficient Mφ • No translocaion in wt or Ptges deficient Mφ Results

34. Visualization of LAMP-1 translocation Confocalmicroscopy/ Mφun-orinfectedwithGFP labeled virulent Mtb • Little amounts of LAMP-1 on the surface of Ptges deficient or wt Mφ • extensive recruitment of LAMP-1 to the surface of Alox5 deficient Mφ Red: luminal domain of LAMP-1 Results

35. Influence of Syt-7 on the death modality Celldeathdetection ELISA / Alox5 deficient MφinfectedwithRvMtbwithandwithoutsilencingof Syt-7 • Without silencing: More apoptosis than necrosis in Alox5 Mφ • After silencing: • more necrosis than apoptosis • => Syt-7:critical for preventing necrosis Results

36. Influence of Syt-7 on bacterial growth Colonycount/ Rvinfectedmouse Mφ • Limited bacerial growth in Alox5 deficient Mφ compared to WT • After silencing: impaired bacterial restriction => Direct involvement of Syt-7 in the innate control of Mtb infection Results

37. Summary • Virulent Mtb perturbs the repair of plasma membrane microdisruptions • Lysosomal and Golgi-derived vesicles are involved in plasma membrane repair • Two distinct calcium sencor proteins regulate lysosomal and Glogi dependent plasma membrane repair • Lysosome-dependent membrane repair was promoted by PGE2 (regulates Syt-7) • In absence of PGE2/Syt-7 Macrophages undergo necrosis and are unable to control Mtb growth Summary

38. Conclusion Inhibiting membrane repair by blocking PGE2 production represents a critical mechanism that allows virulent bacteria to replicate, to induce necrosis and escape from the host macrophage and infect other cells Conclusion

39. Thank you for your attention! The end…

40. Tuberculosis: unsealing the apoptotic envelope Steven A Porcelli & William R Jacobs Jr nature immunology volume 9 number 10 october 2008 Avirulent mycobacteria, including mutant forms of MTb that have lost their ability to cause disease, stimulate the macrophage to undergo apoptosis, which results in a 'cellular corpse' with an impermeable envelope that prevents bacteria from escaping. This process leads to containment and killing of the bacteria and is also associated with rapid priming of antigen-specific T cell responses. In contrast, virulent mycobacteria such as wild-type strains of MTb cause macrophage death by a process that proceeds to necrosis, which produces a permeable cell membrane that enables bacteria to escape and spread.

42. eicosanoids • comprise Prostaglandins and related compounds • Mostly produced from arachidonic acid (a 20-carbon polyunsaturated fatty acid) • considered "local hormones" • effects on target cells close to their site of formation • Are rapidly degraded, so they are not transported to distal sites within the body • participate in intercellular signaling and intracellular signal cascades • various roles in inflammation, immune system modulation, control of reproductive processes and tissue growth cyclopentane ring

43. Total amount of proteins