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Mycobacterium tuberculosis evades macrophage
defenses 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
091012 Johanna Eberhard
Plasma membrane repair
Based on exocytosis of endomembranes• Derived from Golgi
vesicles or lysosomes• Calcium dependant• Calcium sensor:
• Synaptotagmin-7 (Syt-7)• Neuronal Calcium sensor-1(NCS-1)
Introduction
Mycobacterium tuberculosis
Virulent: blocks phagosome maturation, leads to macrophage death by necrosis
Avirulent: induces apoptosis of macrophages
Introduction
Is phagocytosed by pulmonary macrophages
Mtb infection
Introduction
Prostaglandin E2 (PGE2 )
Lipoxin A4
(LXA4)
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
Virulent Mtb causes persistent microdisruptions
-FDX influx-
Results
Flow cytometry/ human macrophages
• Less FDX influx in avirulent infected Mφ than in virulent infected Mφ
Recruitment of lysosomal membranes
-Translocation of LAMP-1-
Results
Flow cytometry/ human macrophages
After 12 h of infection:
• Translocation of LAMP-1 stronger in H37Ra infected Mφ than in virulent infected Mφ
Results
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
Recruitment of Golgi derived membranes
-Translocation of mannosidase II, not of BiP-
Results
Flow cytometry/ human macrophages
• Higher translocation of Mannosidase II after avirulent infection• No BiP translocation, no involvement of ER derived membranes
Summary I
virulent Mtb causes persistent membrane disruptions
Membranes both from the lysosomal and Golgi compartments are involved in macrophage plasma membrane repair
Results
What is the function of calcium sensors in the recruitment of these membranes?
Calcium-sensors in plasma membrane repair-Silencing of Syt-7 expression-
Results
Immunoblot/ human macrophages
Flow cytometry/ human macrophages
• impaired LAMP-1 translocation but enhanced Mannosidase II, Posphatidylserine and annexin-1 expression
Inhibition of Golgi membrane recruitment- blocking with Brefeldin A-
Results
Flow cytometry/ human macrophages
• Inhibition of mannosidase II, phosphatidylserine and annexin-1 translocation, but not of LAMP-1
Calcium sensors in plasma membrane repair-Silencing of NCS-1 expression-
Results
Immunoblot/ human Mφ Flow cytometry/ human Mφ
• Inhibition of the translocation of Golgi membranes, Phosphatetidylserine and annexin-1
Inhibition of plasma membrane repair-FDX influx and Necrosis (7-AminoActinomycinD)-
Results
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
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
Results
Is the upregulation of cAMP sufficient to trigger membrane repair?
Effect of cAMP upregulation on membrane repair-Acivation of adenylate cyclase with forskolin-
Results
Flow cytometry/ human Mφ
• Greater translocation of LAMP-1 and Syt-7 • no effect on translocation of Golgi membranes
Effect of PGE2 on membrane repair
Results
Flow cytometry/ human Mφ
• Reconstitution of plasma membrane repair after addition of PGE2
• No increase of Golgi membrane recruitment
Kinases involved in PGE2 activated membrane repair
- Inhibition of PKA and PI(3)K -
Results
Flow cytometry/ human Mφ
• PGE2 requires PI(3)K activation
PKA inhibitorPI(3)K inhibitor
Effect of PGE2 on membrane repair- Infection of Mφ from Wt and Ptges -/- mice -
Results
• No recruitment of lysosomal membranes in Mφ deficient in Prostaglandin E synthase
• No effect on Golgi membrane recruitment
Flow cytometry/ Ra infected mouse Mφ
Summary III
• recruitment of lysosomal membranes is PGE2 dependent
• recruitment of Golgi-derived membranes is independent of PGE2
Results
How do PGE2 and LXA4 affect the outcome of Mtb infection?
Influence of LXA4 and PGE on the death modality of Mφ in Mtb infecion
Results
Cell death detection ELISA / mouse Mφ
• More necrosis and less apoptosis in Ptges deficient Mφ
• More apoptosis and less necrosis in Alox5 deficient Mφ
Influence of LXA4 and PGE2 in control of Mtb growth
Results
Colony count / mouse Mφ
• enhanced Mtb growth in Ptges deficient Mφ
• Lower growth of Mtb in Alox5 deficient Mφ
Innate control of infection in vivo-virulent infection of ko and wt mice -
Results
Pulmonary lavage /cell death detection ELISA/ mouse APCs
• More apoposis in the cells of Alox5 deficient mice
Innate control of infection in vivo- adoptive transfer of Mφ into Rag1 deficient mice-
Results
Colony count/ transferred mouse Mφ
• Bacterial burden higher in Rag-1 deficient mice with Ptges deficient Mφ
• lower with Alox5 deficient Mφ
Bacterial burden in spleen and lung
Results
Colony count/ transferred mouse Mφ
• Durable effect in spleen and lung
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
Results
Is PGE2 involved in Syt-7 synthesis?
Induction of Syt-7 transcriptionby exogenous PGE2
Results
• PGE2 induces Syt-7 expression in uninfected mouse Mφ
Real –time PCR /uninfected mouse Mφ
Results
No Induction of LAMP-1 transcription Real –time PCR /uninfected mouse Mφ
• No effect of exogeneous PGE2 on LAMP-1 transcription
Results
Induction of Syt-7 transcription in virulent Mtb infection
Real –time PCR/ uninfected and infected Mφ
• Synergistical effect of exogenous PGE2 and virulent Mtb
Results
Real –time PCR/ Rv infected Mφ
Induction of Syt-7 transcription in virulent Mtb infection
• Higher Syt -7 expression in Alox 5 deficient Mφ than in wt or Ptges deficient Mφ
In vivo induction of Syt-7 transcription
Results
Real –time PCR/ lungs of wt mice 7d after aerosol infection
• Greater abundance of Syt-7 transcripts in avirulent infected mice than in wt or virulent infected mice
• Virulent Mtb evades innate immunity by suppressing the production of PGE2
Results
Summary V
What is the direct link among Syt-7 function , the death modality of Mtb infected Mφ and the outcome of infection?
Translocation of LAMP-1
Results
Flow cytometry/ uninfected and Rv infected mouse Mφ
• Translocation of Lamp-1 in Alox-5 deficient Mφ
• No translocaion in wt or Ptges deficient Mφ
Visualization of LAMP-1 translocation
Results
Confocal microscopy/ Mφ un-or infected with GFP labeled virulent Mtb
Red: luminal domain of LAMP-1
• 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
Influence of Syt-7 on the death modality
Results
Cell death detection ELISA / Alox5 deficient Mφ infected with Rv Mtb with and without silencing of Syt-7
• Without silencing: More apoptosis than necrosis in Alox5 Mφ
• After silencing: more necrosis than apoptosis
=> Syt-7:critical for preventing necrosis
Influence of Syt-7 on bacterial growth
Results
Colony count/ Rv infected mouse 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
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
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
Thank you for your attention!
The end…
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.
Tuberculosis: unsealing the apoptotic envelopeSteven A Porcelli & William R Jacobs Jr
nature immunology volume 9 number 10 october 2008
• 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
eicosanoids
cyclopentane ring
Total amount of proteins