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