Execution of macrophage apoptosis by PE_PGRS33 of Mycobacterium tuberculosis
Abstract number: 1732_207
Basu S., Pathak Suschil, Banerjee A., Pathak Shresh, Bhattacharyya A., Yang Z., Talarico S., Kundu M., Basu J.
Objectives: The role of the multi-gene PE family of proteins unique to mycobacteria, in the pathogenesis of tuberculosis is still poorly understood. Tumour necrosis factor-alpha (TNF-a) is essential for successfully combatting tuberculosis. Our objective was to understand how PE_PGRS33, a surface-exposed protein known to undergo variation among strains, influences TNF-a release from macrophages, and how this is linked to apoptosis of macrophages.
Methods: Recombinant PE_PGRS33 was tested for its ability to release TNF-a from the macrophage cell line RAW264.7 as well as from human monocyte-derived macrophages. Its binding to cell surface TLR2 was analysed by flow cytometry and the role of TLR2 was studied by transfecting cells with dominant-negative TLR2 prior to analysing PE_PGRS33-induced TNF-a release. The signaling pathways activated were studied by assaying for the mitogen-activated protein kinase (MAPK) kinase kinase ASK1 and downstream MAPKs. Cell death was analysed using histone ELISA and also by assaying surface-associated annexin-FITC.
Results: PE_PGRS33, a surface exposed protein, elicits TNF-a release from macrophages in a Toll-like receptor 2 (TLR2)-dependent manner. Apoptosis signal-regulating kinase 1 (ASK1) is activated downstream of TLR2. ASK1 activates the mitogen-activated protein kinases (MAPKs) p38 and JNK. PE_PGRS33-induced signaling leads to enhanced expression of TNF-a and TNF receptor I (TNFRI) genes. Mycobacterium smegmatis expressing PE_PGRS33 elicits the same effects as purified PE_PGRS33. Neutralising TNF-a antibodies showed that release of TNF-a is required for triggering apoptosis in macrophages challenged with PE_PGRS33. The death receptor-dependent signals are amplified through classical caspase 8-dependent mitochondrial release of cytochrome c, leading to the activation of caspases 9 and 3.
Conclusions: The important aspect of our findings is that deletions within the PGRS domain (simulating those occurring in clinical strains) attenuate the TNF-a-inducing ability of PE_PGRS33. These results provide the first evidence that variations in the polymorphic repeats of the PGRS domain modulate the innate immune response.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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