Mechanistic investigations of Granzyme A-mediated TB protective effects

Abstract

Abstract One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb), the pathogen that causes Tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. To elucidate the mechanism(s) involved in GzmA-mediated mycobacterial pathogen inhibition using a relevant in vitro human system. This GzmA was utilized to treat mycobacteria-infected primary monocytes from multiple volunteers, and their cell lysates were analyzed using 2D-DIGE global proteomics. A catalytically inactive GzmA variant was produced to study the enzymatic role in the Mycobacterial Growth Inhibition Assay (MGIA). In parallel, confocal microscopy experiments looked at GzmA internalization. We have evidence that Granzyme A’s intact active site is not necessary to inhibit the intracellular replication of mycobacteria as the S195A variant inhibits mycobacterial infection in MGIA as Wild Type (WT) GzmA. The proteomic approach identified two pathways: ER-stress response and purinergic channel receptor activation are important for GzmA-mediated intracellular mycobacterial inhibition. These results were validated in separate volunteers by western blot. Confocal microscopy experiments show that GzmA is internalized within the infected cell and clears the infection. Modulation of key proteins in the discovered pathways are being targeted to confirm our proteomic results and to test potential novel therapies that emulate GzmA. It will also be investigated if GzmA co-localizes in these cellular compartments. These studies will lead to a better understanding of GzmA biology during infection and to the development of novel host-directed therapeutics for control of Mtb infection.