Mechanistic Investigation of Granzyme A inhibitory effects on intracellular Mycobacterium tuberculosis

Abstract

One fourth of the world population is infected with Tuberculosis (TB). Our lab has identified γ9δ2 T cells that secrete Granzyme A (GzmA) with TB protective effects. In this study, we investigated the mechanism(s) by which human GzmA inhibits the intracellular replication of mycobacteria within infected human primary monocytes. GzmA was added to mycobacteria-infected monocytes for downstream analyses using 2D-DIGE and shotgun proteomics. We generated WT, enzymatically inactive (S195A), and monomeric only (C93S) recombinant GzmA and performed: flow cytometry studies using viable mycobacteria; intracellular inhibition assays neutralizing CD14, TLR4 and TLR2; and GzmA immunoprecipitation experiments. The 2D-DIGE proteomic analyses found the ER-stress response and ATP metabolism pathways as important for GzmA-mediated inhibition. Separately, shotgun proteomics uncovered the upregulation of Rab11FIP1 (important for phagocytosis). Both GzmA-WT and S195A proteins inhibited intracellular mycobacteria, but C93S did not. Neutralization of CD14 and TLR4, but not TLR2, reversed GzmA-inhibitory activity. GzmA-WT, S195A, and C93S all bound mycobacteria. However, GzmA-WT and S195A, but not GzmA-C93S, stably bound to TLR4 and CD14. Collectively, these studies demonstrate key structural, functional, and inter-/intra-molecular features required for GzmA-mediated inhibition of intracellular mycobacteria including interactions between GzmA, mycobacteria, TLR4 and CD14. These interactions result in the ER stress response, altered ATP metabolism, enhanced phagocytosis, and inhibition of mycobacteria. Thus, GzmA’s potential role as opsonin could lead to novel host-directed therapeutics for TB infections. Supported by grants from NIH (F30HL151136-01, R01AI048391-12)