This proposal will determine the mechanism responsible for Granzyme A (GzmA)-mediated inhibition of Mycobacterium tuberculosis (Mtb) growth, to better understand the host immune response. One fourth of the world?s population is infected with Mtb. Currently, BCG strains are the only vaccines available to protect against tuberculosis (TB). ?9?2 T cells are found at higher frequencies in lungs, which is the primary site of Mtb infection. Our lab has previously shown that TB specific ?9?2 T cells secrete GzmA upon stimulation with mycobacterial antigens. Furthermore, our lab has demonstrated that GzmA, when released by TB-specific ?? T cells, inhibits the intracellular replication of the pathogen. GzmA is a serine protease found within cytotoxic granules of NK cells, CD8+ CTL, and ?? T cells. It has tryptase activity cleaving basic residues after Arg or Lys, and was originally thought to be an inducer of apoptosis in the target cell in a perforin-dependent manner similar to Granzyme B. However, more recent literature has shown that GzmA at a physiologic concentration (nM) does not cause apoptosis. Instead, it induces monocytes/macrophages to produce pro-inflammatory cytokines. Our lab has shown that GzmA in a perforin-independent manner induces the infected macrophage to secrete pro-inflammatory cytokines, such as TNF-alpha and IL-1?, and inhibits the intracellular growth of mycobacteria. A transcriptomal analysis was performed, which failed to identify the mechanism of GzmA- mediated inhibition. We hypothesize that given the proteolytic nature of GzmA, a rational approach to investigate this mechanism would be to perform a proteomic analysis. GzmA route of entry and enzyme catalysis will be studied in Aim 1 to better understand the necessary steps for inhibition.
In Aim 2, the Endoplasmic Reticulum (ER) stress response and purinergic channel activation will be interrogated: these two pathways were discovered during preliminary proteomic experiments, and will be modulated by knocking out genes or inhibiting key factors in cell lines and/or primary cells. Finally, additional global proteomic screens will be conducted to detect other differentially expressed proteins in Aim 3. Overall, this project will help in the design of novel immunotherapeutic and/or chemotherapeutic solutions for TB protection and will aid the WHO?s current goal to decrease TB deaths of 90% by 2030. A proposed comprehensive training plan includes these exciting studies that will allow me to develop needed skills for my career goal to become a successful independent physician-scientist.
This project will discover pathways associated with Granzyme A-mediated Mycobacterium tuberculosis intracellular inhibition. These studies will determine if these newly discovered cellular processes can be potentiated in a tuberculosis infection to enhance lung host immune responses. New chemotherapeutic and/or immunotherapeutic targets will curtail the number one infectious killer worldwide.
