Mechanistic investigations of Granzyme A-mediated TB protective effects
Valerio Rasi1,2, Mei Xia1, David Wood3, Christopher Eickhoff1, Daniel Hoft1,2.
1Division of Infectious Diseases, Allergy & Immunology, Department of Internal Medicine, Saint Louis University, Saint Louis, MO, USA.
2Department of Molecular Microbiology & Immunology, Saint Louis University, Saint Louis, MO, USA.
3Department of Biochemistry & Molecular Biology, Saint Louis University, Saint Louis, MO, USA.

Introduction and Background: One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb). Currently, Bacillus Calmette–Guérin (BCG) strains are the only vaccines available to protect against tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. GzmA released by TB-specific γδ T cells has been shown to inhibit the intracellular replication of the pathogen.
Specific Experimental Aims: To elucidate the mechanism(s) involved in GzmA-mediated mycobacterial pathogen inhibition using a relevant in vitro human system.
Methods and Design: Our lab purified human GzmA to infer species-specific mechanisms. This GzmA was utilized to treat mycobacteria-infected primary monocytes from multiple volunteers, and cell lysates were analyzed for differentially abundant proteins using 2D-DIGE global proteomics. A catalytically inactive GzmA variant was also produced to understand the enzymatic role of this protein in the Mycobacterial Growth Inhibition Assay (MGIA). In parallel, confocal microscopy experiments have been conducted to study GzmA internalization and subcellular co-localization within infected monocytes.
Results: We have evidence that Granzyme A’s intact active site is necessary to inhibit the intracellular replication of mycobacteria. The substitution of serine to alanine in the active site appears to render the protein unable to control the mycobacterial infection as measured by MGIA. The 2D-DIGE proteomic approach identified two pathways involved in control of mycobacterial replication using GzmA. This analysis indicates that ER-stress response (HSPA5, HSP90B1, P4HB, PDIA3) and purinergic channel receptor activation (ATP5B/C1/D/H/O) may be important for GzmA-mediated intracellular mycobacterial inhibition. Preliminary confocal microscopy experiments show that GzmA is internalized within the infected cell.
Conclusion and Future Directions: Modulation of key proteins in these pathways are currently being targeted by pharmaceutical intervention and gene alteration to test the effects on GzmA-mediated inhibitory effects. It will be investigated if GzmA co-localizes in these cellular compartments. Confirmation that these pathways are involved in pathogen clearance will lead to the development of novel host-directed therapies for control of Mtb infection.

Valerio Rasi BS

Saint Louis University

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