Granzyme A Produced by γ9δ2 T Cells Activates ER Stress Responses and ATP Production, and Protects Against Intracellular Mycobacterial Replication Independent of Enzymatic Activity

Abstract

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world’s population currently infected. Data indicate that γ9δ2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ9δ2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA’s action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

Improved Purification of Human Granzyme A/B and Granulysin Using a Mammalian Expression System

“…Proteins are then concentrated, undergo endotoxin removal using Endotrap columns. As shown by silver stain and western blot in Figures 4A, B , GzmA is highly pure and able to form homodimers (as well as multimers – previously reported in ( 14 , 17 ) and of unknown significance). For Figure 4C , the differences between native, recombinant GzmA purified using old protocol vs new protocol are analyzed for purity by silver stain.…”

“…These changes have significantly improved the yield of our protein purifications as shown in Figure 4G and allowed for the purification of recombinant human GNLY (Figure 5). Our recombinant system also facilitates site-directed mutagenesis and has been successfully employed to mutate a key amino acid within the active site of Granzyme A (GzmA-S195A) (17). There were previous reports of GNLY expression in bacteria (1,49), yeast (32), and insect cells (33), but to the best of our knowledge, this is the first time that GNLY has been successfully purified in a mammalian expression system.…”

“…Optional: site-directed mutagenesis for modifications within original construct to obtain substitution at key amino acid [performed for GzmA-WT vs GzmA-S195A for ( 17 )].…”

“…Death induction by GzmA involves a complex sequence of events, ultimately leading to the activation and nuclear transfer of two nucleases (NM23-H1 and Trex1) that trigger lethal DNA damage (9)(10)(11)(12)(13). GzmA also induces monocytes to produce pro-inflammatory cytokines (14)(15)(16) and to inhibit the intracellular replication of mycobacterial growth within infected primary human monocytes (15,17). Due to its proinflammatory potential, there have been recent reports of its involvement during bacterial sepsis (16,18,19).…”

Oxidative and Non-Oxidative Antimicrobial Activities of the Granzymes

“…For the residual, "orphan" Gzms, caspase-dependency to induce death or even if the induction of apoptosis is their major function is still not clear and needs further study (73,74). However, there are multiple lines of evidence suggesting that GzmM, GzmH and GzmK, as well as the non-orphan GzmA, have well defined proinflammatory and antimicrobial roles as further discussed below (75)(76)(77)(78).…”