Homodimeric Granzyme A Opsonizes Mycobacterium tuberculosis and Inhibits Its Intracellular Growth in Human Monocytes via Toll-Like Receptor 4 and CD14

Abstract

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete granzyme A (GzmA) protective against intracellular Mtb growth. However, GzmA-enzymatic activity is unnecessary for pathogen inhibition, and the mechanisms of GzmA-mediated protection remain unknown. We show that GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes, and subsequent pathogen clearance within the phagolysosome. Although monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Without access to surface-expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1 was associated with inhibitory activity. Furthermore, GzmA colocalized with and was regulated by protein disulfide isomerase AI (PDIA1), which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify a previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis, and elimination of Mtb in human monocytes, and they highlight PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

Keywords: CD14; PDIA1; TLR4; granzyme A; mycobacteria.

Figure 1.

The granzyme A (GzmA)-C93S variant forms only monomers, and GzmA-monomer cannot inhibit the intracellular mycobacterial growth. (A) and (B) The GzmA-wild type (WT), but not GzmA-C93S, can form homodimers (54 kDa) under nonreducing conditions as shown by silver stain and anti-GzmA Western blot. (C) The GzmA-C93S retains enzymatic activity as measured by the benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) cleavage assay (n = 3 independent experiments, mean ± standard deviation). (D) The GzmA-WT and GzmA-S195A inhibit intracellular mycobacterial replication, whereas GzmA-C93S does not (n = 8 subjects from 3 independent experiments; mean ± standard error of the mean [SEM]; Wilcoxon matched-pairs signed-rank test). (E) Mixing experiment combining GzmA-WT and GzmA-C93S shows partial inhibition of GzmA-WT activity (n = 6 subjects from 3 independent experiments; mean and SEM; Wilcoxon matched-pairs signed-rank test) (***, P < .0001; **, P < .001; *, P < .01; ns, not significant).

Figure 2.

CD14 and Toll-like receptor (TLR) 4 receptors are important for granzyme A (GzmA)-mediated inhibition of intracellular mycobacterial Bacillus Calmette-Guérin growth. (A) Neutralization of CD14 receptor by neutralizing antibody abolishes GzmA-wild type (WT) and GzmA-S195A inhibitory activities (n = 8 subjects from 3 independent experiments; mean ± standard error of the mean [SEM]; Wilcoxon matched-pairs signed-rank test). (B) The TLR4 was neutralized using neutralizing antibody similar to (A) (n = 8 subjects from 3 independent experiments; mean and SEM; Wilcoxon matched-pairs signed-rank test). (C) Neutralization of TLR2 does not reverse GzmA-WT or GzmA-S195A inhibitory function (n = 8 subjects from 3 independent experiments; mean and SEM; Wilcoxon matched-pairs signed-rank test). (D) The GzmA-WT and GzmA-S195A, but not GzmA-C93S, induced inhibition of mycobacteria within immortalized mouse macrophage (iMac)-WT mouse cells (n = 3 independent experiments; mean and standard error of the mean [SEM]; Student t test). (E) The iMac-WT responding to GzmA-WT mediates intracellular mycobacterial inhibition within iMac-WT, but not CD14KO and iMac TLR4KO cells (n = 3 independent experiments; mean and SEM; Student t test) (**, P < .001; ns, not significant).

Figure 3.

Granzyme A (GzmA) binds to Bacillus Calmette-Guérin (BCG), and its opsonization enhances monocyte inhibitory and phagocytic capacity. (A) Results demonstrating that GzmA-wild type (WT), GzmA-S195A, and GzmA-C93S bind to BCG, whereas negative control protein rTS cannot (n = 3 independent experiments; mean and standard error of the mean [SEM]; Student t test). (B) The GzmA needs to be added to cells before and/or during infection to induce inhibitory activity (n = 8 subjects from 3 independent experiments; mean and SEM; Wilcoxon matched-pairs signed-rank test). (C) The GzmA-WT enhances phagocytosis of BCG-green fluorescence protein (GFP) compared to untreated cells at 2 hours postinfection (n = 3 independent experiments; mean and SEM; Student t test). (D) Rab11-FIP1 protein is upregulated in GzmA-treated and BCG-infected cells as measured by quantitative shotgun proteomics (n = 4 subjects from 4 independent experiments; mean and standard deviation). (E) The GzmA-WT opsonizes BCG, and after its excess is removed it continues to inhibit the intracellular replication of the pathogen, whereas GzmA-C93S cannot (n = 8 subjects from 3 independent experiments; mean and SEM) (***, P < .0001; **, P < .001; ns, not significant).

Figure 4.

Granzyme A wild type (GzmA-WT), but not GzmA-C93S, interacts with CD14 and Toll-like receptor 4 (TLR4), kinetics of GzmA action, evidence of enhanced phagolysosome fusion, and translation of key findings to virulent Mycobacterium tuberculosis (Mtb) model of infection. (A) and (B) Different amounts of protein lysates made from Bacillus Calmette-Guérin (BCG)-infected human monocytes were incubated with α-GzmA to perform immunoprecipitation of bound proteins. These proteins were then probed for CD14 (A) or TLR4 (B), which showed that GzmA-WT binds to CD14 and TLR4, whereas GzmA-C93S does not (representative figures from 2 independent experiments). (C) The BCG-green fluorescence protein (GFP) colocalizes with LAMP1 after GzmA-WT treatment, demonstrating that opsonized mycobacteria enter the phagolysosome leading to better neutralization. The GzmA-C93S fails to direct mycobacteria into the phagolysosome (quantification of integrated density from the confocal microscopy experiments for the colocalization of mycobacteria and LAMP1 marker analyzed: values are mean ± standard deviation of ∼40 cells per each experiment, from 2 independent experiments). (D) All components of GzmA effects shown with BCG-infected cells (shown in Figures 1 and 2) were also confirmed with Mtb-infected monocytes (n = 6 subjects, from 2 independent experiments; mean and standard error of the mean; Wilcoxon matched-pairs signed-rank test) (***, P < .0001; **, P < .001; ns, not significant).

Figure 5.

Protein disulfide isomerase A1 (PDIA1) cleaves granzyme A (GzmA) homodimers into monomers, and PDIA1 colocalizes with GzmA after treatment. (A and B) Silver-stained and anti-GzmA Western blot showing results from incubating heat-inactivated or fresh PDIA1 with GzmA-wild type (WT) and the appearance of GzmA monomer at 27 kDa. (C) Confocal microscopy image of human monocytes infected with mycobacteria and treated with GzmA, and its colocalization with PDIA1. (D) Deconvoluted stimulation emission depletion images showing visual colocalization of GzmA and PDIA1 (representative figure from 3 independent experiments). (E) Percentage of colocalization by quantifying objective Pearson correlation for GzmA and ATP5H (control) or PDIA1 (data from 3 independent experiments; mean ± standard deviation). (F) Cell-permeable inhibitor PACMA-31 (200 nM) enhances GzmA-inhibitory activity (n = 8 subjects from 3 independent experiments; mean and standard error of the mean [SEM]; Wilcoxon matched-pairs signed-rank test). (G) Similar to (F), but cell-impermeable inhibitor rutin (50 μM) was used and showed GzmA enhancement in Bacillus Calmette-Guérin (BCG) and Mycobacterium tuberculosis (Mtb)-infected monocytes (n = 8 subjects from 3 independent experiments; mean and SEM; Wilcoxon matched-pairs signed-rank test) (**, P < .001; *, P < .01; ns, not significant).

Figure 6.

(A) Summary figures depicting our understanding of granzyme A (GzmA) in the inhibition of intracellular Mycobacterium tuberculosis (Mtb) growth. The GzmA either used as host-directed therapy (HDT) or secreted by γ₉δ₂ T cells opsonizes mycobacteria. Only GzmA-wild type can interact with Toll-like receptor 4 (TLR4) and CD14 leading to inhibition. (B) The GzmA-treated cells have enhanced phagocytosis and Rab11-FIP1 protein aids in the recycling of the endocytic vesicle. (C) Diagram showing that homodimeric GzmA is feature necessary for mediating mycobacterial growth inhibition. Monomeric GzmA cannot inhibit mycobacterial growth. (D) Protein disulfide isomerase A1 (PDIA1) converts GzmA homodimer into monomer, and its inhibition potentiates GzmA-inhibitory activity. (E) Homodimeric GzmA-treated cells show enhancement of phagolysosome fusion (figure created with BioRender.com).