The multifunctional PE_PGRS11 protein from Mycobacterium tuberculosis plays a role in regulating resistance to oxidative stress

Abstract

Mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. Infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. We demonstrate here that hypothetical PE_PGRS11 ORF encodes a functional phosphoglycerate mutase. The transcriptional analysis revealed that PE_PGRS11 is a hypoxia-responsive gene, and enforced expression of PE_PGRS11 by recombinant adenovirus or Mycobacterium smegmatis imparted resistance to alveolar epithelial cells against oxidative stress. PE_PGRS11-induced resistance to oxidative stress necessitated the modulation of genetic signatures like induced expression of Bcl2 or COX-2. This modulation of specific antiapoptotic molecular signatures involved recognition of PE_PGRS11 by TLR2 and subsequent activation of the PI3K-ERK1/2-NF-κB signaling axis. Furthermore, PE_PGRS11 markedly diminished H(2)O(2)-induced p38 MAPK activation. Interestingly, PE_PGRS11 protein was exposed at the mycobacterial cell surface and was involved in survival of mycobacteria under oxidative stress. Furthermore, PE_PGRS11 displayed differential B cell responses during tuberculosis infection. Taken together, our investigation identified PE_PGRS11 as an in vivo expressed immunodominant antigen that plays a crucial role in modulating cellular life span restrictions imposed during oxidative stress by triggering TLR2-dependent expression of COX-2 and Bcl2. These observations clearly provide a mechanistic basis for the rescue of pathogenic Mycobacterium-infected lung epithelial cells from oxidative stress.

FIGURE 1.

In silico analyses and functional characterization of multifunctional protein PE_PGRS11. A, i, the presence of the phosphoglycerate mutase domain toward the C terminus of PE_PGRS11 protein upon the search against the domain database. Phosphoglycerate mutase matched with region 287–374 of PE_PGRS11 upon comparison with four Protein Data Bank structures, 1K6M, 1C81, 1TIP, and 1V7Q. All the active site residues of PE_PGRS11 (by active site prediction methods) were identical with these templates. ii, a three-dimensional model of the PE_PGRS11 phosphoglycerate mutase domain constructed on the basis of template 1K6M. iii, representation of the active site residues of phosphoglycerate mutase with 3-phosphoglyceric acid as a ligand upon automated docking simulations with the AutoDock 3.0 software suite using Silicon Graphics station Octane. B, a typical hyperbolic substrate saturation plot for the phosphoglycerate mutase enzyme activity of PE_PGRS11. The inset shows a Lineweaver-Burk plot to derive K_m and the V_max values for the phosphoglycerate mutase enzyme activity of PE_PGRS11. C, quantitative real time RT-PCR analysis of PE_PGRS11 or HspX transcripts assessed in the RNA isolated from M. tuberculosis grown under the indicated growth conditions. Error bars represent mean ± S.E. (n = 3). The data represent three independent experiments.

FIGURE 2.

PE_PGRS11 imparts resistance to lung epithelial cells against oxidative stress. A, MTT assay to determine cell viability of A549 cells infected with Ad-PE_PGRS11 or Ad-LacZ upon oxidative stress (25 μm H₂O₂ and 50 μm H₂O₂). B, determination of cell viability of A549 cells infected with recombinant M. smegmatis pJAM2-PE_PGRS11 or M. smegmatis pJAM2 vector alone after 48 h of oxidative stress (25 μm H₂O₂ and 50 μm H₂O₂). C, percent cell viability for A549 cells under oxidative stress (50 μm H₂O₂) upon treatment with recombinant wild-type PE_PGRS11 (WT PE_PGRS11) or the phosphoglycerate mutase domain of PE_PGRS11 (PGM). Error bars represent mean ± S.D. The data are representative of three independent experiments. MOI, multiplicity of infection.

FIGURE 3.

PE_PGRS11 triggers TLR2-dependent activation of PI3K-ERK1/2-NF-κB signaling axis. A, TLR2- or vector-transfected HEK-293 cells were treated with PE_PGRS11. The cells were further stained with antibody specific to PE_PGRS11 followed by Cy5-labeled secondary antibody. The immunofluorescence staining of cells was analyzed by confocal microscopy. B, HEK-293 cells, transiently transfected with vector or TLR2 cDNA construct along with NF-κB reporter construct, were treated with different doses of PE_PGRS11 protein followed by analysis of NF-κB reporter activity (mean ± S.E., n = 3). C, A549 cells were treated with PE_PGRS11 protein for the indicated time points followed by immunoblotting of nuclear and cytosolic fractions of cells for NF-κB. D, activation of p85 (PI3K), ERK1/2, and 4EBP1 by PE_PGRS11. E, transfection of cells with TLR2 dominant-negative (D/N) cDNA construct abrogates PE_PGRS11-induced activation of ERK1/2 and 4EBP1. F, A549 cells were pretreated with LY294002 or AKT inhibitor (Inhi.) followed by analysis of PE_PGRS11-induced activation of ERK1/2. The results are representative of two independent experiments. PCNA, proliferating cell nuclear antigen; p, phospho; Med, medium.

FIGURE 4.

PE_PGRS11 induced rescue from oxidative stress: activation of antiapoptotic and inhibition of apoptotic signaling axis. A, induced expression of COX-2 and Bcl2 upon PE_PGRS11 treatment as analyzed by immunoblotting. B, dose-dependent increase in Bcl2 expression upon PGE₂ treatment. C, A549 cells were pretreated with NS-398, and PE_PGRS11-induced Bcl2 expression was analyzed by immunoblotting. D, pretreatment of A549 cells with U0126 and LY294002 leads to abrogation of PE_PGRS11-induced COX-2 expression. E, activation of p38 MAPK upon exposure of cells to H₂O₂. F, PE_PGRS11 abolishes H₂O₂-mediated activation of p38 MAPK. The blots are representative of three independent experiments. p, phospho; Med, medium.

FIGURE 5.

Subcellular localization and role of PE_PGRS11 in protection of mycobacteria against oxidative stress. A, i, recombinant M. smegmatis expressing PE_PGRS11 under an acetamide-inducible promoter was constructed, and different cellular fractions of M. smegmatis transformed with either pJAM2 or pJAM2-PE_PGRS11 were immunoblotted with antiserum to PE_PGRS11. Lane 1, cell wall fraction of induced M. smegmatis (pJAM2); lane 2, cytosol fraction of induced M. smegmatis (pJAM2); lane 3, cell membrane fractions of induced M. smegmatis (pJAM2); lane 4, cell wall fraction of induced M. smegmatis (pJAM2-PE_PGRS11); lane 5, cytosol fraction of induced M. smegmatis (pJAM2-PE_PGRS11); lane 6, cell membrane fraction of induced M. smegmatis (pJAM2-PE_PGRS11); lane 7, purified PE_PGRS11 protein. ii, expression of PE_PGRS11 was analyzed by immunoblotting in various cellular fractions of M. tuberculosis H37Rv cells. Lane 1, whole cell lysate of M. tuberculosis; lane 2, cell wall fraction of M. tuberculosis; lane 3, culture filtrate proteins of M. tuberculosis; lane 4, cytosol fraction of M. tuberculosis; lane 5, purified PE_PGRS11 protein. B, M. smegmatis transformed with pJAM2 or pJAM2-PE_PGRS11 were subjected to partial digestion by either proteinase K or trypsin for the indicated time points, and cell lysates were analyzed for integrity of PE_PGRS11 by immunoblotting. C, growth and survival of M. smegmatis transformed with pJAM2 or pJAM2-PE_PGRS11 in the presence of oxidative stress (5 mm hydrogen peroxide). D, analysis of zone of inhibition for M. smegmatis transformed with pJAM2 or pJAM2-PE_PGRS11 upon oxidative stress (30% H₂O₂) (mean ± S.E., n = 3). E, micrographs representing colony morphology of M. smegmatis transformed with pJAM2 or pJAM2-PE_PGRS11. F, electron micrographs of M. bovis BCG transformed with pMV361 sense PE_PGRS11 (M. bovis BCG S-PE_PGRS11) or pMV361 antisense PE_PGRS11 (M. bovis BCG AS-PE_PGRS11). The data represent two independent experiments.

FIGURE 6.

Differential immunoreactivity of PE_PGRS11 protein with sera or CSF from different categories of TB patients. A and B, PE_PGRS11 protein showed significant reactivity in all the groups in child TB patients for IgG antibody (B), whereas adult patients showed no reactivity except group 1 patients (A). C and D, when assessed for IgM antibody, PE_PGRS11 showed significant reactivity in all the categories of adult patients (C) but not in the case of any group of child TB patients (D). E, the IgG antibody reactivity to M. tuberculosis H37Rv whole cell lysate (WCL) and PE_PGRS11 in CSF from TBM patients in comparison with healthy controls and other meningitis disease controls. Gr 1, pulmonary infection; Gr 2, relapsed infection; Gr 3, extrasputum infection; HC, healthy control; ODC, other disease control.