Mycobacterium tuberculosis Rv3402c enhances mycobacterial survival within macrophages and modulates the host pro-inflammatory cytokines production via NF-kappa B/ERK/p38 signaling

Abstract

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H2O2 exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1β compared with controls. Rv3402c-induced TNF-α and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.

Figure 1. Expression of Rv3402c in recombinant M. smegmatis.

(A). Ms_Rv3402c and Ms_Vec were grown at 37°C in MB 7H9 liquid medium to an OD₆₀₀ of 0.6–1.0. Total bacterial RNA was isolated after 16 h induction and subjected to RT-PCR to detect expression of the rv3402c and rrsB genes. (B). Lysates were prepared from bacterial cells that were cultured as in (A) and subjected to Western blot analysis to detect Myc-tagged Rv3402c protein using mouse anti-Myc antibody.

Figure 2. Rv3402c is a cell envelope-associated protein.

(A). Subcellular fractionation of M. smegmatis induced to express Rv3402c-myc showed localization to the cell wall and membrane fractions. Subcellular fractions were separated by SDS-PAGE and proteins were detected with an anti-Myc antibody. Native GroEL2 was detected as a cytoplasmic control. WCL, whole cell lysates (66 μg total protein); CW, cell wall fraction (24 μg total protein); CM, cell membrane fraction (47 μg total protein); SOL, soluble fraction (60 μg total protein). (B and C). M. smegmatis transformed with pALACE, pALACE-GFP or pALACE-Rv3402c were incubated with proteinase K or trypsin at different time points. Whole-cell lysates obtained by the recombinant bacteria were separated on SDS-PAGE. Immunoblots were performed using anti-His antibodies and anti-GFP antibodies. Similar results were obtained in two independent experiments.

Figure 3. Intracellular survival of recombinant M. smegmatis in macrophages.

U-937 cells (A) and RAW267.4 cells (B) were infected with MS_Vec and MS_Rv3402c as described in the methods section, respectively. Aliquots of infected cells were lysed with 0.025% SDS at indicated times, and serial dilutions were plated on 7H10 agar plates containing kanamycin. Recovered CFUs were enumerated after the incubation for 3–4 days at 37°C. Numbers of intracellular bacteria are shown as a percentage of the numbers detected at t = 6 h (U-937) or 4 h (RAW267.4) (% survival). Data are shown as means ± SD of triplicate wells. Similar results were obtained in three independent experiments.

Figure 4. Growth of MS_Vec and MS_Rv3402c under stress conditions.

(A). Growth of MS-Vec and MS-Rv3402c at 37°C in MB 7H9 liquid medium was monitored by determining OD₆₀₀ at intervals of 3 h. (B). Growth curve of MS-Vec and MS-Rv3402c in iron-depleted medium. Two recombinant M. smegmatis were grown in MB 7H9 medium supplemented with 100 μM 2′ 2′ dipyridyl. The growth of the strains were monitored by measuring OD₆₀₀ at intervals of 6 h. (C). In vitro growth of MS-Vec and MS-Rv3402c at different pH. The bacteria were collected by centrifugation and resuspended to an OD₆₀₀ of 0.5 in 5 ml MB 7H9 (pH 5 or 3). All cultures were again incubated at 37°C and 0.1 ml removed for viable count enumeration after 0, 3 and 6 h. (D). Survival of recombinant M. smegmatis after exposure to hydrogen peroxide. Aliquots (5 ml) of cultures at OD₆₀₀ of 0.5 were exposed to 5 mM hydrogen peroxide (H₂O₂) for 6 h at 37°C. The cultures were serially diluted and plated onto MB 7H10 plates and the colonies counted after 3–4 days of incubation at 37°C.

Figure 5. Assay of cell death in macrophages infected with recombinant M. Smegmatis.

Culture supernatants were collected from mono-layers of U-937 infected at a MOI of 10∶1 with MS_Vec or MS_Rv3402c and the release of LDH as a measure of macrophage cell death was estimated at various time points. Data are shown as means ± SD of triplicate wells. Similar results were obtained in three independent experiments.

Figure 6. Rv3402c induces the secretion of TNF-α and IL-1β in infected macrophages.

Culture supernatants were harvested after 6 or 72-α (A) and IL-1β (C) were determined. Cells were harvested after 12 h of infection and semi-quantitative RT-PCR analysis of TNF-α (B) and IL-1β (D) mRNA level was performed. Each three lanes in the RT-PCR analysis are replicates of a single time point. Similar results were obtained in three independent experiments.

Figure 7. Rv3402c alters the activation levels of NF-κB, ERK and p38 in macrophages infected with M. smegmatis.

Culture supernatants were harvested after 12 μg/ml and the concentrations of TNF-α (A) and IL-1β (B) were determined. U-937 cells were pre-treated with 30 μM TPCK (a NF-κB inhibitor) or with 20 μM PD 98059 (an ERK1/2 inhibitor) or with 10 μM SB 202190 (a p38 inhibitor). Treatment with DMSO served as a control for the inhibitor treatments. After 1 h, the macrophages were infected with MS_Vec at an MOI of 10 or treated with rRv3402c at the concentration of 5 μg/ml. Protease K (K) used to digest the recombinant protein. Culture supernatants were harvested after 24 h of infection and the concentrations of TNF-α (C, E) and IL-1β (D) were determined. (A), (B) and (E) are performed with recombinant protein; (C) and (D) are performed with M. smegmatis strains. The data are representative of two independent experiments.