Mycobacterium tuberculosis PE31 (Rv3477) Attenuates Host Cell Apoptosis and Promotes Recombinant M. smegmatis Intracellular Survival via Up-regulating GTPase Guanylate Binding Protein-1

Abstract

The Mycobacterium (M.) tuberculosis comprising proline-glutamic acid (PE) subfamily proteins associate with virulence, pathogenesis, and host-immune modulations. While the functions of most of this family members are not yet explored. Here, we explore the functions of "PE only" subfamily member PE31 (Rv3477) in virulence and host-pathogen interactions. We have expressed the M. tuberculosis PE31 in non-pathogenic Mycobacterium smegmatis strain (Ms_PE31) and demonstrated that PE31 significantly altered the cell facet features including colony morphology and biofilm formation. PE31 expressing M. smegmatis showed more resistant to the low pH, diamide, H₂O₂ and surface stress. Moreover, Ms_PE31 showed higher intracellular survival in macrophage THP-1 cells. Ms_PE31 significantly down-regulated the production of IL-12p40 and IL-6, while up-regulates the production of IL-10 in macrophages. Ms_PE31 also induced the expression of guanylate-binding protein-1 (GBP-1) in macrophages. Further analysis demonstrates that Ms_PE31 inhibits the caspase-3 activation and reduces the macrophages apoptosis. Besides, the NF-κB signaling pathway involves the interplay between Ms_PE31 and macrophages. Collectively, our finding identified that PE31 act as a functionally relevant virulence factor of M. tuberculosis.

Keywords: PE subfamily; apoptosis; cell surface; cytokines; guanylate-binding protein-1.

Figure 1

Ms_PE31 can alter the surface characteristics of M. smegmatis. (A) PE31 (Rv3477) sequence amplified by PCR, applying M. tuberculosis genome, ~297 bp in length (lane 1 = DNA ladder and lane 2 = amplified gene). (B) The Ace-induced recombinant cell lysates were prepared and employed the Western blot for confirmation of His-tagged PE31 protein expression. (C) Both recombinant strains cultured on plates containing 7H9 solid media with hyg and Ace (0.02%, w/v), the developed single colony (D) Recombinant strains cultured in Ace (0.02%, w/v) added 7H9 liquid on polystyrene plates to induce the development of biofilm. (E) The quantification of biofilm formation was confirmed by Tetrahydrofuran (THF) assay (n = 3). Results were determined by Student's t-test, ^*P < 0.05. Error bars represent the standard deviation of mean.

Figure 2

Ms_PE31 increase growth under multiple stress. (A) The growth rate of recombinant strains was measured under in-vitro low pH condition. Cultured bacteria were harvested and re-cultured in 7H9 (pH = 5) to maintained the OD₆₀₀ =0.8. Then, incubated in 37°C and 100 μl taken from it after different time intervals for workable enumeration (n = 3). (B) The reactive nitrogen stress was examined by spot test, the supplementation of 2 and 5 mM diamide to the 7H9 solid medium to grown the Ms_Vec and Ms_PE31 strains. Growth of bacteria on 7H9 solid media which contained mentioned concentrations of diamide (n = 3). (C,D) Ms_Ves and Ms_PE31 survival upon exposure to H2O2 and SDS, respectively, examined by disk diffusion technique. Whatman disks used to mottled the different concentrations of H2O2 (10 μl) and SDS (10 μl) (n = 3). Area of the zone of inhibition was calculated after 3−4 days incubation at 37°C. The results were determined by Student's t-test, ^*P < 0.05 and ^**P < 0.01. Error bars represent the standard deviation of mean.

Figure 3

Survival rate of M. smegmatis recombinant strains. (A) Recombinant strains infected macrophages were laved and lysed SDS (0.025%, w/v) at indicated intervals. 10-fold serial diluted lysed cells were mottled on hyg containing 7H9 solid medium plates. After 3–4 days, CFU was computed (n = 3). (B) Growth kinetics of recombinant strains were determined by the growth of bacteria in 7H9 liquid added Ace (1%, w/v), Tw (0.05%, v/v) and hyg (100 μg/ml) (n = 3). Results were determined by Student's t-test, ^***P < 0.001. Error bars represent the standard deviation of mean.

Figure 4

M. tuberculosis PE31 expressed recombinant M. smegmatis regulated the cytokines profile. Ms_PE31 and Ms_Vec infected differentiated human macrophages, total RNA collected to carried out qRT-PCR and analyzed the relative mRNA of (A) IL-10 (n = 4), (B) IL-6 (n = 4), (C) and IL-12p40 (n = 4). For all RT-PCRs, β-actin of macrophages for internal control. Infected cells supernatant collected, and ELISA was accomplished to detect the production of (D) IL-10 (n = 3) (E) IL-6 (n = 3) (F) and IL-12p40 (n = 3). Results were determined by Student's t-test, ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. Error bars represent the standard deviation of mean.

Figure 5

Ms_PE31 reduced the cell apoptosis of macrophage. Recombinant strains infected PMA differentiated macrophages were stained by Annexin V-FITC/ PI, and collected to measure the apoptosis levels by using (A) fluorescence microscope (B) flow cytometry (n = 3). Results were determined by Student's t-test, ^*P < 0.001. Error bars represent the standard deviation of mean.

Figure 6

Ms_PE31 limits the apoptosis of macrophage via GBP-1 dependent manner. Recombinant strains infected PMA differentiated macrophages at post-infection (A) washed and detected the expression of caspase-3, activated caspase-3 and hGBP-1 proteins, by western blot. (B) qRT-PCR was executed, by using isolated RNA, to analyzed mRNA level of hGBP-1 (n = 3). In both experiments, β-actin considered as internal control. Results were determined by Student's t-test, ^***P < 0.001. Error bars represent the standard deviation of mean.

Figure 7

Ms_PE31 regulates the inflammatory molecules in macrophages via NF-κB signaling. TPCK (NF-κB inhibitor) pre-treated differentiated THP-1 cells were infected with recombinant strains. Total RNA was collected to carry out qRT-PCR to detect the mRNA level of (A) IL-10 (n = 3) (B) hGBP-1 (n = 3) (C) IL-6 (n = 3) (D) and IL-12p40 (n = 3). The β-actin gene of macrophages cells was used for internal control. Results were determined by Student's t-test, ^**P < 0.01. Error bars represent the standard deviation of mean.

Figure 8

Schematic delineation of Ms_PE31 and interaction with macrophage proteins. Ms_PE31 induced the IL-10, GTPase family protein GBP-1 and down-regulates the IL-12p40 and IL-6 feasibly by the NF-κB pathway. These proteins inhibit the activation of caspase-3 to reduce the macrophages apoptosis level and boost the recombinant M. smegmatis survivorship in macrophages.