Mycobacterium tuberculosis Methyltransferase Rv1515c Can Suppress Host Defense Mechanisms by Modulating Immune Functions Utilizing a Multipronged Mechanism.

Mycobacterium tuberculosis (M. tb) gene Rv1515c encodes a conserved hypothetical protein exclusively present within organisms of MTB complex and absent in non-pathogenic mycobacteria. In silico analysis revealed that Rv1515c contain S-adenosylmethionine binding site and methyltransferase domain. The DNA binding and DNA methyltransferase activity of Rv1515c was confirmed in vitro. Knock-in of Rv1515c in a model mycobacteria M. smegmatis (M. s_Rv1515c) resulted in remarkable physiological and morphological changes and conferred the recombinant strain with an ability to adapt to various stress conditions, including resistance to TB drugs. M. s_Rv1515c was phagocytosed at a greater rate and displayed extended intra-macrophage survival in vitro. Recombinant M. s_Rv1515c contributed to enhanced virulence by suppressing the host defense mechanisms including RNS and ROS production, and apoptotic clearance. M. s_Rv1515c, while suppressing the phagolysosomal maturation, modulated pro-inflammatory cytokine production and also inhibited antigen presentation by downregulating the expression of MHC-I/MHC-II and co-stimulatory signals CD80 and CD86. Mice infected with M. s_Rv1515c produced more Treg cells than vector control (M. s_Vc) and exhibited reduced effector T cell responses, along-with reduced expression of macrophage activation markers in the chronic phase of infection. M. s_Rv1515c was able to survive in the major organs of mice up to 7 weeks post-infection. These results indicate a crucial role of Rv1515c in M. tb pathogenesis.

PGRS Domain of Rv0297 of Mycobacterium tuberculosis Functions in A Calcium Dependent Manner.

Mycobacterium tuberculosis (M.tb), the pathogen causing tuberculosis, is a major threat to human health worldwide. Nearly 10% of M.tb genome encodes for a unique family of PE/PPE/PGRS proteins present exclusively in the genus Mycobacterium. The functions of most of these proteins are yet unexplored. The PGRS domains of these proteins have been hypothesized to consist of Ca2+ binding motifs that help these intrinsically disordered proteins to modulate the host cellular responses. Ca2+ is an important secondary messenger that is involved in the pathogenesis of tuberculosis in diverse ways. This study presents the calcium-dependent function of the PGRS domain of Rv0297 (PE_PGRS5) in M.tb virulence and pathogenesis. Tandem repeat search revealed the presence of repetitive Ca2+ binding motifs in the PGRS domain of the Rv0297 protein (Rv0297PGRS). Molecular Dynamics simulations and fluorescence spectroscopy revealed Ca2+ dependent stabilization of the Rv0297PGRS protein. Calcium stabilized Rv0297PGRS enhances the interaction of Rv0297PGRS with surface localized Toll like receptor 4 (TLR4) of macrophages. The Ca2+ stabilized binding of Rv0297PGRS with the surface receptor of macrophages enhances its downstream consequences in terms of Nitric Oxide (NO) production and cytokine release. Thus, this study points to hitherto unidentified roles of calcium-modulated PE_PGRS proteins in the virulence of M.tb. Understanding the pathogenic potential of Ca2+ dependent PE_PGRS proteins can aid in targeting these proteins for therapeutic interventions.

Mycobacterium tuberculosis Specific Protein Rv1509 Evokes Efficient Innate and Adaptive Immune Response Indicative of Protective Th1 Immune Signature.

Dissecting the function(s) of proteins present exclusively in Mycobacterium tuberculosis (M.tb) will provide important clues regarding the role of these proteins in mycobacterial pathogenesis. Using extensive computational approaches, we shortlisted ORFs/proteins unique to M.tb among 13 different species of mycobacteria and identified a hypothetical protein Rv1509 as a 'signature protein' of M.tb. This unique protein was found to be present only in M.tb and absent in all other mycobacterial species, including BCG. In silico analysis identified numerous putative T cell and B cell epitopes in Rv1509. Initial in vitro experiments using innate immune cells demonstrated Rv1509 to be immunogenic with potential to modulate innate immune responses. Macrophages treated with Rv1509 exhibited higher activation status along with substantial release of pro-inflammatory cytokines. Besides, Rv1509 protein boosts dendritic cell maturation by increasing the expression of activation markers such as CD80, HLA-DR and decreasing DC-SIGN expression and this interaction was mediated by innate immune receptor TLR2. Further, in vivo experiments in mice demonstrated that Rv1509 protein promotes the expansion of multifunctional CD4+ and CD8+T cells and induces effector memory response along with evoking a canonical Th1 type of immune response. Rv1509 also induces substantial B cell response as revealed by increased IgG reactivity in sera of immunized animals. This allowed us to demonstrate the diagnostic efficacy of this protein in sera of human TB patients compared to the healthy controls. Taken together, our results reveal that Rv1509 signature protein has immunomodulatory functions evoking immunological memory response with possible implications in serodiagnosis and TB vaccine development.

PGRS Domain of Rv0297 of Mycobacterium tuberculosis Is Involved in Modulation of Macrophage Functions to Favor Bacterial Persistence.

Mycobacterium tuberculosis (M. tb) Rv0297-encoded PE_PGRS5 has been known to be expressed at the later stages of infection and in acidified phagosomes during transcriptome and proteomic studies. The possible role of Rv0297 in the modulation of phagosomal maturation and in providing protection against a microbicidal environment has been hypothesized. We show that Rv0297PGRS is involved in modulating the calcium homeostasis of macrophages followed by impedance of the phagolysosomal acidification process. This is evident from the downregulation of the late endosomal markers (Rab7 and cathepsin D) in the macrophages infected with recombinant Mycobacterium smegmatis (rM.smeg)-M.smeg_Rv0297 and M.smeg_Rv0297PGRS-or treated with recombinant Rv0297PGRS protein. Macrophages infected with rM.smeg expressing Rv0297 produce nitric oxide and undergo apoptosis, which may aid in the dissemination of pathogen in the later stages of infection. Rv0297 was also found to be involved in rescuing the bacterium from oxidative and hypoxic stress employed by macrophages and augmented the survivability of the recombinant bacterium. These results attribute to the functional significance of this protein in M.tb virulence mechanism. The fact that this protein gets expressed at the later stages of lung granulomas during M.tb infection suggests that the bacterium possibly employs Rv0297 as its dissemination and survival strategy.

The PGRS Domain of Mycobacterium tuberculosis PE_PGRS Protein Rv0297 Is Involved in Endoplasmic Reticulum Stress-Mediated Apoptosis through Toll-Like Receptor 4.

The genome of Mycobacterium tuberculosis, the causal organism of tuberculosis (TB), encodes a unique protein family known as the PE/PPE/PGRS family, present exclusively in the genus Mycobacterium and nowhere else in the living kingdom, with largely unexplored functions. We describe the functional significance of the PGRS domain of Rv0297, a member of this family. In silico analyses revealed the presence of intrinsically disordered stretches and putative endoplasmic reticulum (ER) localization signals in the PGRS domain of Rv0297 (Rv0297PGRS). The PGRS domain aids in ER localization, which was shown by infecting macrophage cells with M. tuberculosis and by overexpressing the protein by transfection in macrophage cells followed by activation of the unfolded protein response, as evident from increased expression of GRP78/GRP94 and CHOP/ATF4, leading to disruption of intracellular Ca2+ homeostasis and increased nitric oxide (NO) and reactive oxygen species (ROS) production. The consequent activation of the effector caspase-8 resulted in apoptosis of macrophages, which was Toll-like receptor 4 (TLR4) dependent. Administration of recombinant Rv0297PGRS (rRv0297PGRS) also exhibited similar effects. These results implicate a hitherto-unknown role of the PGRS domain of the PE_PGRS protein family in ER stress-mediated cell death through TLR4. Since this protein is already known to be present at later stages of infection in human granulomas it points to the possibility of it being employed by M. tuberculosis for its dissemination via an apoptotic mechanism.IMPORTANCE Apoptosis is generally thought to be a defense mechanism in protecting the host against Mycobacterium tuberculosis in early stages of infection. However, apoptosis during later stages in lung granulomas may favor the bacterium in disseminating the disease. ER stress has been found to induce apoptosis in TB granulomas, in zones where apoptotic macrophages accumulate in mice and humans. In this study, we report ER stress-mediated apoptosis of host cells by the Rv0297-encoded PE_PGRS5 protein of M. tuberculosis exceptionally present in the pathogenic Mycobacterium genus. The PGRS domain of Rv0297 aids the protein in localizing to the ER and induces the unfolded protein response followed by apoptosis of macrophages. The effect of the Rv0297PGRS domain was found to be TLR4 dependent. This study presents novel insights on the strategies employed by M. tuberculosis to disseminate the disease.