Expression of a unique M. tuberculosis DNA MTase Rv1509 in M. smegmatis alters the gene expression pattern and enhances virulence.

Mycobacterium tuberculosis (M. tb) genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using in-silico comparative genome analysis, we identified one of the M. tb genes, Rv1509, as a signature protein exclusively present in M. tb. To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in Mycobacterium smegmatis (M. smegmatis) constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, in-vitro and in-vivo studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. In-vivo infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathological manifestations in the pancreas after intraperitoneal infection. Long-time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in M. tb pathogenesis.

Chaperone function in antigen presentation by MHC class I molecules-tapasin in the PLC and TAPBPR beyond.

Peptide loading of MHC-I molecules plays a critical role in the T cell response to infections and tumors as well as to interactions with inhibitory receptors on natural killer (NK) cells. To facilitate and optimize peptide acquisition, vertebrates have evolved specialized chaperones to stabilize MHC-I molecules during their biosynthesis and to catalyze peptide exchange favoring high affinity or optimal peptides to permit transport to the cell surface where stable peptide/MHC-I (pMHC-I) complexes are displayed and are available for interaction with T cell receptors and any of a host of inhibitory and activating receptors. Although components of the endoplasmic reticulum (ER) resident peptide loading complex (PLC) were identified some 30 years ago, the detailed biophysical parameters that govern peptide selection, binding, and surface display have recently been understood better with advances in structural methods including X-ray crystallography, cryogenic electron microscopy (cryo-EM), and computational modeling. These approaches have provided refined mechanistic illustration of the molecular events involved in the folding of the MHC-I heavy chain, its coordinate glycosylation, assembly with its light chain, ß2-microglobulin (ß2m), its association with the PLC, and its binding of peptides. Our current view of this important cellular process as it relates to antigen presentation to CD8+ T cells is based on many different approaches: biochemical, genetic, structural, computational, cell biological, and immunological. In this review, taking advantage of recent X-ray and cryo-EM structural evidence and molecular dynamics simulations, examined in the context of past experiments, we attempt a dispassionate evaluation of the details of peptide loading in the MHC-I pathway. By critical evaluation of several decades of investigation, we outline aspects of the peptide loading process that are well-understood and indicate those that demand further detailed investigation. Further studies should contribute not only to basic understanding, but also to applications for immunization and therapy of tumors and infections.

Structural mechanism of tapasin-mediated MHC-I peptide loading in antigen presentation.

Loading of MHC-I molecules with peptide by the catalytic chaperone tapasin in the peptide loading complex plays a critical role in antigen presentation and immune recognition. Mechanistic insight has been hampered by the lack of detailed structural information concerning tapasin-MHC-I. We present here crystal structures of human tapasin complexed with the MHC-I molecule HLA-B*44:05, and with each of two anti-tapasin antibodies. The tapasin-stabilized peptide-receptive state of HLA-B*44:05 is characterized by distortion of the peptide binding groove and destabilization of the ß2-microglobulin interaction, leading to release of peptide. Movements of the membrane proximal Ig-like domains of tapasin, HLA-B*44:05, and ß2-microglobulin accompany the transition to a peptide-receptive state. Together this ensemble of crystal structures provides insights into a distinct mechanism of tapasin-mediated peptide exchange.

Chaperones and Catalysts: How Antigen Presentation Pathways Cope With Biological Necessity.

Immune recognition by T lymphocytes and natural killer (NK) cells is in large part dependent on the identification of cell surface MHC molecules bearing peptides generated from either endogenous (MHC I) or exogenous (MHC II) dependent pathways. This review focuses on MHC I molecules that coordinately fold to bind self or foreign peptides for such surface display. Peptide loading occurs in an antigen presentation pathway that includes either the multimolecular peptide loading complex (PLC) or a single chain chaperone/catalyst, TAP binding protein, related, TAPBPR, that mimics a key component of the PLC, tapasin. Recent structural and dynamic studies of TAPBPR reveal details of its function and reflect on mechanisms common to tapasin. Regions of structural conservation among species suggest that TAPBPR and tapasin have evolved to satisfy functional complexities demanded by the enormous polymorphism of MHC I molecules. Recent studies suggest that these two chaperone/catalysts exploit structural flexibility and dynamics to stabilize MHC molecules and facilitate peptide loading.

Mycobacterium smegmatis Bacteria Expressing Mycobacterium tuberculosis-Specific Rv1954A Induce Macrophage Activation and Modulate the Immune Response.

Mycobacterium tuberculosis (M. tb), the intracellular pathogen causing tuberculosis, has developed mechanisms that endow infectivity and allow it to modulate host immune response for its survival. Genomic and proteomic analyses of non-pathogenic and pathogenic mycobacteria showed presence of genes and proteins that are specific to M. tb. In silico studies predicted that M.tb Rv1954A is a hypothetical secretory protein that exhibits intrinsically disordered regions and possess B cell/T cell epitopes. Treatment of macrophages with Rv1954A led to TLR4-mediated activation with concomitant increase in secretion of pro-inflammatory cytokines, IL-12 and TNF-α. In vitro studies showed that rRv1954A protein or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) activates macrophages by enhancing the expression of CD80 and CD86. An upregulation in the expression of CD40 and MHC I/II was noted in the presence of Rv1954A, pointing to its role in enhancing the association of APCs with T cells and in the modulation of antigen presentation, respectively. Ms_Rv1954A showed increased infectivity, induction of ROS and RNS, and apoptosis in RAW264.7 macrophage cells. Rv1954A imparted protection against oxidative and nitrosative stress, thereby enhancing the survival of Ms_Rv1954A inside macrophages. Mice immunized with Ms_Rv1954A showed that splenomegaly and primed splenocytes restimulated with Rv1954A elicited a Th1 response. Infection of Ms_Rv1954A in mice through intratracheal instillation leads to enhanced infiltration of lymphocytes in the lungs without formation of granuloma. While Rv1954A is immunogenic, it did not cause adverse pathology. Purified Rv1954A or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) elicited a nearly two-fold higher titer of IgG response in mice, and PTB patients possess a higher IgG titer against Rv1954A, also pointing to its utility as a diagnostic marker for TB. The observed modulation of innate and adaptive immunity renders Rv1954A a vital protein in the pathophysiology of this pathogen.

Immunodominant Mycobacterium tuberculosis Protein Rv1507A Elicits Th1 Response and Modulates Host Macrophage Effector Functions.

Mycobacterium tuberculosis (M. tb) persists as latent infection in nearly a quarter of the global population and remains the leading cause of death among infectious diseases. While BCG is the only vaccine for TB, its inability to provide complete protection makes it imperative to engineer BCG such that it expresses immunodominant antigens that can enhance its protective potential. In-silico comparative genomic analysis of Mycobacterium species identified M. tb Rv1507A as a "signature protein" found exclusively in M. tb. In-vitro (cell lines) and in-vivo experiments carried out in mice, using purified recombinant Rv1507A revealed it to be a pro-inflammatory molecule, eliciting significantly high levels of IL-6, TNF-α, and IL-12. There was increased expression of activation markers CD69, CD80, CD86, antigen presentation molecules (MHC I/MHCII), and associated Th1 type of immune response. Rv1507A knocked-in M. smegmatis also induced significantly higher pro-inflammatory Th1 response and higher survivability under stress conditions, both in-vitro (macrophage RAW264.7 cells) and in-vivo (mice). Sera derived from human TB patients showed significantly enhanced B-cell response against M. tb Rv1507A. The ability of M. tb Rv1507A to induce immuno-modulatory effect, B cell response, and significant memory response, renders it a putative vaccine candidate that demands further exploration.

Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein.

Pathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion-an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis.IMPORTANCE To hijack the human host cell machinery to enable survival inside macrophages, the pathogen Mycobacterium tuberculosis requires a repertoire of proteins that can mimic host protein function and modulate host cell machinery. Here, we have shown how a single protein can play multiple functions and hijack the host cell for the benefit of the pathogen. Full-length membrane-anchored PPE37 protein is cleaved into N- and C-terminal domains under iron-depleted conditions. The N-terminal domain facilitates the propathogen semimature tolerogenic state of dendritic cells, whereas the C-terminal segment is localized into host cell nucleus and induces apoptosis. The immune implications of these in vitro observations were assessed and validated in mice and also human TB patients. This study presents novel mechanistic insight adopted by M. tuberculosis to survive inside host cells.

TNF-alpha -308G/A and -238G/A polymorphisms and its protein network associated with type 2 diabetes mellitus.

Several reports document the role of tumor necrosis factor alpha (TNF-α) and lipid metabolism in the context of acute inflammation as a causative factor in obesity-associated insulin resistance and as one of the causative parameter of type 2 diabetes mellitus (T2DM). Our aim was to investigate the association between -308G/A and -238G/A polymorphisms located in the promoter region of the TNF-α gene in T2DM in the Indian population with bioinformatics analysis of TNF-α protein networking with an aim to find new target sites for the treatment of T2DM. Demographics of 100 diabetes patients and 100 healthy volunteers were collected in a structured proforma and 3 ml blood samples were obtained from the study group, after approval of Institutional Ethics Committee of the hospital (IEC). The information on clinical parameters was obtained from medical records. Genomic DNA was extracted; PCR-RFLP was performed using TNF-α primers specific to detect the presence of SNPs. Various bioinformatics tools such as STRING software were used to determine its network with other associated genes. The PCR-RFLP studies showed that among the -238G/A types the GG genotype was 87%, GA genotype was 12% and AA genotype was 1%. Almost a similar pattern of results was obtained with TNF-α -308G/A polymorphism. The results obtained were evaluated statistically to determine the significance. By constructing TNF-α protein interaction network we could analyze ontology and hubness of the network to identify the networking of this gene which may influence the functioning of other genes in promoting T2DM. We could identify new targets in T2DM which may function in association with TNF-α. Through hub analysis of TNF-α protein network we have identified three novel proteins RIPK1, BIRC2 and BIRC3 which may contribute to TNF-mediated T2DM pathogenesis. In conclusion, our study indicated that some of the genotypes of TNF-α -308G/A, -238G/A were not significantly associated to type 2 diabetes mellitus, but TNF-α -308G/A polymorphism was reported to be a potent risk factor for diabetes in higher age (>45) groups. Also, the novel hub proteins may serve as new targets against TNF-α T2DM pathogenesis.