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.

Experimental Structures of Antibody/MHC-I Complexes Reveal Details of Epitopes Overlooked by Computational Prediction.

mAbs to MHC class I (MHC-I) molecules have proved to be crucial reagents for tissue typing and fundamental studies of immune recognition. To augment our understanding of epitopic sites seen by a set of anti-MHC-I mAb, we determined X-ray crystal structures of four complexes of anti-MHC-I Fabs bound to peptide/MHC-I/ß2-microglobulin (pMHC-I). An anti-H2-Dd mAb, two anti-MHC-I α3 domain mAbs, and an anti-ß2-microglobulin mAb bind pMHC-I at sites consistent with earlier mutational and functional experiments, and the structures explain allelomorph specificity. Comparison of the experimentally determined structures with computationally derived models using AlphaFold Multimer showed that although predictions of the individual pMHC-I heterodimers were quite acceptable, the computational models failed to properly identify the docking sites of the mAb on pMHC-I. The experimental and predicted structures provide insight into strengths and weaknesses of purely computational approaches and suggest areas that merit additional attention.

Experimental structures of antibody/MHC-I complexes reveal details of epitopes overlooked by computational prediction.

Monoclonal antibodies (mAb) to major histocompatibility complex class I (MHC-I) molecules have proved to be crucial reagents for tissue typing and fundamental studies of immune recognition. To augment our understanding of epitopic sites seen by a set of anti-MHC-I mAb, we determined X-ray crystal structures of four complexes of anti-MHC-I antigen-binding fragments (Fab) bound to peptide/MHC-I/ß2m (pMHC-I). An anti-H2-Dd mAb, two anti-MHC-I α3 domain mAb, and an anti-ß2-microglobulin (ß2m) mAb bind pMHC-I at sites consistent with earlier mutational and functional experiments, and the structures explain allelomorph specificity. Comparison of the experimentally determined structures with computationally derived models using AlphaFold Multimer (AF-M) showed that although predictions of the individual pMHC-I heterodimers were quite acceptable, the computational models failed to properly identify the docking sites of the mAb on pMHC-I. The experimental and predicted structures provide insight into strengths and weaknesses of purely computational approaches and suggest areas that merit additional attention.

SARS-CoV-2 antibodies recognize 23 distinct epitopic sites on the receptor binding domain.

The COVID-19 pandemic and SARS-CoV-2 variants have dramatically illustrated the need for a better understanding of antigen (epitope)-antibody (paratope) interactions. To gain insight into the immunogenic characteristics of epitopic sites (ES), we systematically investigated the structures of 340 Abs and 83 nanobodies (Nbs) complexed with the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein. We identified 23 distinct ES on the RBD surface and determined the frequencies of amino acid usage in the corresponding CDR paratopes. We describe a clustering method for analysis of ES similarities that reveals binding motifs of the paratopes and that provides insights for vaccine design and therapies for SARS-CoV-2, as well as a broader understanding of the structural basis of Ab-protein antigen (Ag) interactions.

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.

SARS-CoV-2 antibodies recognize 23 distinct epitopic sites on the receptor binding domain.

The COVID-19 pandemic and SARS-CoV-2 variants have dramatically illustrated the need for a better understanding of antigen (epitope)-antibody (paratope) interactions. To gain insight into the immunogenic characteristics of epitopic sites (ES), we systematically investigated the structures of 340 Abs and 83 nanobodies (Nbs) complexed with the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein. We identified 23 distinct ES on the RBD surface and determined the frequencies of amino acid usage in the corresponding CDR paratopes. We describe a clustering method for analysis of ES similarities that reveals binding motifs of the paratopes and that provides insights for vaccine design and therapies for SARS-CoV-2, as well as a broader understanding of the structural basis of Ab-protein antigen (Ag) interactions.

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.

Outcomes of in-hospital cardiac arrest in COVID-19 patients: A proportional prevalence meta-analysis.

BACKGROUND: Limited epidemiological data are available on the outcomes of in-hospital cardiac arrest (CA) in COVID-19 patients. METHODS: We performed literature search of PubMed, EMBASE, Cochrane, and Ovid to identify research articles that studied outcomes of in-hospital cardiac arrest in COVID-19 patients. The primary outcome was survival at discharge. Secondary outcomes included return of spontaneous circulation (ROSC) and types of cardiac arrest. Pooled percentages with a 95% confidence interval (CI) were calculated for the prevalence of outcomes. RESULTS: A total of 7,891 COVID patients were included in the study. There were 621 (pooled prevalence 8%, 95% CI 4-13%) cardiac arrest patients. There were 52 (pooled prevalence 3.0%; 95% CI 0.0-10.0%) patients that survived at the time of discharge. ROSC was achieved in 202 (pooled prevalence 39%;95% CI 21.0-59.0%) patients. Mean time to ROSC was 7.74 (95% CI 7.51-7.98) min. The commonest rhythm at the time of cardiac arrest was pulseless electrical activity (pooled prevalence 46%; 95% 13-80%), followed by asystole (pooled prevalence 40%; 95% CI 6-80%). Unstable ventricular arrhythmia occurred in a minority of patients (pooled prevalence 8%; 95% CI 4-13%). CONCLUSION: This pooled analysis of studies showed that the survival post in-hospital cardiac arrest in COVID patients is dismal despite adequate ROSC obtained at the time of resuscitation. Nonshockable rhythm cardiac arrest is commoner suggesting a non-cardiac cause while cardiac related etiology is uncommon. Future studies are needed to improve the survival in these patients.

Structures of synthetic nanobody-SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction.

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.

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.

Structures of synthetic nanobody-SARS-CoV-2-RBD complexes reveal distinct sites of interaction and recognition of variants.

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of new variants demands understanding the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here we report five X-ray crystal structures of sybodies (synthetic nanobodies) including binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68; and Sb16 unliganded. These reveal that Sb14, Sb16, and Sb45 bind the RBD at the ACE2 interface and that the Sb16 interaction is accompanied by a large CDR2 shift. In contrast, Sb68 interacts at the periphery of the interface. We also determined cryo-EM structures of Sb45 bound to spike (S). Superposition of the X-ray structures of sybodies onto the trimeric S protein cryo-EM map indicates some may bind both "up" and "down" configurations, but others may not. Sensitivity of sybody binding to several recently identified RBD mutants is consistent with these structures.

Development and Validation of Signature Sequence-Based PCR for Improved Molecular Diagnosis of Tuberculosis.

Reliable, fast, and affordable diagnosis for tuberculosis (TB) remains a challenge to reduce disease incidence in resource-poor countries. Tests based on nucleotide sequences that are signature to Mycobacterium tuberculosis have the potential to make a positive impact on case detection rates, which can eventually help control TB. Using extensive comparative bioinformatics approach, we mined the genome for M. tuberculosis-specific genes and identified four genes so-called signature sequence (SS). With <25% homology with other known genes/proteins of mycobacterial/nonmycobacterial origin in various databases, these SS genes are ideal targets for species-specific identification. Sputum from suspected patients was liquefied using novel complete liquefying reagent, and DNA was isolated. Samples from patients (n = 417), reporting to TB clinics at two different hospitals, which met our inclusion criteria, were collected for this study. A small number (n = 143) was used for initial standardization, and the remaining patient samples (n = 274) were evaluated by SS and compared with smear microscopy, GeneXpert, culture, and clinical outcome. An overwhelming sensitivity of 97.0%, significantly higher than GeneXpert (95.0%), was seen. SS could pick all smear-negative, but culture-positive samples, along with other culture-negative samples; some of the latter were declared clinically positive. Our results yielded superior sensitivity and specificity through conventional PCR.

Synthetic nanobody-SARS-CoV-2 receptor-binding domain structures identify distinct epitopes.

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands unprecedented attention. We report four X-ray crystal structures of three synthetic nanobodies (sybodies) (Sb16, Sb45 and Sb68) bind to the receptor-binding domain (RBD) of SARS-CoV-2: binary complexes of Sb16-RBD and Sb45-RBD; a ternary complex of Sb45-RBD-Sb68; and Sb16 unliganded. Sb16 and Sb45 bind the RBD at the ACE2 interface, positioning their CDR2 and CDR3 loops diametrically. Sb16 reveals a large CDR2 shift when binding the RBD. Sb68 interacts peripherally at the ACE2 interface; steric clashes with glycans explain its mechanism of viral neutralization. Superposing these structures onto trimeric spike (S) protein models indicates these sybodies bind conformations of the mature S protein differently, which may aid therapeutic design. ONE SENTENCE SUMMARY: X-ray structures of synthetic nanobodies complexed with the receptor-binding domain of the spike protein of SARS-CoV-2 reveal details of CDR loop interactions in recognition of distinct epitopic sites.

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.

Disorder-to-order transition in PE-PPE proteins of Mycobacterium tuberculosis augments the pro-pathogen immune response.

A growing body of evidence supports the hypothesis that intrinsically disordered proteins often mediate host-pathogen interactions and modulate host functions for pathogen survival and virulence. Mycobacterium tuberculosis (M.tb) has evolved largely through reductive evolution, with a few exceptions such as the glycine-alanine-rich PE-PPE/PGRS protein family, which has been expanding in pathogenic mycobacteria. Here, our analyses of the M.tb proteome and secretome revealed that the PE-PGRS subfamily is enriched for disordered regions and disordered binding sites, pointing to their importance in host-pathogen interactions. As a case study, the secondary structure of PE35-PPE68 and PE32-PPE65 of the pathogenesis-related RD1 and RD8 regions was analyzed through Fourier-transform infrared spectroscopy. These disordered proteins displayed a considerable structural shift from disordered to ordered while engaged in the formation of complexes. While these proteins are immunogenic individually and enhance the pro-pathogen response, their corresponding complexes enhanced the responses manifold as displayed here by PE35 and PPE68. It is likely that M.tb exploits such disorder-order structural dynamics as a strategy to mount a pro-pathogen response and subvert host defense for productive infection. This functional gain also serves as a means to compensate genomic content loss due to reductive evolution.

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.

Comparative analyses of nonpathogenic, opportunistic, and totally pathogenic mycobacteria reveal genomic and biochemical variabilities and highlight the survival attributes of Mycobacterium tuberculosis.

UNLABELLED: Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size--their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets. IMPORTANCE: The complete sequence analysis of Mycobacterium indicus pranii, a novel species of Mycobacterium shown earlier to have strong immunomodulatory properties and currently in use for the treatment of leprosy, places it evolutionarily at the point of transition to pathogenicity. With the purpose of establishing the importance of M. indicus pranii in providing insight into the virulence mechanism of tuberculous and nontuberculous mycobacteria, we carried out comparative genomic and proteomic analyses of 44 mycobacterial species representing nonpathogenic (NP), opportunistic (OP), and totally pathogenic (TP) mycobacteria. Our results clearly placed M. indicus pranii as an ancestor of the M. avium complex. Analyses of comparative metabolic pathways between M. indicus pranii (NP), M. tuberculosis (TP), and M. intracellulare (OP) pointed to the presence of novel alternative pathways in M. tuberculosis with implications for pathogenesis and survival in the human host and identification of new drug targets.