RESUMO
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.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Regulação Bacteriana da Expressão Gênica , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium tuberculosis/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Humanos , Macrófagos/microbiologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/crescimento & desenvolvimento , Conformação Proteica , Homologia de SequênciaRESUMO
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.
RESUMO
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.
Assuntos
Antígenos de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Células Th1/imunologia , Tuberculose/imunologia , Imunidade Adaptativa , Animais , Antígenos de Bactérias/isolamento & purificação , Humanos , Imunidade Inata , Camundongos , Células RAW 264.7 , Desenvolvimento de VacinasRESUMO
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.
Assuntos
Mycobacterium tuberculosis , Proteínas de Bactérias/genética , Macrófagos , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , ProteômicaRESUMO
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.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Apoptose , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Estresse do Retículo Endoplasmático , Macrófagos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mycobacterium tuberculosis/fisiologia , Receptor 4 Toll-Like/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/citologia , Macrófagos/microbiologia , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Camundongos , Mycobacterium tuberculosis/patogenicidade , Ligação Proteica , Sinais Direcionadores de Proteínas , Células RAW 264.7 , Receptor 4 Toll-Like/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Background: Patients with Human Immunodeficiency Virus (HIV) infection are predisposed to numerous opportunistic infections due to decreased cell mediated immunity, Tuberculosis being most common. Low CD4 count is associated with low immunity and higher risk of tuberculosis.Methods: Author conducted a retrospective study in the department of Pulmonary medicine in a tertiary care teaching hospital during January to December 2017. Author collected data of all the patients with HIV diagnosed with Tuberculosis from the ART centre. Author collected demographic details including age, sex, symptoms at presentation, details of diagnosis of TB including type of tuberculosis, CBNAAT results, CD4 count at the diagnosis of TB, details of ART therapy and ATT therapy and outcomes of treatment.Results: Eighty one patients with HIV-TB co- infection were included in the study. Males (70.37%) were more affected than females. Mean age of the study group was 39.97� years. Sixty one patients (75.4%) were diagnosed with Pulmonary Tuberculosis and 20 (24.6%) patients were diagnosed with extra pulmonary TB. Mean CD4 counts of the cohort was 226�0/祃. Eighty percent of patients developed Tuberculosis with CD4 count <250/祃.Conclusions: Author found in this study higher proportions of tuberculosis (80.2%) in patients with HIV infection with CD4 count <200/祃. Author also found higher proportion of pulmonary Koch抯 in patients with low CD4 count (CD4 <200/祃).
RESUMO
Ovarian cancer often goes undiagnosed or misdiagnosed in the early stages. The present study aimed to validate a modified version of the Goff Symptom Index [GSI] in an Indian population. This prospective case-control study was conducted between July 2010 and June 2012 in a university hospital in Manipal, Karnataka, India. A total of 305 inpatients admitted for ovarian pathology investigations and outpatients undergoing routine gynaecological check-ups were included in the study. The modified GSI [MGSI] was used to investigate the presence, severity, frequency and duration of 10 ovarian cancer symptoms on a scale of 1-5. Four additional symptoms were included with those of the original GSI [two symptoms from a previous MGSI and two new symptoms]. Patients were regarded as positive for ovarian cancer if symptoms occurred >12 times per month and time since onset was <1 year. Histopathology confirmed the diagnosis of ovarian tumours. A total of 13 patients were excluded. The final sample [n = 292] was divided into a test group [n = 74] and a control group [n = 218] based on histopathology. Within the controls, 144 women were found to have benign tumours. The MGSI was positive in 71.6% of the test group as opposed to only 11.5% of the control group. The addition of two symptoms [loss of appetite and weight] to the GSI increased the test's sensitivity from 71.6% to 77% without compromising specificity [88.5%].Based on these findings, the addition of two new symptoms [loss of appetite and weight] to the GSI is proposed in order to increase the test's sensitivity. However, the addition of urinary symptoms to the GSI requires further validation