Role of M.tuberculosis protein Rv2005c in the aminoglycosides resistance.

Tuberculosis is an airborne infectious disease caused by Mycobacterium tuberculosis which threatens the globe. Aminoglycosides {Amikacin (AK) & Kanamycin (KM)} are WHO recommended second-line anti-TB drugs used against the treatment of drug-resistant tuberculosis. Aminoglycosides target the steps of protein translation machinery of M.tuberculosis. Several mechanisms have been put forward to elucidate the phenomena of aminoglycosides resistance but our knowledge is still insufficient. The aim of the study was to understand the involvement of Mycobacterium tuberculosis universal stress protein (Rv2005c) in aminoglycosides resistance and virulence. To establish the relationship of universal stress protein Rv2005c with AK & KM resistance, Rv2005c was cloned, expressed in E.coli BL21 using pQE2 expression vector and antimicrobial drug susceptibility testing (DST) was carried out. STRING-10 was also used to predict the interacting protein partners of Rv2005c. DST showed that the minimum inhibitory concentration of induced recombinant cells (Rv2005c) were five and four folds shifted with AK and KM E-strips, respectively. STRING-10 showed the interacting protein partners of Rv2005c. Overexpression of Rv2005c leads to shifting in MIC which might be signifying its involvement in the survival/resistance of Mycobacteria by inhibiting/modulating the effects of AK and KM released from the E-strips. Interactome also suggests that Rv2005c and its interacting protein partners are cumulatively involved in M.tuberculosis resistance, stresses, and latency.

Transcription factors STAT-4, STAT-6 and CREB regulate Th1/Th2 response in leprosy patients: effect of M. leprae antigens.

BACKGROUND: Leprosy is an ideal human disease to study T cell regulation as patients show correlation between cytokine skewed Th1-Th2 responses and clinical forms of the disease. The Role of transcription factors on the modulation of Th1 and Th2 responses by M. leprae antigens has not been adequately studied. In the present study, we studied the effect of M. leprae antigens on transcription factors STAT-4, STAT-6 and CREB and their correlation with Th1/Th2 cell mediated immune responses in leprosy. METHODS: Leprosy patients of both categories of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were selected from the OPD of NJ1L & OMD, (ICMR), Agra and healthy individuals (H) were chosen from the staff and students working in the institute. Peripheral blood mononuclear cells (PBMCs) of the study subjects were stimulated with M. leprae antigens (WCL, MLSA, and PGL-1). Sandwich ELISA was done in the culture supernatants of healthy and leprosy patients to detect IL-4, IL-10 and IFN-γ. Further, expression of IFN-γ and IL-4 and activation of STAT4, STAT6 and CREB transcription factors in CD4+ T cell with or without stimulation of M. leprae antigens was investigated by flow cytometry. RESULTS: Lepromatous leprosy patients showed significantly lower IFN-γ and higher IL-4 levels in culture supernatant and significantly low expression of IFN-γ and higher expression of IL-4 by CD4+ T cells than healthy individuals with or without antigenic stimulation. Antigenic stimulation significantly increased IL-10 in BL/LL patients but not in BT/TT patients or healthy individuals. PGL-1 stimulation led to significantly higher activation of STAT-6 in BT/TT and BL/LL patients in comparison to healthy individuals. All the three antigens led to activation of CREB in healthy and BT/TT patients but not in BL/LL patients. CONCLUSION: Our findings show that M. leprae antigens differentially modulate activation of T cell transcription factors STAT-4/STAT-6 and CREB. These transcription factors are well known to regulate Th1 and Th2 mediated immune response which in turn could play vital role in the clinical manifestations of leprosy. These observations may help to determine how these T cell transcription factors affect the development of immune dysfunction and whether these new pathways have a role in immunomodulation in intracellular diseases like leprosy and TB.

M. tuberculosis ferritin (Rv3841): Potential involvement in Amikacin (AK) & Kanamycin (KM) resistance.

Tuberculosis is an infectious disease, caused by one of the most successful human pathogen, Mycobacterium tuberculosis. Aminoglycosides, Amikacin (AK) & Kanamycin (KM) are commonly used to treat drug resistant tuberculosis. They target the protein synthesis machinery by interacting with several steps of translation. Several explanations have been proposed to explain the mechanism of aminoglycoside resistance but still our information is inadequate. Iron storing/interacting proteins were found to be overexpressed in aminoglycosides resistant isolates. Iron assimilation and utilization in M. tuberculosis plays a crucial role in growth, virulence and latency. To establish the relationship of ferritin with AK & KM resistance ferritin (Rv3841/bfrB) was cloned, expressed and antimicrobial drug susceptibility testing (DST) was carried out. Rv3841/bfrB gene was cloned and expressed in E. coli BL21 using pQE2 expression vector. Etest results for DST against AK & KM showed that the minimum inhibitory concentration (MIC) of ferritin recombinant cells was changed. Recombinants showed two fold changes in MIC with AK and three fold with KM E-strips. Overexpression of ferritin reflect the MIC shift which might be playing a critical role in the survival of mycobacteria by inhibiting/modulating the effects of AK & KM. String analysis also suggests that ferritin interacted with few proteins which are directly and indirectly involved in M. tuberculosis growth, Iron assimilation, virulence, resistance, stresses and latency.

Mycobacterium tuberculosis secretory proteins downregulate T cell activation by interfering with proximal and downstream T cell signalling events.

BACKGROUND: Mycobacterium tuberculosis (M. tuberculosis) modulates host immune response, mainly T cell responses for its own survival leading to disease or latent infection. The molecules and mechanisms utilized to accomplish immune subversion by M. tuberculosis are not fully understood. Understanding the molecular mechanism of T cell response to M. tuberculosis is important for development of efficacious vaccine against TB. METHODS: Here, we investigated effect of M. tuberculosis antigens Ag85A and ESAT-6 on T cell signalling events in CD3/CD28 induced Peripheral blood mononuclear cells (PBMCs) of PPD+ve healthy individuals and pulmonary TB patients. We studied CD3 induced intracellular calcium mobilization in PBMCs of healthy individuals and TB patients by spectrofluorimetry, CD3 and CD28 induced activation of mitogen activated protein kinases (MAPKs) in PBMCs of healthy individuals and TB patients by western blotting and binding of transcription factors NFAT and NFκB by Electrophorectic mobility shift assay (EMSA). RESULTS: We observed CD3 triggered modulations in free intracellular calcium concentrations in PPD+ve healthy individuals and pulmonary TB patients after the treatment of M. tuberculosis antigens. As regards the downstream signalling events, phosphorylation of MAPKs, Extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 was curtailed by M. tuberculosis antigens in TB patients whereas, in PPD+ve healthy individuals only ERK1/2 phosphorylation was inhibited. Besides, the terminal signalling events like binding of transcription factors NFAT and NFκB was also altered by M. tuberculosis antigens. Altogether, our results suggest that M. tuberculosis antigens, specifically ESAT-6, interfere with TCR/CD28-induced upstream as well as downstream signalling events which might be responsible for defective IL-2 production which further contributed in T-cell unresponsiveness, implicated in the progression of disease. CONCLUSION: To the best of our knowledge, this is the first study to investigate effect of Ag85A and ESAT-6 on TCR- and TCR/CD28- induced upstream and downstream signalling events of T-cell activation in TB patients. This study showed the effect of secretory antigens of M. tuberculosis in the modulation of T cell signalling pathways. This inflection is accomplished by altering the proximal and distal events of signalling cascade which could be involved in T-cell dysfunctioning during the progression of the disease.

The role of TLR9 polymorphism in susceptibility to pulmonary tuberculosis.

Mycobacterium tuberculosis (MTB) is the causative agent of pulmonary tuberculosis (PTB), a major health problem that leads to 1.5 million deaths annually. Host genetic factors play a significant role in disease resistance/susceptibility by altering immunity against MTB. Toll-like receptor (TLR) sensors such as TLR2, TLR4, TLR8, and TLR9 are known to play a pivotal role in PTB via modulating sensor expression and/or effector responses. Single-nucleotide polymorphism (SNP) rs187084 (T-1486C) of the TLR9 promoter is associated with various autoimmune disorders and cancers. A recent bioinformatic analysis predicted that the T-1486C SNP is involved in PTB, although its potential role is unclear. To investigate the role of T-1486C in PTB, we stimulated PBMCs with the H37Rv whole cell lysate. We found that the presence of the "C" allele increases the transcriptional activity of the TLR9, which in turn induces high levels of Interferon gamma-induced protein 10 (IP-10), a biomarker for PTB. However, the expression of protective cytokines such as IFNγ and TNFα was observed significantly less with "C" allele in comparison to "T" allele. We further selected three different tribe populations showing differential susceptibility to PTB and performed genotypic analyses for the TLR9 promoter. We found a significantly lower minor allele frequency (MAF) of T-1486C in the Baiga tribe, wherein fewer PTB cases were reported, than that in the Gond and Korku tribes. Collectively, these data suggest that the minor "C" allele at rs187084 locus may be associated with susceptibility to PTB, which may explain the relatively lower PTB rates observed in Baiga tribe members.

Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates.

BACKGROUND: Tuberculosis (TB) is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR) TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates. RESULTS: On comparing the two dimensional (2-DE) gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein) were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase) of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain) of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase) of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin) of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A) of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032) of intracellular MDR and two proteins (Rv2896c and Rv2558c) of sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival. CONCLUSIONS: Mass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug-resistant and sensitive subpopulations of M. tuberculosis.

Phenolic-glycolipid-1 and lipoarabinomannan preferentially modulate TCR- and CD28-triggered proximal biochemical events, leading to T-cell unresponsiveness in mycobacterial diseases.

BACKGROUND: Advanced stages of leprosy show T cell unresponsiveness and lipids of mycobacterial origin are speculated to modulate immune responses in these patients. Present study elucidates the role of phenolicglycolipid (PGL-1) and Mannose-capped lipoarabinomannan (Man-LAM) on TCR- and TCR/CD28- mediated signalling. RESULTS: We observed that lipid antigens significantly inhibit proximal early signalling events like Zap-70 phosphorylation and calcium mobilization. Interestingly, these antigens preferentially curtailed TCR-triggered early downstream signalling events like p38 phosphorylation whereas potentiated that of Erk1/2. Further, at later stages inhibition of NFAT binding, IL-2 message, CD25 expression and T-cell blastogenesis by PGL-1 and Man-LAM was noted. CONCLUSION: Altogether, we report that Man-LAM and PGL-1 preferentially interfere with TCR/CD28-triggered upstream cell signalling events, leading to reduced IL-2 secretion and T-cell blastogenesis which potentially could lead to immunosupression and thus, disease exacerbation, as noted in disease spectrum.

Whole cell & culture filtrate proteins from prevalent genotypes of Mycobacterium tuberculosis provoke better antibody & T cell response than laboratory strain H 37 Rv.

BACKGROUND & OBJECTIVES: The immune responses to different antigens of Mycobacterium tuberculosis H 37 Rv vary from patient to patient with tuberculosis (TB). Therefore, significant difference might be documented between the H 37 Rv with long histories of passages and recent clinical isolates of M. tuberculosis. In the present study, immune response of TB patients and healthy controls against 39 clinical M. tuberculosis isolates was correlated with laboratory strain H 37 Rv. METHODS: The antibody response was studied coating whole cell extracts and culture filtrate proteins of M. tuberculosis isolates and laboratory strain H 37Rv by enzyme linked immunosorbent assay (ELISA). Lymphoproliferation was studied by incorporation of tritiated thymidine and cytokines (IFN-γ and IL-4) by using commercially available kits. RESULTS: Sero-reactivity to whole cell extract (WCE) of 11 clinical isolates was higher with pooled serum and individual's serum from tuberculosis patients showed significant reactivity (P<0.05) to ten of these isolates using ELISA. Of the WCE of 39 clinical isolates, 10 were found to be potent inducer of lymphoproliferation as well as cytokine secretion (P<0.05) in peripheral blood mononuclear cells from PPD+ healthy controls. Six culture filtrate proteins (CFPs) from these selected clinical isolates were also better inducers of antibody and T-cell response. INTERPRETATION & CONCLUSION: Overall, our results revealed that the clinical isolates belonging to prevalent genotypes; CAS1_Del (ST-26), East African-Indian (ST-11) and Beijing family (ST-1) induced better antibody and T cell responses compared to H 37 Rv laboratory strain. Further studies need to be done to purify and identify the dominant protein (s) using whole cell extract and culture filtrates from these immunologically relevant clinical M. tuberculosis isolates, which will be worthwhile to find out pathogenic factors, potential diagnostic markers and protective molecules for tuberculosis.

Activated TLR2/4-positive T cells boost cell exhaustion during lepromatous leprosy infection via PD-1 upregulation.

The most important stage in activating an appropriate immune response during an infection is pathogen detection. Pattern recognition receptors (PRRs) are innate sensors used for pathogen detection that mould and link the innate and adaptive immune responses by the host. Toll Like receptors (TLRs) specifically TLR2 and TLR4, are PRRs, which have gained prominence due to their exceptional capacity to recognize unique molecular patterns from invading pathogens. They also play a critical role in maintaining the balance between Th1 and Th2 responses, which are necessary for the host's survival. Leprosy is a spectral disease with a wide range of immunological manifestations in the host. Cells of both the innate and adaptive branches play crucial roles in this polarized immune state. Here, we have analysed the proportional expression patterns of TLR2 and TLR4 on the surface of CD3+, CD4+, CD8+, CD19+ and CD161+ lymphocytes and CD14+ monocytes in different groups of leprosy patients. Further, these TLRs positive cells were correlated with the surface markers of cell exhaustion such as Programmed Death-1 (PD-1) and its ligand (PD-L1), which indicated their role in immunosuppression. Additionally, blocking the interaction of PD-1 with PD-L1 in lymphocytes demonstrated visible improvement in their immune activation status through release of pro-inflammatory cytokines (IFN-γ and TNF-α).

Exploring modulations in T-cell receptor-mediated T-cell signaling events in systemic circulation and at local disease site of patients with tubercular pleural effusion: An attempt to understand tuberculosis pathogenesis at the local disease site.

Introduction: T cells are crucial for pathogenesis as well as control for tuberculosis (TB). Although much is known about the signaling pathways which are required for the activation of T cells during acute infection but the way these cells respond during persistent of infection still remained elusive. Therefore, it is rationale to understand T cell activation during tuberculous pleural effusion (TPE), which is similar to bacterial persistency system. Methods: Herein, we will employ T cell receptor (TCR) based approaches for studying events of T cell activation pathways in cells of blood and pleural fluid among patients with TPE. We performed spectrofluorimetric analysis to study effect of M. tuberculosis antigens, ESAT-6 and Ag85A stimulation on intracellular calcium levels, Phosphorylation levels of ZAP-70 (Zeta-chain-associated protein kinase 70), PKC-θ (Protein kinase C theta), Erk1/2 (Extracellular signal-regulated kinase 1 and 2) and p-38 two important members of MAPKs (Mitogen activated Protein kinases) in CD3 and CD28 induced cells of blood and pleural fluid of same patients with TPE by western blotting. Patients with non-TPE were also included as matching disease controls in this study. Results: We observed significantly higher intracellular calcium levels, Phosphorylation levels of ZAP-70, Erk1/2 and p-38 in CD3 and CD28 induced cells of pleural fluid as compared to the blood cells of same patients with TPE. Alteration in the activation of these events has also been noted after stimulation of ESAT-6 and Ag85A. Discussion: Present study demonstrated up-regulated activation of TCR mediated T cell signaling events at local disease site (Pleural fluid) as compared to the blood sample of TB pleurisy patients which could be involved in T-cell dysfunctioning during the progression of the disease and also could be responsible for Th 1 dominance at local disease site in patients with TPE.

The multifunctional PE_PGRS11 protein from Mycobacterium tuberculosis plays a role in regulating resistance to oxidative stress.

Mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. Infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. We demonstrate here that hypothetical PE_PGRS11 ORF encodes a functional phosphoglycerate mutase. The transcriptional analysis revealed that PE_PGRS11 is a hypoxia-responsive gene, and enforced expression of PE_PGRS11 by recombinant adenovirus or Mycobacterium smegmatis imparted resistance to alveolar epithelial cells against oxidative stress. PE_PGRS11-induced resistance to oxidative stress necessitated the modulation of genetic signatures like induced expression of Bcl2 or COX-2. This modulation of specific antiapoptotic molecular signatures involved recognition of PE_PGRS11 by TLR2 and subsequent activation of the PI3K-ERK1/2-NF-κB signaling axis. Furthermore, PE_PGRS11 markedly diminished H(2)O(2)-induced p38 MAPK activation. Interestingly, PE_PGRS11 protein was exposed at the mycobacterial cell surface and was involved in survival of mycobacteria under oxidative stress. Furthermore, PE_PGRS11 displayed differential B cell responses during tuberculosis infection. Taken together, our investigation identified PE_PGRS11 as an in vivo expressed immunodominant antigen that plays a crucial role in modulating cellular life span restrictions imposed during oxidative stress by triggering TLR2-dependent expression of COX-2 and Bcl2. These observations clearly provide a mechanistic basis for the rescue of pathogenic Mycobacterium-infected lung epithelial cells from oxidative stress.

A simple and rapid method of sample preparation from culture filtrate of M. tuberculosis for two-dimensional gel electrophoresis.

Sample preparation for Two-dimensional gel electrophoresis (2DE) is tedious and not sufficient to provide a comparative profile of secreted proteins for various strains of M. tuberculosis. High lipid content in mycobacteria limits the use of common methods as it can hinder the 2DE run. This study highlights the significance of SDS-TCA procedure over common used methods for the preparation of sample from culture filtrate as well as other proteinaceous fluids.

Investigating role of Mycobacterium tuberculosis secretory antigens in altering activation of T cell signaling events in Jurkat T cells.

Background: Mycobacterium tuberculosis is able to survive and persist as an intracellular pathogen by modulating its own metabolism and host immunity. The molecules and mechanisms utilized to accomplish this modulation are not fully understood. The present study elucidates the effects of M. tuberculosis secretory antigens on T-cell-receptor (TCR)/CD28-triggered signaling in Jurkat T-cells. Method: In the present study, intracellular calcium mobilization was investigated in CD3-activated cells in response to M. tuberculosis antigens, Ag85A, early secretory antigenic target-6 (ESAT-6), and H37Rv. The activation of mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p-38 was also analyzed in CD3- and CD28-activated cells by western blotting. Results: Our results showed CD3-triggered modulations in free intracellular calcium levels in Jurkat T-cells in response to M. tuberculosis antigens. In addition, we also noted M. tuberculosis antigens induced downregulation in phosphorylation of ERK1/2 and p-38. Overall, our results proposed that M. tuberculosis secretory antigens, particularly ESAT-6, impede TCR/CD28-induced signaling events which could be responsible for T-cell unresponsiveness, implicated in the progression of disease. Conclusion: The present study demonstrated M. tuberculosis secretory antigens induced alteration of T-cell signaling pathways in unsensitized Jurkat T-cell line which might be implied in T-cell dysfunctioning during the progression of the disease.

Notch signaling induces lymphoproliferation, T helper cell activation and Th1/Th2 differentiation in leprosy.

The present study evaluates role of Notch1 signaling in the regulation of T cell immunity in leprosy. Peripheral blood mononuclear cells from leprosy patients and healthy controls were activated with Mycobacterium leprae antigens along with activation of Notch1 signaling pathway and then lymphoproliferation was analyzed by lymphocytes transformation test and the expression of Notch1 and its ligands DLL1, Jagged1 and Jagged 2, T cell activation marker and Th1-Th2 cytokines on Th cells in PBMCs of study subjects were analyzed by flow cytometry. Further, these parameters were also analyzed after inhibition of Notch1 signaling pathway. Higher percentage of Notch1expressing Th cells were noted in TT/BT cases and higher percentage of DLL1 expressing Th cells in TT/BT and BL/LL cases. M. leprae antigens were found to induce the expression of Jagged1 on Th cells. Interestingly activation of Notch1 signaling pathway induced lymphoproliferation in BL/LL cases in response of PGL-1. Activation of Notch1 signaling was also found to induce the expression of T cell activation markers CD25, CD69 and Th1 cytokine IFN-γ in response to M. leprae antigens. Immunomodulation through Notch1 signaling seen in our study could be helpful in augmenting Th1 response in leprosy.

Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY.

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(deltaPE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY(mar), in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY(mar) in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY(mar) was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(deltaPE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(deltaPE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(deltaPE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.

Functional characterization of the phospholipase C activity of Rv3487c and its localization on the cell wall of Mycobacterium tuberculosis.

Mycobacterium tuberculosis survives and persists for prolonged periods within its host in an asymptomatic,latent state and can reactivate years later if the host's immune system weakens. The dormant bacilli synthesize and accumulate triacylglycerol, reputed to be an energy source during latency. Among the phospholipases, phospholipase C plays an important role in the pathogenesis. Mutations in a known phospholipase C, plcC, of M.tuberculosis attenuate its growth during the late phase of infection in mice. Hydrolysis of phospholipids by phospholipase C generates diacylglycerol, a well-known signalling molecule that participates in the activation of extracellular signal-regulated kinases (ERK) through protein kinase C leading to macrophage activation. In the present study, we show that M.tuberculosis possesses an additional cell wall-associated protein, Rv3487c, with phospholipase C activity. The recombinant Rv3487c hydrolyses the substrate phosphatidylcholine and generates diacylglycerol by removing the phosphocholine. Furthermore, Rv3487c is expressed during infection as it exhibits significant humoral immunoreactivity with sera from children with tuberculosis, but not with that from adult patients.

Phytofabrication of Silver nanoparticles: Novel Drug to overcome hepatocellular ailments.

This study aimed to treat hepatocellular ailments with biologically prepared silver nanoparticle (AgNPs). AgNPs were formulated using Morus alba leaf extract and their synthesis and characterization were determined by UV-visible spectroscopy, Transmission Electron Microscope (TEM), Scanning Electron microscope (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and Zeta analysis. In vitro studies on HepG2 cell lines for cytotoxic effect and in vivo studies in a rat model for hepatoprotective effect were carried out using biologically prepared AgNPs as curing agents. Dose response cytotoxicity on hepatic cancer (HepG2) cells was confirmed by 3-(4, 5-dimethyl thiazole-2-yl)-2, 5-diphenyl tetrazolium (MTT) assay. The inhibitory concentrations (IC50) were found to be 20 µg/mL and 80 µg/mL for AgNPs and M. alba leaf extract respectively against HepG2 cells at 24 h incubation. In addition, hepatotoxicity in Wistar rats (180 ±â€¯10 g) was induced by intraperitoneal injection of N-nitrosodiethylamine (NDEA) and were treated with different doses of AgNPs (25, 50, 100 µg/kg). NDEA administration showed a significant rise in the biochemical parameters whereas the levels of enzymic antioxidants were decreased. Obtained results revealed that the elevated levels of Liver Function Test (LFTs) biomarkers were significantly reversed and the antioxidant levels were significantly recouped towards normal after the conjoint treatment of AgNPs in a dose-dependent manner. Thus green synthesized AgNPs showed a promising curing effect on hepatocellular ailments.

IL-10 down-regulates the expression of survival associated gene hspX of Mycobacterium tuberculosis in murine macrophage.

Mycobacterium tuberculosis (MTB) adopts a special survival strategy to overcome the killing mechanism(s) of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3) of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ) and recombinant murine interleukin-10 (rmIL-10) on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50ng/ml) of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50ng/ml) significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.