Thiruvananthapuram, India
Thiruvananthapuram, India

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PubMed | Mycobacterium Research Group, Neurobiology Group and Indian Institute of Science
Type: Journal Article | Journal: The FEBS journal | Year: 2016

We isolated an 8 kDa mycobacterial hypothetical protein, Rv3423.1, from the chromatin of human macrophages infected with Mycobacterium tuberculosis H37Rv. Bioinformatics predictions followed by in vitro biochemical assays with purified recombinant protein showed that Rv3423.1 is a novel histone acetyltransferase that acetylates histone H3 at the K9/K14 positions. Transient transfection of macrophages containing GFP-tagged histone H1 with RFP-tagged Rv3423.1 revealed that the protein co-localizes with the chromatin in the nucleus. Co-immunoprecipitation assays confirmed that the Rv3423.1-histone interaction is specific. Rv3423.1 protein was detected in the culture filtrate of virulent but not avirulent M. tuberculosis. Infection of macrophages with recombinant Mycobacterium smegmatis constitutively expressing Rv3423.1 resulted in a significant increase in the number of intracellular bacteria. However, the protein did not seem to offer any growth advantage to free-living recombinant M. smegmatis. It is highly likely that, by binding to the host chromatin, this histone acetyltransferase from M. tuberculosis may manipulate the expression of host genes involved in anti-inflammatory responses to evade clearance and to survive in the intracellular environment.


Augustine N.,Rajiv Gandhi Center for Biotechnology | Goel A.K.,Defence Research and Development Establishment | Sivakumar K.C.,Distributed Information Sub Center | Ajay Kumar R.,Mycobacterium Research Group | Thomas S.,Rajiv Gandhi Center for Biotechnology
Phytomedicine | Year: 2014

Resveratrol, a phytochemical commonly found in the skin of grapes and berries, was tested for its biofilm inhibitory activity against Vibrio cholerae. Biofilm inhibition was assessed using crystal violet assay. MTT assay was performed to check the viability of the treated bacterial cells and the biofilm architecture was analysed using confocal laser scanning microscopy. The possible target of the compound was determined by docking analysis. Results showed that subinhibitory concentrations of the compound could significantly inhibit biofilm formation in V. cholerae in a concentration-dependent manner. AphB was found to be the putative target of resveratrol using docking analysis. The results generated in this study proved that resveratrol is a potent biofilm inhibitor of V. cholerae and can be used as a novel therapeutic agent against cholera. To our knowledge, this is the first report of resveratrol showing antibiofilm activity against V. cholerae. © 2013 Elsevier GmbH.


PubMed | Distributed Information Sub Center, Mycobacterium Research Group, Defence Research & Development Establishment and Rajiv Gandhi Center for Biotechnology
Type: Journal Article | Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology | Year: 2014

Resveratrol, a phytochemical commonly found in the skin of grapes and berries, was tested for its biofilm inhibitory activity against Vibrio cholerae. Biofilm inhibition was assessed using crystal violet assay. MTT assay was performed to check the viability of the treated bacterial cells and the biofilm architecture was analysed using confocal laser scanning microscopy. The possible target of the compound was determined by docking analysis. Results showed that subinhibitory concentrations of the compound could significantly inhibit biofilm formation in V. cholerae in a concentration-dependent manner. AphB was found to be the putative target of resveratrol using docking analysis. The results generated in this study proved that resveratrol is a potent biofilm inhibitor of V. cholerae and can be used as a novel therapeutic agent against cholera. To our knowledge, this is the first report of resveratrol showing antibiofilm activity against V. cholerae.


PubMed | Mycobacterium Research Group and University of Delhi
Type: | Journal: Frontiers in cellular and infection microbiology | Year: 2015

Downregulation of host gene expression is one of the many strategies employed by intracellular pathogens such as Mycobacterium tuberculosis (MTB) to survive inside the macrophages and cause disease. The underlying molecular mechanism behind the downregulation of host defense gene expression is largely unknown. In this study we explored the role of histone deacetylation in macrophages in response to infection by virulent MTB H37Rv in manipulating host gene expression. We show a significant increase in the levels of HDAC1 with a concomitant and marked reduction in the levels of histone H3-acetylation in macrophages containing live, but not killed, virulent MTB. Additionally, we show that HDAC1 is recruited to the promoter of IL-12B in macrophages infected with live, virulent MTB, and the subsequent hypoacetylation of histone H3 suppresses the expression of this gene which plays a key role in initiating Th1 responses. By inhibiting immunologically relevant kinases, and by knockdown of crucial transcriptional regulators, we demonstrate that protein kinase-A (PKA), CREB, and c-Jun play an important role in regulating HDAC1 level in live MTB-infected macrophages. By chromatin immunoprecipitation (ChIP) analysis, we prove that HDAC1 expression is positively regulated by the recruitment of c-Jun to its promoter. Knockdown of HDAC1 in macrophages significantly reduced the survival of intracellular MTB. These observations indicate a novel HDAC1-mediated epigenetic modification induced by live, virulent MTB to subvert the immune system to survive and replicate in the host.


Gomez R.L.,Mycobacterium Research Group | Jose L.,Mycobacterium Research Group | Ramachandran R.,Mycobacterium Research Group | Raghunandanan S.,Mycobacterium Research Group | And 5 more authors.
FEBS Journal | Year: 2016

Rv3334 protein of Mycobacterium tuberculosis belongs to the MerR family of transcriptional regulators and is upregulated during hypoxia and other stress conditions. Employing GFP reporter constructs, mobility shift assays and ChIP assays, we demonstrate that Rv3334 binds to its own promoter and acts as an autorepressor. We were able to locate a 22 bp palindrome in its promoter that we show to be the cognate binding sequence of Rv3334. Using chase experiments, we could conclusively prove the requirement of this palindrome for Rv3334 binding. Recombinant Rv3334 readily formed homodimers in vitro, which could be necessary for its transcriptional regulatory role in vivo. Although the DNA-binding activity of the protein was abrogated by the presence of certain divalent metal cations, the homodimer formation remained unaffected. In silico predictions and subsequent assays using GFP reporter constructs and mobility shift assays revealed that the expression of ketosteroid regulator gene (kstR), involved in lipid catabolism, is positively regulated by Rv3334. ChIP assays with aerobically grown M. tuberculosis as well as dormant bacteria unambiguously prove that Rv3334 specifically upregulates expression of kstR during dormancy. Our study throws light on the possible role of Rv3334 as a master regulator of lipid catabolism during hypoxia-induced dormancy. © 2016 Federation of European Biochemical Societies


Sivakumar K.C.,Rajiv Gandhi Center for Biotechnology | Dhanesh S.B.,Rajiv Gandhi Center for Biotechnology | Shobana S.,Rajiv Gandhi Center for Biotechnology | James J.,Rajiv Gandhi Center for Biotechnology | And 2 more authors.
OMICS A Journal of Integrative Biology | Year: 2011

The Notch, Sonic Hedgehog (Shh), Wnt, and EGF pathways have long been known to influence cell fate specification in the developing nervous system. Here we attempted to evaluate the contemporary knowledge about neural stem cell differentiation promoted by various drug-based regulations through a systems biology approach. Our model showed the phenomenon of DAPT-mediated antagonism of Enhancer of split [E(spl)] genes and enhancement of Shh target genes by a SAG agonist that were effectively demonstrated computationally and were consistent with experimental studies. However, in the case of model simulation of Wnt and EGF pathways, the model network did not supply any concurrent results with experimental data despite the fact that drugs were added at the appropriate positions. This paves insight into the potential of crosstalks between pathways considered in our study. Therefore, we manually developed a map of signaling crosstalk, which included the species connected by representatives from Notch, Shh, Wnt, and EGF pathways and highlighted the regulation of a single target gene, Hes-1, based on drug-induced simulations. These simulations provided results that matched with experimental studies. Therefore, these signaling crosstalk models complement as a tool toward the discovery of novel regulatory processes involved in neural stem cell maintenance, proliferation, and differentiation during mammalian central nervous system development. To our knowledge, this is the first report of a simple crosstalk map that highlights the differential regulation of neural stem cell differentiation and underscores the flow of positive and negative regulatory signals modulated by drugs. © 2011 Mary Ann Liebert, Inc.


Anilkumar A.K.,Mycobacterium Research Group | Madhavilatha G.K.,Mycobacterium Research Group | Paul L.K.,Mycobacterium Research Group | Radhakrishnan I.,Mycobacterium Research Group | And 2 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2012

The clinical presentation of pulmonary tuberculosis by members of Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) cannot be differentiated using the available standard diagnostic procedures. A single-tube tetraplex polymerase chain reaction (T-PCR) was designed to simultaneously amplify 4 well-known DNA targets of MTC. Taguchi's protocol was followed for the optimization of the conditions and was then tested on 288 pulmonary TB patient samples. The analytical sensitivity of the T-PCR was 100 fg of purified mycobacterial DNA, and specificity was found to be 100% in being able to distinguish MTC and NTM in all the cases tested. The results correlated well when validated with hsp65 PCR restriction analysis and sequencing of the 16S-23S internal transcribed spacer region, hsp65, and rpoB. The T-PCR described here is a quick, valuable, and cost-effective tool for determining whether the causative organism is MTC or NTM, and thus is useful for disease surveillance. © 2012 Elsevier Inc..


Dhanasooraj D.,Mycobacterium Research Group | Ajay Kumar R.,Mycobacterium Research Group | Mundayoor S.,Mycobacterium Research Group
International Journal of Nanomedicine | Year: 2013

Nano-sized hepatitis B virus core virus-like particles (HBc-VLP) are suitable for uptake by antigen-presenting cells. Mycobacterium tuberculosis antigen culture filtrate protein 10 (CFP-10) is an important vaccine candidate against tuberculosis. The purified antigen shows low immune response without adjuvant and tends to have low protective efficacy. The present study is based on the assumption that expression of these proteins on HBc nanoparticles would provide higher protection when compared to the native antigen alone. The cfp-10 gene was expressed as a fusion on the major immunodominant region of HBc-VLP, and the immune response in Balb/c mice was studied and compared to pure proteins, a mixture of antigens, and fusion protein-VLP, all without using any adjuvant. The humoral, cytokine, and splenocyte cell proliferation responses suggested that the HBc-VLP bearing CFP-10 generated an antigen-specific immune response in a Th1-dependent manner. By virtue of its self-adjuvant nature and ability to form nano-sized particles, HBc-VLPs are an excellent vaccine delivery system for use with subunit protein antigens identified in the course of recent vaccine research. © 2013 Dhanasooraj et al, publisher and licensee Dove Medical Press Ltd.


Vadakkan M.V.,Rajiv Gandhi Center for Biotechnology | Annapoorna K.,Mycobacterium Research Group | Sivakumar K.C.,Rajiv Gandhi Center for Biotechnology | Mundayoor S.,Mycobacterium Research Group | Kumar G.S.V.,Rajiv Gandhi Center for Biotechnology
International Journal of Nanomedicine | Year: 2013

Excipients having self-assembling properties are less explored in the field of dry powder inhalation (DPI) technology. An amphiphilic lipopolymer system was developed using stearic acid (SA) and branched polyethyleneimine (BPEI) (1800 Dalton), at different proportions by covalent conjugation. A molecular dynamic (MD) simulation tool was employed for predicting the carrier behavior in a polar in vivo condition. The structural characterization was carried out using nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared (FTIR) spectroscopy. The physical nature of the lipopolymer was analyzed by differential scanning calorimetry. Determination of zeta potential and diameter of the micelles showed existence of cationic particles in the nano size range when a lower number of primary amino groups of BPEI was grafted with SA. The rifampicin (RIF)-loaded lipopolymer was also formulated further into spray-dried microparticles. Powder X-ray diffraction (PXRD) studies revealed that the RIF API (active pharmaceutical ingredient) exists as molecular dispersion in spray-dried microparticles. Topological analysis of the spray-dried nanomicelle was carried out using scanning electron microscopy (SEM). A large population of the drug-carrying particles were found to be under the inhalable size range (fine particle fraction 67.88% ± 3%). In vitro drug release kinetics from spray-dried nanomicelles were carried out at lung fluid pH. © 2013 Vadakkan et al, publisher and licensee Dove Medical Press Ltd.


The clinical presentation of pulmonary tuberculosis by members of Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) cannot be differentiated using the available standard diagnostic procedures. A single-tube tetraplex polymerase chain reaction (T-PCR) was designed to simultaneously amplify 4 well-known DNA targets of MTC. Taguchis protocol was followed for the optimization of the conditions and was then tested on 288 pulmonary TB patient samples. The analytical sensitivity of the T-PCR was 100 fg of purified mycobacterial DNA, and specificity was found to be 100% in being able to distinguish MTC and NTM in all the cases tested. The results correlated well when validated with hsp65 PCR restriction analysis and sequencing of the 16S-23S internal transcribed spacer region, hsp65, and rpoB. The T-PCR described here is a quick, valuable, and cost-effective tool for determining whether the causative organism is MTC or NTM, and thus is useful for disease surveillance.

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