[Show abstract][Hide abstract] ABSTRACT: The present study was carried out to prepare & screen the extracts of Salicornia brachiata plant for anti tubercular activity. In-vitro and in-vivo bioassays of Salicornia plant extracts were studied by inhibition of Mycobacterium tuberculosis H37Rv. The water extract of roots was found more active than alcoholic & water: alcohol extracts. The water extract inhibited growth of Mycobacterium tuberculosis H37Rv in-vitro with 50µg/mL MIC (Minimum Inhibitory Concentration). In-vivo tests showed 10-fold less load of bacilli in the lungs of mice receiving treatment. The revalidation of the anti tubercular activity was also studied for the repeat collected & freshly prepared crude water extract of roots.
The anti tubercular activity of the most potent extract was enhanced by activity guided fractionation and sub-fractionation. The active crude extract was fractionated into five fractions F1, F2, F3, F4 and F5 using different concentrations of n-butanol, chloroform, methanol and water on CombiFlash Separation System. Out of five fractions, fraction F2 (MIC 25µg/mL) was further sub-fractionated into seven sub-fractions SF1 to SF7 using HPLC (High Performance Liquid Chromatography) at 220 nm, RP-18 Column and acetonitrile: water isocratic eluting phase. The sub-fraction SF3 was further processed on HPLC to obtain purified sub-fraction SF3-K (MIC 3.125µg/mL). The in-vivo study in two different mice model was carried out for SF3-K at a dosage of 50mg/Kg body weight of mouse. The sub-fraction SF3-K treated mice survived longer and mean survival time was enhanced by 5 days as compared to control. The sub-fraction SF3-K was tested for cytotoxicity (IC50) using VERO cell at different concentration 0 to 100µg/mL and it was found non-cytotoxic even up to 100µg/mL. The toxicity study of sub-fraction SF3-K was also conducted on healthy mice and it was found to be non-toxic. The purified sub-fraction SF3-K was characterized using various spectroscopic methods. From the spectroscopic studies, it was observed that SF3-K is consisting of sucrose as a major (80-85%) and few other minor constituents
Joint International Training Workshop on Herbal Medicine: Drug Discovery from Herbs Approaches, Innovations and Approaches, Mysore; 03/2015
[Show abstract][Hide abstract] ABSTRACT: We have investigated the role of Rv3097c-encoded lipase (LipY) on the virulence of Mycobacterium tuberculosis. It has been shown that the overexpression of LipY in strain H37Rv induced increase in virulence of recombinant H37Rv::LipY strain. Compared to H37Rv, infection with H37Rv::LipY caused enhanced mortality, weight loss, bacterial load in lungs, splenomegaly, worsening lung morphology and pathology. Mice immunized with recombinant LipY antigen were protected against challenge with H37Rv::LipY, which correlated with enhanced survival of challenged mice and striking decrease in pathological features observed in unimmunized mice. To probe the cause of increase in virulence of H37Rv::LipY, the immune status of the host infected with H37Rv and H37Rv::LipY was compared. It was found that overexpression of LipY compromised immune responses resulting in attenuation of Th1 and Th17 responses, significant increase in IL-10, decrease in number of macrophages and T cells, and increase in numbers of Treg, and DCs in the lungs whereas in mice immunized with LipY an increased pool of T cells and DCs was observed. This led us to conclude that the increase in the virulence of H37Rv::LipY was due to downregulation of the host’s protective immunity and the Rv3097c encoded LipY lipase is a virulence factor of M. tuberculosis.
[Show abstract][Hide abstract] ABSTRACT: Cholera is caused by intestinal infection of Vibrio cholerae O1 or O139 through oral route by ingestion of contaminated food and water. Symptoms of cholera include acute watery diarrhea, vomiting, cramps and dehydration. If not treated in time, patient can die of dehydration and hypovolemic shock. Effective therapy is rehydration replacement of salts and electrolytes orally and/or intravenously. Once diarrhea has commenced antibiotic administration only help reduce the bacterial burden and duration of illness. Pathogenesis of cholera is characterized by entry of vibrios in small intestine, adherence to epithelial surface, colonization and release of enterotoxin followed by secretion of isotonic fluid. Mucinase, motility, adherence, multiplication, colonization and secretion of one or more types of toxin are among important factors contributing to disease process. Lipopolysaccharide and flagellar antigens, cholera lectins, haemagglutinins and major outer membrane proteins have been identified as mediating adherence and colonization. Immune response after infection is exclusively humoral. Antibodies to cholera antigens have been detected in sera of experimental cholera and convalescent patients. Antibacterial and antitoxin immunities are synergistic and contribute in protection. A vaccine providing durable immunity has remained elusive despite tremendous efforts. However, judicious application of knowledge of mucosal immunity, pathogenesis, antigen formulations and vaccine delivery will yield the right cholera vaccine.
Water and Health, 01/2014: pages 75-92; , ISBN: 978-81-322-1028-3
[Show abstract][Hide abstract] ABSTRACT: The mycobacterial Rv3097c-encoded lipase LipY is considered as a true lipase involved in the hydrolysis of triacylglycerol stored in lipid inclusion bodies for the survival of dormant mycobacteria. To date, orlistat is the only known LipY inhibitor. In view of the important emerging role of this enzyme, a search for small-molecule inhibitors of LipY was made, leading to the identification of some new compounds (8a-8d, 8f, 8h and 8i) with potent inhibitory activities against recombinant LipY, with no cytotoxicity [50% inhibitory concentration (CC50) ≥500μg/mL]. The compounds 6a, 8c and 8f potently inhibited (>90%) the growth of Mycobacterium tuberculosis H37Rv grown under hypoxia (oxygen-depleted condition) but had no effect on aerobically grown bacilli, suggesting that these new small molecules are highly selective towards the growth inhibition of hypoxic cultures of M. tuberculosis and hence provide new leads for combating latent tuberculosis.
International journal of antimicrobial agents 05/2013; 42(1). DOI:10.1016/j.ijantimicag.2013.03.007 · 4.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium fortuitum causes opportunist non-tubercular infection in humans. Chronic infection of M. fortuitum has been clinically documented and requires prolonged chemotherapy. The objectives of this study were to characterize acute and persistent infection of M. fortuitum in a murine infection model and to screen thiophene-containing trisubstituted methanes active against both acute and persistent infection.
A murine infection model of M. fortuitum was used. Bacillary count, bioluminescence, disease symptoms, host immune response, drug susceptibility and mortality were measured. Reactivation of persistent bacilli was induced by dexamethasone. Trisubstituted methanes containing thiophene rings were synthesized and screened in vitro by agar dilution and BACTEC assay and in mice. Cytotoxicity was tested with Vero monkey kidney cells using a resazurin assay.
The acute infection in mice was marked by a 3 log rise in viable counts, the appearance of disease symptoms and a rise in the Th1 immune response. Bacilli were susceptible to fluoroquinolones. This was followed by persistent infection, in which disappearance of disease symptoms, a decline in Th1 response and non-susceptibility to fluoroquinolones was observed. When the mice were immunocompromised on day 40 post-infection (persistent state) by dexamethasone, a rise in viable counts, symptoms and susceptibility to fluoroquinolones and a prominent Th1 response reappeared. Two lead compounds were found that cleared the mice of bacilli in acute infection and caused a 2.29-2.99 log reduction in cfu of persistent bacilli.
The study established acute and persistent infection in mice and identified two promising anti-M. fortuitum compounds with a selectivity index >10.
[Show abstract][Hide abstract] ABSTRACT: The nonadhesive mutant CD11 of Vibrio cholerae El Tor, defective in expression of mannose-sensitive haemagglutinin, lacks a protein when compared with its parent strain. Determination of the amino acid sequence revealed the identity of the protein as the product of VC1929, which is annotated to encode a protein, DctP, involved in the transport of C₄-dicarboxylates. We cloned the dctP gene in pUC19 vector and expressed it in mutant CD11. Expression of DctP in the resulting complemented strain restored virulence, adhesive and colonizing capabilities, mannose-sensitive haemagglutination (MSHA) and ability to grow in medium containing sialic acid as a sole carbon source. The mutation in CD11 was caused by insertion of an adenine nucleotide in the reading frame of dctP. Recombinant purified DctP protein showed MSHA of human red blood cells, and protected rabbits against infection by V. cholerae. The protein was localized in membrane and cell wall fractions. The mutant, recombinant CD11 expressing DctP and parent strains were grown in M9 minimal medium in the presence of various carbohydrates (glucose, malate, fumarate, succinate or N-acetylneuraminic acid). The mutant was unable to grow in minimal medium containing N-acetylneuraminic acid (sialic acid) as the sole carbon source whereas the recombinant and parent strains utilized all the sugars tested. It is concluded that DctP is a mannose-sensitive haemagglutinin and a virulence factor and is involved in the utilization of sialic acid.
[Show abstract][Hide abstract] ABSTRACT: Rv3097c of Mycobacterium tuberculosis encoding lipase (LipY) was overexpressed in Mycobacterium bovis BCG. Efficacy of recombinant BCG to protect against infection of M. tuberculosis was evaluated in mice. Whereas the parent BCG vaccine protected the mice against infection, recombinant BCG overexpressing LipY offered no protection as judged by viable counts of tubercule bacilli in lungs, weight of infected mice, pathology of lungs and survival of challenged mice. Downregulation of overexpression of LipY by antisense approach considerably restored protection of infected mice as observed with parent BCG vaccine. Overexpression of lipase in BCG caused extensive hydrolysis of triacylglycerol (TG) as identified by TLC, HPLC and NMR spectroscopy. A good correlation could be inferred between hydrolysis of TG and decrease in Th1 secreted IFNγ and IL-2, proinflammatory cytokines and survival of infected mice. Mice immunized with purified LipY antigen were protected and both proinflammatory and Th1 specific cytokines were augmented. TG was found to be a poor vaccine providing no protection, which appears to be due to attenuation of Th1 and proinflammatory immune responses. In conclusion this is the first experimental report to show that immunogenicity of BCG vaccine was impaired by LipY-induced hydrolysis of specific lipids leading to suppression of host immune responses.
[Show abstract][Hide abstract] ABSTRACT: The alarming resurgence of tuberculosis (TB) underlines the urgent need for development of new and potent anti-TB drugs. Towards this goal we herein report the design and synthesis of 2,3-dideoxy hex-2-enopyranosid-4-uloses as promising new anti-tubercular agents. These easily accessible, small molecules were found to exhibit in vitro activity against Mycobacterium tuberculosis H37Rv in a MIC range of 0.78 μg/mL to 25 μg/mL. A detailed SAR study on these hex-2-enopyranosid-4-uloses led to the identification of compound 5g (S007-724) which on the basis of low MIC (0.78 μg/mL-M. tuberculosis H37Rv; 1.56 μg/mL-MDR, SDR strains of M. tuberculosis; 0.78 μg/mL-inhibition of intracellular replication of M. tuberculosis) and SI value of 13.5 has been identified as a promising lead molecule.
European Journal of Medicinal Chemistry 03/2011; 46(6):2217-23. DOI:10.1016/j.ejmech.2011.03.002 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dihydroxyacid dehydratase (DHAD), a key enzyme involved in branched-chain amino acid (BCAA) biosynthesis, catalyses the synthesis of 2-ketoacids from dihydroxyacids. In Mycobacterium tuberculosis, DHAD is encoded by gene Rv0189c, and it shares 40% amino acid sequence identity and conserved motifs with DHAD of Escherichia coli encoded by ilvD. In this study, Rv0189c was overexpressed in E. coli and the resultant protein was characterized as a homodimer (~155 kDa). Functional characterization of Rv0189c was established by biochemical testing and by genetic complementation of an intron-disrupted ilvD-auxotrophic mutant of E. coli to prototrophy. Growth of M. tuberculosis, E. coli BL21(DE3) and recombinant E. coli BL21(DE3) ΔilvD carrying Rv0189c was inhibited by transient nitric oxide (NO) exposure in minimal medium but growth was restored if the medium was supplemented with BCAA (isoleucine, leucine and valine). This suggested that inactivation of Rv0189c by NO probably inhibited bacterial growth. The role of Rv0189c in M. tuberculosis was elucidated by antisense and sense RNA constructs. Growth of M. tuberculosis transformed with a plasmid encoding antisense mRNA was markedly poor in the lungs of infected mice and in Middlebrook 7H9 broth compared to that of sense and vector-alone transformants, but growth was normal when the medium was supplemented with BCAA. Upregulation of Rv0189c was observed during the early exponential phase of growth, under acid stress and ex vivo, suggesting that Rv0189c has a role in the survival of M. tuberculosis during normal and stress conditions. It may be concluded that the DHAD encoded by Rv0189c is essential for the survival of M. tuberculosis and could be a potential drug/vaccine target, as it is absent in mammals.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis H37Rv possesses five resuscitation-promoting factors, RpfA-E, which are required for the resuscitation of dormancy in mycobacteria induced by prolonged incubation of the culture in stationary phase. This study explores the transcriptional profile of all the rpf-like genes of M. tuberculosis H37Rv in the exponential phase, stationary phase, non-culturable phase and Rpf-mediated resuscitation phase. The relative expression was also monitored under acid stress, nutrient starvation and low-oxygen (hypoxia) conditions by real-time quantitative PCR. We show differential relative expression of the rpf genes during different stages of growth and under stress. During early resuscitation, all rpf genes were expressed with maximal expression ratios for rpfA and rpfD. rpfC was consistently expressed during all stages of growth and nutrient starvation. Acid stress induced higher relative expression of rpfD and rpfE and hypoxia of rpfC and rpfE. These results therefore provide further evidence that the rpf genes perform distinct roles during cell growth and cell survival under different physiological stresses, and are consistent with the rpf-like genes being differentially regulated.
[Show abstract][Hide abstract] ABSTRACT: Acetohydroxyacid synthase (AHAS) is a biosynthetic enzyme essential for de novo synthesis of branched-chain amino acids. The genome sequence of Mycobacterium tuberculosis revealed genes encoding four catalytic subunits, ilvB1 (Rv3003c), ilvB2 (Rv3470c), ilvG (Rv1820) and ilvX (Rv3509c), and one regulatory subunit, ilvN (Rv3002c), of AHAS. All these genes were found to be expressed in M. tuberculosis growing in vitro. Each AHAS subunit gene was cloned and expressed in Escherichia coli. AHAS activity of IlvB1 and IlvG was found in cell-free lysates and with recombinant purified proteins. Kinetic studies with purified IlvG revealed positive cooperativity towards substrate and cofactors. To understand the role of the catalytic subunits in the biology of M. tuberculosis, expression of AHAS genes was analysed in different physiological conditions. ilvB1, ilvB2 and ilvG were differentially expressed. The role of ilvB1 in persistence is known, but the upregulation of ilvB2 and ilvG in extended stationary phase, ex vivo, and in acid stress and hypoxic environments, suggests the relevance of AHAS enzymes in the metabolism and survival of M. tuberculosis by functioning as catabolic AHAS. These enzymes are therefore potential targets for drug development.