The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Dipankar Chatterji - One of the best experts on this subject based on the ideXlab platform.
-
synthetic p ppgpp analogue is an inhibitor of Stringent Response in mycobacteria
Antimicrobial Agents and Chemotherapy, 2017Co-Authors: Kirtimaan Syal, Christina L Stallings, Krishnagopal Maiti, Narayanaswamy Jayaraman, Kelly Flentie, Neerupma Bhardwaj, Dipankar ChatterjiAbstract:ABSTRACT Bacteria elicit an adaptive Response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on Stringent Response pathways. (p)ppGpp, considered to be the master regulator of the Stringent Response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress Response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by Rel Msm in the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo , (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis. The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.
-
synthetic glycolipids and p ppgpp analogs development of inhibitors for mycobacterial growth biofilm and Stringent Response
Advances in Experimental Medicine and Biology, 2015Co-Authors: Kirtimaan Syal, Dipankar Chatterji, Krishnagopal Maiti, Kottari Naresh, Narayanaswamy JayaramanAbstract:Bacterial pathogens are the major cause of mortality across the globe, as current use of antibiotics and vaccines is unable to prevent the spread of bacterial pathogens. Rapid evolution allowed bacteria to overcome the hostile environmental conditions. Survival strategies, such as, biofilm formation, Stringent Response, sporulation, cyst formation and horizontal transfer of resistance genes have made it even more difficult to treat the bacterial disease. Rate of bacterial evolution has subdued the pace of discovery of new antibiotics. Most of the antibiotics target the lag and log phases of bacterial growth. It is realized that for complete omission of bacterial pathogens, survival strategies have to be restricted. Under stress, bacteria give rise to Stringent Response which initiates various signalling cascades escalating the activation of different survival strategies, including biofilm formation. Synthetic glycolipids and (p)ppGpp analogs hold promise to potential therapeutics for impeding the survival strategies. We have noticed earlier that various analogs of Stringent Response modulator (p)ppGpp hold promise for therapeutic intervention during stress. In addition, several small molecules, which are overproduced during biofilm formation, may also be targeted. In this article, such attempts are discussed.
-
ppgpp Stringent Response and survival
Journal of Microbiology, 2006Co-Authors: Vikas Jain, Manish Kumar, Dipankar ChatterjiAbstract:Adaptation to any undesirable change in the environment dictates the survivability of many microorganisms, with such changes generating a quick and suitable Response, which guides the physiology of bacteria. During nutritional deprivation, bacteria show a Stringent Response, as characterized by the accumulation of (p)ppGpp, resulting in the repression of stable RNA species, such as rRNA and tRNA, with a concomitant change in colony morphology. However, genes involved in amino acid biosynthesis become over-expressed to help bacteria survive under such conditions. The survivability of pathogenic bacteria inside a host cell also depends upon the Stringent Response demonstrated. Therefore, an understanding of the physiology of Stringent conditions becomes very interesting in regulation of the growth and persistence of such invading pathogens.
-
revisiting the Stringent Response ppgpp and starvation signaling
Current Opinion in Microbiology, 2001Co-Authors: Dipankar Chatterji, Anil K OjhaAbstract:Microbial adaptation to environmental stress plays an important role in survival. It is necessary to understand the mechanisms underlying the survival of microbes under stress, as they may eventually aid in the successful control of the growth and persistence of these organisms. During nutrient starvation, Escherichia coli elicits a Stringent Response to conserve energy. The hallmark of the Stringent Response is the accumulation of guanosine tetra- (ppGpp) and pentaphosphates (pppGpp), which probably bind RNA polymerase to regulate gene expression at certain promoters. Recently, there has been renewed interest in the Stringent Responses of other microbes, with a view to correlating it with sporulation, virulence and long-term persistence.
Petros C Karakousis - One of the best experts on this subject based on the ideXlab platform.
-
Stringent Response factors ppx1 and ppk2 play an important role in mycobacterium tuberculosis metabolism biofilm formation and sensitivity to isoniazid in vivo
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Yu Min Chuang, Noton K Dutta, Harvey Rubin, Chien Fu Hung, Petros C KarakousisAbstract:Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis Stringent Response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis in M. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2, together with two other members of the M. tuberculosis Stringent Response, M. tuberculosis rel and sigE, did not show protective activity against aerosol challenge with M. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis Stringent Response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo.
-
The Stringent Response Is Required for Full Virulence of Mycobacterium tuberculosis in Guinea Pigs
2016Co-Authors: Lee G Klinkenberg, Jonghee Lee, William R Bishai, Petros C KarakousisAbstract:During human latent tuberculosis infection, Mycobacterium tuberculosis likely resides within the nutrient-starved environment of caseous lung granulomas. The Stringent Response alarmone (p)ppGpp is synthesized by Rel in Response to nutrient starvation, thus enabling tubercle bacilli to restrict growth and shut down metabolism in a coordinated fashion. In this study, we investigated the virulence of a rel-deficient M. tuberculosis mutant in the guinea pig model. Quantitative reverse-transcription polymerase chain reaction was used to study the effect of (p)ppGpp deficiency on expression of key cytokine and chemokine genes in guinea pig lungs. The rel-deficient mutant showed impaired initial growth and survival relative to the wild-type strain. Loss of Rel was associated with the striking absence of tubercle lesions grossly and of caseous granulomas histologically. The attenuated phenotype of the rel-deficient mutant was not associated with increased ex-pression of genes encoding the proinflammatory cytokines interferon-g and tumor necrosis factor a in the lungs 28 days after infection. One of the major challenges facing global tuberculosis (TB) eradication efforts is the fact that 2 billion people are latently infected with Mycobacterium tuberculosis, many of whom, especially in the setting of human im
-
the Stringent Response is required for full virulence of mycobacterium tuberculosis in guinea pigs
The Journal of Infectious Diseases, 2010Co-Authors: Lee G Klinkenberg, Petros C Karakousis, Jonghee Lee, William R BishaiAbstract:During human latent tuberculosis infection, Mycobacterium tuberculosis likely resides within the nutrient‐starved environment of caseous lung granulomas. The Stringent Response alarmone (p)ppGpp is synthesized by Rel in Response to nutrient starvation, thus enabling tubercle bacilli to restrict growth and shut down metabolism in a coordinated fashion. In this study, we investigated the virulence of a rel‐deficient M. tuberculosis mutant in the guinea pig model. Quantitative reverse‐transcription polymerase chain reaction was used to study the effect of (p)ppGpp deficiency on expression of key cytokine and chemokine genes in guinea pig lungs. The rel‐deficient mutant showed impaired initial growth and survival relative to the wild‐type strain. Loss of Rel was associated with the striking absence of tubercle lesions grossly and of caseous granulomas histologically. The attenuated phenotype of the rel‐deficient mutant was not associated with increased expression of genes encoding the proinflammatory cytokines interferon‐γ and tumor necrosis factor α in the lungs 28 days after infection.
Liis Andresen - One of the best experts on this subject based on the ideXlab platform.
-
Cationic bactericidal peptide 1018 does not specifically target the Stringent Response alarmone (p)ppGpp
Scientific Reports, 2016Co-Authors: Liis Andresen, Tanel Tenson, Vasili HauryliukAbstract:The bacterial Stringent Response is a key regulator of bacterial virulence, biofilm formation and antibiotic tolerance, and is a promising target for the development of new antibacterial compounds. The intracellular nucleotide (p)ppGpp acts as a messenger orchestrating the Stringent Response. A synthetic peptide 1018 was recently proposed to specifically disrupt biofilms by inhibiting the Stringent Response via direct interaction with (p)ppGpp (de la Fuente-Núñez et al . (2014) PLoS Pathogens). We have interrogated the specificity of the proposed molecular mechanism. When inhibition of Pseudomonas aeruginosa planktonic and biofilm growth is tested simultaneously in the same assay, peptides 1018 and the control peptide 8101 generated by an inversion of the amino acid sequence of 1018 are equally potent, and, importantly, do not display a preferential activity against biofilm. 1018 inhibits planktonic growth of Escherichia coli equally efficiently either when the alleged target, (p)ppGpp, is essential (MOPS media lacking amino acid L-valine), or dispensable for growth (MOPS media supplemented with L-valine). Genetic disruption of the genes relA and spoT responsible for (p)ppGpp synthesis moderately sensitizes – rather than protects – E. coli to 1018. We suggest that the antimicrobial activity of 1018 does not rely on specific recognition of the Stringent Response messenger (p)ppGpp.
-
auxotrophy based high throughput screening assay for the identification of bacillus subtilis Stringent Response inhibitors
Scientific Reports, 2016Co-Authors: Liis Andresen, Vallo Varik, Yuzuru Tozawa, Steffi Jimmy, Stina Lindberg, Tanel TensonAbstract:The Stringent Response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the Stringent Response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the Stringent Response hold great promise as molecular tools for disarming and pacifying bacterial pathogens. By taking advantage of the valine amino acid auxotrophy of the Bacillus subtilis Stringent Response-deficient strain, we have set up a High Throughput Screening assay for the identification of Stringent Response inhibitors. By screening 17,500 compounds, we have identified a novel class of antibacterials based on the 4-(6-(phenoxy)alkyl)-3,5-dimethyl-1H-pyrazole core. Detailed characterization of the hit compounds as well as two previously identified promising Stringent Response inhibitors – a ppGpp-mimic nucleotide Relacin and cationic peptide 1018 – showed that neither of the compounds is sufficiently specific, thus motivating future application of our screening assay to larger and more diverse molecular libraries.
-
auxotrophy based high throughput screening assay for the identification of bacillus subtilis Stringent Response inhibitors
Scientific Reports, 2016Co-Authors: Liis Andresen, Vallo Varik, Yuzuru Tozawa, Steffi Jimmy, Stina Lindberg, Tanel TensonAbstract:The Stringent Response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the Stringent respons ...
Ramachandran Sarojini Santhosh - One of the best experts on this subject based on the ideXlab platform.
-
What is the link between Stringent Response, endoribonuclease encoding Type II Toxin-Antitoxin systems and persistence?
Frontiers in microbiology, 2016Co-Authors: Bhaskar Chandra Mohan Ramisetty, Dimpy Ghosh, Maoumita Roy Chowdhury, Ramachandran Sarojini SanthoshAbstract:Persistence is a transient and non-inheritable tolerance to antibiotics by a small fraction of a bacterial population. One of the proposed determinants of bacterial persistence is toxin-antitoxin systems (TAS) which are also implicated in a wide range of stress-related phenomena. Maisonneuve E, Castro-Camargo M, Gerdes K. 2013. Cell 154:1140-1150 reported an interesting link between ppGpp mediated Stringent Response, TAS, and persistence. It is proposed that accumulation of ppGpp enhances the accumulation of inorganic polyphosphate which modulates Lon protease to degrade antitoxins. The decrease in the concentration of antitoxins supposedly activated the toxin to increase in the number of persisters during antibiotic treatment. In this study, we show that inorganic polyphosphate is not required for transcriptional activation of yefM/yoeB TAS, which is an indirect indication of Lon-dependent degradation of YefM antitoxin. The Δ10 strain, an Escherichia coli MG1655 derivative in which the ten TAS are deleted, is more sensitive to ciprofloxacin compared to wild-type MG1655. Furthermore, we show that the Δ10 strain has relatively lower fitness compared to the wild type and hence, we argue that the persistence related implications based on Δ10 strain are void. We conclude that the transcriptional regulation and endoribonuclease activity of YefM/YoeB TAS is independent of ppGpp and inorganic polyphosphate. Therefore we urge for thorough inspection and debate on the link between chromosomal endoribonuclease TAS and persistence.
-
What is the link between Stringent Response, endoribonuclease encoding Type II Toxin-Antitoxin systems and persistence?
2016Co-Authors: Bhaskar Chandra Mohan Ramisetty, Dimpy Ghosh, Maoumita Roy Chowdhury, Ramachandran Sarojini SanthoshAbstract:Persistence is a transient and non-inheritable tolerance to antibiotics by a small fraction of a bacterial population. One of the proposed determinants of bacterial persistence is Toxin-Antitoxin systems (TAS) which are also implicated in a wide range of stress-related phenomena. In a report (Maisonneuve E, Castro-Camargo M, Gerdes K. 2013. Cell 154:1140-1150) an interesting link between ppGpp mediated Stringent Response, TAS and persistence is proposed. It is proposed that accumulation of ppGpp enhances the accumulation of inorganic polyphosphate which modulates Lon protease to degrade antitoxins. The decrease in the concentration of antitoxins supposedly activated the toxin to increase in the number of persisters during antibiotic treatment. In this study, we show that inorganic polyphosphate is not required for Lon-dependent degradation of YefM, the antitoxin of YefM/YoeB TAS. The Δ10 strain, an Escherichia coli MG1655 derivative in which the ten TAS are deleted, is more sensitive to Ciprofloxacin and Ampicillin compared to wild-type MG1655. Furthermore, we show that the Δ10 strain has relatively lower fitness compared to the wild type and hence, we argue that the implications based on this strain are void. We conclude that there is no direct and specific link between Stringent Response and the regulation of TAS. The link between TAS and persistence is inconclusive due to altered fitness of Δ10 strain and hence requires thorough inspection and debate.
David M Raskin - One of the best experts on this subject based on the ideXlab platform.
-
Stringent Response Regulation of Biofilm Formation in Vibrio cholerae
Journal of Bacteriology, 2012Co-Authors: Huajun He, Jennifer N. Cooper, Arunima Mishra, David M RaskinAbstract:Biofilm formation is a key factor in Vibrio cholerae environmental survival and host colonization. Production of biofilm enables V. cholerae to survive and persist in aquatic environments and aids in the passage through the gastric acid barrier to allow access to the small intestine. The genes involved in biofilm formation are regulated by the transcriptional activators vpsR and vpsT, which are in turn transcriptionally regulated by a number of environmental signals. In this study, the role of the Stringent Response in biofilm formation was examined. V. cholerae mutants deficient in Stringent Response had a reduced ability to form biofilms, although they were not completely deficient in biofilm formation. There are three (p)ppGpp synthases in V. cholerae: RelA, SpoT, and RelV. All three synthases were necessary for vpsR transcription, with RelV showing the strongest effect. RelA was the only synthase that was necessary for vpsT expression. Stringent Response regulation of vpsR and vpsT was shown to partially occur through rpoS. Biofilm formation in V. cholerae is controlled by a complex regulatory apparatus, with negative regulators of biofilm gene expression, such as quorum sensing, and positive regulators of biofilm genes, including Stringent Response, interacting to ensure that biofilm formation is coordinated with the environment.
-
Regulation of the Stringent Response is the essential function of the conserved bacterial G protein CgtA in Vibrio cholerae
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: David M Raskin, Nicholas Judson, John J. MekalanosAbstract:The gene encoding the conserved bacterial G protein CgtA (Obg) is essential for viability in every organism in which it has been studied. CgtA has been reported to be involved in several diverse bacterial functions, including ribosome assembly, DNA repair, sporulation, and morphological development. However, none of these functions have been identified as essential. Here we show that depletion of CgtA in Vibrio cholerae causes global changes in gene expression that are consistent with induction of a classical low nutrient stress Response or “Stringent” Response. We show that depletion of CgtA leads to increased ppGpp levels that correlate with induction of the global stress Response and cessation of growth. The enzyme RelA is responsible for synthesis of the alarmone ppGpp during the Stringent Response. We show that CgtA is no longer essential in a relA deletion mutant and thus conclude that the essentiality of CgtA is directly linked to its ability to affect ppGpp levels. The enzyme SpoT degrades ppGpp, and here we show that SpoT is essential in a RelA+ CgtA+ background but not in a relA deletion mutant. We also confirmed that CgtA interacts with SpoT in a two-hybrid assay. We suggest that the essential function of CgtA is as a repressor of the Stringent Response that acts by regulating SpoT activity to maintain low ppGpp levels when bacteria are growing in a nutrient-rich environment.
