scholarly journals Developing a Cell Based Screen for Inhibitors of Two Component Signal Transduction in Mycobacteria

2021 ◽  
Author(s):  
◽  
Sandi Grainne Dempsey
Keyword(s):  
Signal Transduction ◽  
Oleic Acid ◽  
Mycobacterium Smegmatis ◽  
Drug Targets ◽  
Antibacterial Drug ◽  
Compound Libraries ◽  
Two Component ◽  
Oxygen Starvation ◽  
Transduction Mechanisms

<p>The growing number of drug resistant strains of Mycobacterium tuberculosis appearing worldwide has had an enormous impact on the ability to control and treat Tuberculosis (TB). Discovering new anti-TB drugs is of paramount importance to the global effort for TB eradication. The success of the pathogen is largely due to its inherent ability to remain in a non-replicating or latent state for extended periods of time. In order to achieve this shift it requires tightly controlled signal transduction mechanisms to respond to its host environment. Two component systems (TCS) are one example of signalling mechanisms employed by prokaryotes and are ideal candidates for antibacterial drug targets. It is understood that many TCS are conserved in a large number of organisms, they are often essential to the virulence and persistence of pathogens and they are virtually exclusive to prokaryotes. In this study three Mycobacterium smegmatis TCS were selected; DevS/DevR, MtrB/MtrA and SenX3/RegX3. Promoters under the control of these systems were cloned into an optimised mycobacterial high copy number GFP reporter plasmid and subject to a number of in vitro stress conditions to ascertain induction conditions for these systems. As expected the DevS/DevR controlled hspX promoter was responsive to oxygen starvation and the SenX3/RegX3 controlled phoA was induced by phosphate starvation. Interestingly, phoA and mtrA were also induced by magnesium chelator EDTA in minimal media. The phoA and mtrA promoter constructs were then used for in vitro high throughput bioassays with a number of compound libraries in order to screen for any inhibitory activity on each of the target systems. A phosphorylation inhibitor included in one of the screens, oleic acid, indicated that this assay could potentially be used to screen for TCS inhibitors, but no novel compounds were found in this study. As a proof of principle, known TCS inhibitors palmitoleic and oleic acid were employed to show a dose dependent inhibition mtrA expression. This method could potentially be expanded to other TCS of Mycobacterium smegmatis and Mycobacterium bovis BCG, or other signal transduction systems such as one component regulators and serine threonine kinases.</p>

scholarly journals Developing a Cell Based Screen for Inhibitors of Two Component Signal Transduction in Mycobacteria

2021 ◽  
Author(s):  
◽  
Sandi Grainne Dempsey
Keyword(s):  
Signal Transduction ◽  
Oleic Acid ◽  
Mycobacterium Smegmatis ◽  
Drug Targets ◽  
Antibacterial Drug ◽  
Compound Libraries ◽  
Two Component ◽  
Oxygen Starvation ◽  
Transduction Mechanisms

<p>The growing number of drug resistant strains of Mycobacterium tuberculosis appearing worldwide has had an enormous impact on the ability to control and treat Tuberculosis (TB). Discovering new anti-TB drugs is of paramount importance to the global effort for TB eradication. The success of the pathogen is largely due to its inherent ability to remain in a non-replicating or latent state for extended periods of time. In order to achieve this shift it requires tightly controlled signal transduction mechanisms to respond to its host environment. Two component systems (TCS) are one example of signalling mechanisms employed by prokaryotes and are ideal candidates for antibacterial drug targets. It is understood that many TCS are conserved in a large number of organisms, they are often essential to the virulence and persistence of pathogens and they are virtually exclusive to prokaryotes. In this study three Mycobacterium smegmatis TCS were selected; DevS/DevR, MtrB/MtrA and SenX3/RegX3. Promoters under the control of these systems were cloned into an optimised mycobacterial high copy number GFP reporter plasmid and subject to a number of in vitro stress conditions to ascertain induction conditions for these systems. As expected the DevS/DevR controlled hspX promoter was responsive to oxygen starvation and the SenX3/RegX3 controlled phoA was induced by phosphate starvation. Interestingly, phoA and mtrA were also induced by magnesium chelator EDTA in minimal media. The phoA and mtrA promoter constructs were then used for in vitro high throughput bioassays with a number of compound libraries in order to screen for any inhibitory activity on each of the target systems. A phosphorylation inhibitor included in one of the screens, oleic acid, indicated that this assay could potentially be used to screen for TCS inhibitors, but no novel compounds were found in this study. As a proof of principle, known TCS inhibitors palmitoleic and oleic acid were employed to show a dose dependent inhibition mtrA expression. This method could potentially be expanded to other TCS of Mycobacterium smegmatis and Mycobacterium bovis BCG, or other signal transduction systems such as one component regulators and serine threonine kinases.</p>

scholarly journals Antifungal Properties and Target Evaluation of Three Putative Bacterial Histidine Kinase Inhibitors

1999 ◽  
Vol 43 (7) ◽  
pp. 1700-1703 ◽  
Author(s):  
Robert J. Deschenes ◽  
Hong Lin ◽  
Addison D. Ault ◽  
Jan S. Fassler
Keyword(s):  
Histidine Kinase ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Inhibitory Concentration ◽  
Drug Efficacy ◽  
Antibacterial Drug ◽  
Histidine Kinases ◽  
Antifungal Properties ◽  
Two Component

ABSTRACT Histidine protein kinases have been explored as potential antibacterial drug targets. The recent identification of two-component histidine kinases in fungi has led us to investigate the antifungal properties of three bacterial histidine kinase inhibitors (RWJ-49815, RWJ-49968, and RWJ-61907). All three compounds were found to inhibit growth of the Saccharomyces cerevisiae and Candida albicans strains, with MICs ranging from 1 to 20 μg/ml. However, deletion of SLN1, the only histidine kinase inS. cerevisiae, did not alter drug efficacy. In vitro kinase assays were performed by using the Sln1 histidine kinase purified from bacteria as a fusion protein to glutathione S-transferase. RWJ-49815 and RWJ-49968 inhibited kinase a 50% inhibitory concentration of 10 μM, whereas RWJ-61907 failed to inhibit at concentrations up to 100 μM. Based on these results, we conclude that these compounds have antifungal properties; however, their mode of action appears to be independent of histidine kinase inhibition.

scholarly journals The key bacterial cell division protein FtsZ as a novel antibacterial drug target

2020 ◽  
Author(s):  
Mujeeb Rahman ◽  
Ping Wang ◽  
Na Wang ◽  
Yaodong Chen
Keyword(s):  
Cell Division ◽  
Bacterial Cell ◽  
Drug Targets ◽  
Multidrug Resistant ◽  
Antibacterial Drug ◽  
Bacterial Cell Division ◽  
Bacterial Strains ◽  
Antimicrobial Drugs ◽  
Novel Antibiotics

The number of multidrug-resistant bacterial strains is currently increasing; thus, the determination of drug targets for the development of novel antimicrobial drugs is urgently needed. FtsZ, the prokaryotic homolog of the eukaryotic tubulin, is a GTP-dependent prokaryotic cytoskeletal protein that is conserved among most bacterial strains. In vitro studies revealed that FtsZ self-assembles into dynamic protofilaments or bundles, and it forms a dynamic Z-ring at the center of the cell, leading to septation and consequent cell division. The potential role of FtsZ in the blockage of cell division makes FtsZ a highly attractive target for developing novel antibiotics. Researchers have been working on synthetic molecules and natural products as inhibitors of FtsZ. Accumulating data suggest that FtsZ may provide the platform for the development of novel antibiotics. In this review, we summarize recent advances on the properties of FtsZ protein and bacterial cell division, as well as on the development of FtsZ inhibitors.

Two-component signal transduction as potential drug targets in pathogenic bacteria

2010 ◽  
Vol 13 (2) ◽  
pp. 232-239 ◽  
Author(s):  
Yasuhiro Gotoh ◽  
Yoko Eguchi ◽  
Takafumi Watanabe ◽  
Sho Okamoto ◽  
Akihiro Doi ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Drug Targets ◽  
Pathogenic Bacteria ◽  
Potential Drug ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
Potential Drug Targets

scholarly journals Mycobacterium tuberculosis PhoY Proteins Promote Persister Formation by Mediating Pst/SenX3-RegX3 Phosphate Sensing

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Sarah B. Namugenyi ◽  
Alisha M. Aagesen ◽  
Sarah R. Elliott ◽  
Anna D. Tischler
Keyword(s):  
Signal Transduction ◽  
Mycobacterium Tuberculosis ◽  
System Component ◽  
Signal Transduction System ◽  
Two Component System ◽  
Component System ◽  
Phosphate Sensing ◽  
Two Component ◽  
Pst System

ABSTRACT The Mycobacterium tuberculosis phosphate-specific transport (Pst) system controls gene expression in response to phosphate availability by inhibiting the activation of the SenX3-RegX3 two-component system under phosphate-rich conditions, but the mechanism of communication between these systems is unknown. In Escherichia coli, inhibition of the two-component system PhoR-PhoB under phosphate-rich conditions requires both the Pst system and PhoU, a putative adaptor protein. E. coli PhoU is also involved in the formation of persisters, a subpopulation of phenotypically antibiotic-tolerant bacteria. M. tuberculosis encodes two PhoU orthologs, PhoY1 and PhoY2. We generated phoY single- and double-deletion mutants and examined the expression of RegX3-regulated genes by quantitative reverse transcription-PCR (qRT-PCR). Gene expression was increased only in the ΔphoY1 ΔphoY2 double mutant and could be restored to the wild-type level by complementation with either phoY1 or phoY2 or by deletion of regX3. These data suggest that the PhoY proteins function redundantly to inhibit SenX3-RegX3 activation. We analyzed the frequencies of antibiotic-tolerant persister variants in the phoY mutants using several antibiotic combinations. Persister frequency was decreased at least 40-fold in the ΔphoY1 ΔphoY2 mutant compared to the frequency in the wild type, and this phenotype was RegX3 dependent. A ΔpstA1 mutant lacking a Pst system transmembrane component exhibited a similar RegX3-dependent decrease in persister frequency. In aerosol-infected mice, the ΔphoY1 ΔphoY2 and ΔpstA1 mutants were more susceptible to treatment with rifampin but not isoniazid. Our data demonstrate that disrupting phosphate sensing mediated by the PhoY proteins and the Pst system enhances the susceptibility of M. tuberculosis to antibiotics both in vitro and during infection. IMPORTANCE Persister variants, subpopulations of bacteria that are phenotypically antibiotic tolerant, contribute to the lengthy treatment times required to cure Mycobacterium tuberculosis infection, but the molecular mechanisms governing their formation and maintenance are poorly characterized. Here, we demonstrate that a phosphate-sensing signal transduction system, comprising the Pst phosphate transporter, the two-component system SenX3-RegX3, and functionally redundant PhoY proteins that mediate signaling between Pst and SenX3-RegX3, influences persister formation. Activation of RegX3 by deletion of the phoY genes or a Pst system component resulted in decreased persister formation in vitro. Activated RegX3 also limited persister formation during growth under phosphate-limiting conditions. Importantly, increased susceptibility to the front-line drug rifampin was also observed in a mouse infection model. Thus, the M. tuberculosis phosphate-sensing signal transduction system contributes to antibiotic tolerance and is a potential target for the development of novel therapeutics that may shorten the duration of tuberculosis treatment. IMPORTANCE Persister variants, subpopulations of bacteria that are phenotypically antibiotic tolerant, contribute to the lengthy treatment times required to cure Mycobacterium tuberculosis infection, but the molecular mechanisms governing their formation and maintenance are poorly characterized. Here, we demonstrate that a phosphate-sensing signal transduction system, comprising the Pst phosphate transporter, the two-component system SenX3-RegX3, and functionally redundant PhoY proteins that mediate signaling between Pst and SenX3-RegX3, influences persister formation. Activation of RegX3 by deletion of the phoY genes or a Pst system component resulted in decreased persister formation in vitro. Activated RegX3 also limited persister formation during growth under phosphate-limiting conditions. Importantly, increased susceptibility to the front-line drug rifampin was also observed in a mouse infection model. Thus, the M. tuberculosis phosphate-sensing signal transduction system contributes to antibiotic tolerance and is a potential target for the development of novel therapeutics that may shorten the duration of tuberculosis treatment.

scholarly journals Redox Signal Transduction by the ArcB Sensor Kinase of Haemophilus influenzae Lacking the PAS Domain

2001 ◽  
Vol 183 (24) ◽  
pp. 7206-7212 ◽  
Author(s):  
Dimitris Georgellis ◽  
Ohsuk Kwon ◽  
Edmund C. C. Lin ◽  
Sandy M. Wong ◽  
Brian J. Akerley
Keyword(s):  
Signal Transduction ◽  
Haemophilus Influenzae ◽  
Bacterial Species ◽  
Redox Conditions ◽  
Sensor Kinase ◽  
Pas Domain ◽  
Signal Transduction System ◽  
E Coli ◽  
Two Component

ABSTRACT The Arc (anoxic redox control) two-component signal transduction system of Escherichia coli, which comprises the tripartite ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the arcA and arcBgenes of Haemophilus influenzae specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro. Moreover, the Arc system of H. influenzae mediates transcriptional control according to the redox condition of growth both autologously in its own host and homologously in E. coli, indicating a high degree of functional conservation of the signal transduction system. The H. influenzae ArcB, however, lacks the PAS domain present in the region of E. coli ArcB linking the transmembrane to the cytosolic catalytic domains. Because the PAS domain participates in signal reception in a variety of sensory proteins, including sensors of molecular oxygen and redox state, a similar role was previously ascribed to it in ArcB. Our results demonstrate that the ArcB protein of H. influenzae mediates signal transduction in response to redox conditions of growth despite the absence of the PAS domain.

scholarly journals New Twists and Turns in Bacterial Locomotion and Signal Transduction

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Kylie J. Watts ◽  
Ady Vaknin ◽  
Clay Fuqua ◽  
Barbara I. Kazmierczak
Keyword(s):  
Signal Transduction ◽  
De Novo ◽  
Electron Tomography ◽  
Bacterial Cells ◽  
Research Areas ◽  
Flagellar Motors ◽  
Two Component ◽  
Two Component Signal Transduction

ABSTRACT Prokaryotic organisms occupy the most diverse set of environments and conditions on our planet. Their ability to sense and respond to a broad range of external cues remain key research areas in modern microbiology, central to behaviors that underlie beneficial and pathogenic interactions of bacteria with multicellular organisms and within complex ecosystems. Advances in our understanding of the one- and two-component signal transduction systems that underlie these sensing pathways have been driven by advances in imaging the behavior of many individual bacterial cells, as well as visualizing individual proteins and protein arrays within living cells. Cryo-electron tomography continues to provide new insights into the structure and function of chemosensory receptors and flagellar motors, while advances in protein labeling and tracking are applied to understand information flow between receptor and motor. Sophisticated microfluidics allow simultaneous analysis of the behavior of thousands of individual cells, increasing our understanding of how variance between individuals is generated, regulated, and employed to maximize fitness of a population. In vitro experiments have been complemented by the study of signal transduction and motility in complex in vivo models, allowing investigators to directly address the contribution of motility, chemotaxis, and aggregation/adhesion on virulence during infection. Finally, systems biology approaches have demonstrated previously uncharted areas of protein space in which novel two-component signal transduction pathways can be designed and constructed de novo. These exciting experimental advances were just some of the many novel findings presented at the 15th Bacterial Locomotion and Signal Transduction conference (BLAST XV) in January 2019.

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