scholarly journals Inhibition of the DevSR Two-Component System by Overexpression of Mycobacterium tuberculosis PknB in Mycobacterium smegmatis

2017 ◽  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Two Component System ◽  
Component System ◽  
Two Component

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.

Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis

2000 ◽  
Vol 80 (3) ◽  
pp. 141-159 ◽  
Author(s):  
N. Dasgupta ◽  
V. Kapur ◽  
K.K. Singh ◽  
T.K. Das ◽  
S. Sachdeva ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Two Component System ◽  
Component System ◽  
Two Component

scholarly journals Corrigendum: MtrA, an essential response regulator of the MtrAB two-component system, regulates the transcription of resuscitation-promoting factor B of Mycobacterium tuberculosis

Microbiology ◽  
2020 ◽  
Vol 166 (7) ◽  
pp. 679-679
Author(s):  
Arun Kumar Sharma ◽  
Ayan Chatterjee ◽  
Shamba Gupta ◽  
Rajdeep Banerjee ◽  
Sukhendu Mandal ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Response Regulator ◽  
Two Component System ◽  
Factor B ◽  
Component System ◽  
Two Component

The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis

2006 ◽  
Vol 60 (2) ◽  
pp. 312-330 ◽  
Author(s):  
Shaun B. Walters ◽  
Eugenie Dubnau ◽  
Irina Kolesnikova ◽  
Francoise Laval ◽  
Mamadou Daffe ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lipid Biosynthesis ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Complex Lipid

Interaction analysis of TcrX/Y two component system from Mycobacterium tuberculosis

Biochimie ◽  
2010 ◽  
Vol 92 (3) ◽  
pp. 263-272 ◽  
Author(s):  
Monolekha Bhattacharya ◽  
Ashis Biswas ◽  
Amit Kumar Das
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Interaction Analysis ◽  
Two Component System ◽  
Component System ◽  
Two Component

DevR–DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 865-875 ◽  
Author(s):  
Deepak Kumar Saini ◽  
Vandana Malhotra ◽  
Deepanwita Dey ◽  
Neha Pant ◽  
Taposh K. Das ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pathogenic Bacteria ◽  
Response Regulator ◽  
Regulatory System ◽  
Site Directed Mutagenesis ◽  
Phosphoryl Transfer ◽  
Dependent Manner ◽  
Two Component System ◽  
Component System ◽  
Two Component

Two-component systems play a central role in the adaptation of pathogenic bacteria to the environment prevailing within host tissues. The genes encoding the response regulator DevR (Rv3133c/DosR) and the cytoplasmic portion (DevS201) of the histidine kinase DevS (Rv3132c/DosS), a putative two-component system of Mycobacterium tuberculosis, were cloned and the protein products were overexpressed, purified and refolded as N-terminally His6-tagged proteins from Escherichia coli. DevS201 underwent autophosphorylation and participated in rapid phosphotransfer to DevR in a Mg2+-dependent manner. Chemical stability analysis and site-directed mutagenesis implicated the highly conserved residues His395 and Asp54 as the sites of phosphorylation in DevS and DevR, respectively. Mutations in Asp8 and Asp9 residues, postulated to form the acidic Mg2+-binding pocket, and the invariant Lys104 of DevR, abrogated phosphoryl transfer from DevS201 to DevR. DevR–DevS was thus established as a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Expression of the Rv3134c–devR–devS operon was induced at the RNA level in hypoxic cultures of M. tuberculosis H37Rv and was associated with an increase in the level of DevR protein. However, in a devR mutant strain expressing the N-terminal domain of DevR, induction was observed at the level of RNA expression but not at that of protein. DevS was translated independently of DevR and induction of devS transcripts was not associated with an increase in protein level in either wild-type or mutant strains, reflecting differential regulation of this locus during hypoxia.

scholarly journals Expression, Autoregulation, and DNA Binding Properties of the Mycobacterium tuberculosis TrcR Response Regulator

2002 ◽  
Vol 184 (8) ◽  
pp. 2192-2203 ◽  
Author(s):  
Shelley E. Haydel ◽  
William H. Benjamin ◽  
Nancy E. Dunlap ◽  
Josephine E. Clark-Curtiss
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Response Regulator ◽  
Two Component System ◽  
Mobility Shift ◽  
Rich Region ◽  
Component System ◽  
Two Component ◽  
Gel Mobility Shift

ABSTRACT The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis. Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M. tuberculosis growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli. Upon induction of the TrcR protein, there was a >500-fold increase in β-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR. This report indicates that the M. tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter.

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