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 MprAB Is a Stress-Responsive Two-Component System That Directly Regulates Expression of Sigma Factors SigB and SigE in Mycobacterium tuberculosis

2006 ◽  
Vol 188 (6) ◽  
pp. 2134-2143 ◽  
Author(s):  
Hongjun He ◽  
Raymond Hovey ◽  
Jason Kane ◽  
Vineet Singh ◽  
Thomas C. Zahrt
Keyword(s):  
Signal Transduction ◽  
Mycobacterium Tuberculosis ◽  
Sigma Factors ◽  
Two Component System ◽  
Direct Role ◽  
Component System ◽  
Two Component ◽  
Two Component Signal Transduction

ABSTRACT The genetic mechanisms mediating the adaptation of Mycobacterium tuberculosis within the host are poorly understood. The best-characterized regulatory systems in this organism include sigma factors and two-component signal transduction systems. mprAB is a two-component system required by M. tuberculosis for growth in vivo during the persistent stage of infection. In this report, we demonstrate that MprAB is stress responsive and regulates the expression of numerous stress-responsive genes in M. tuberculosis. With DNA microarrays and quantitative real-time reverse transcription-PCR, genes regulated by MprA in M. tuberculosis that included two stress-responsive sigma factors were identified. Response regulator MprA bound to conserved motifs in the upstream regions of both sigB and sigE in vitro and regulated the in vivo expression of sigB and sigE in M. tuberculosis. In addition, mprA itself was induced following exposure to stress, establishing a direct role for this regulatory system in stress response pathways of M. tuberculosis. Induction of mprA and sigE by MprA in response to stress was mediated through the cognate sensor kinase MprB and required expression of the extracytoplasmic loop domain. These results provide the first evidence that recognition of and adaptation to specific stress in M. tuberculosis are mediated through activation of a two-component signal transduction system that directly regulates the expression of stress-responsive determinants.

scholarly journals Mycobacterium tuberculosis Requires Phosphate-Responsive Gene Regulation To Resist Host Immunity

2012 ◽  
Vol 81 (1) ◽  
pp. 317-328 ◽  
Author(s):  
Anna D. Tischler ◽  
Rachel L. Leistikow ◽  
Meghan A. Kirksey ◽  
Martin I. Voskuil ◽  
John D. McKinney
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Immune Response ◽  
Mycobacterium Tuberculosis ◽  
Signal Transduction System ◽  
Content Type ◽  
Two Component ◽  
Ifn Γ ◽  
Responsive Gene ◽  
Pst System

Mycobacterium tuberculosispersists in the tissues of mammalian hosts despite inducing a robust immune response dominated by the macrophage-activating cytokine gamma interferon (IFN-γ). We identified theM. tuberculosisphosphate-specific transport (Pst) system component PstA1 as a factor required to resist IFN-γ-dependent immunity. A ΔpstA1mutant was fully virulent in IFN-γ−/−mice but attenuated in wild-type (WT) mice and mice lacking specific IFN-γ-inducible immune mechanisms: nitric oxide synthase (NOS2), phagosome-associated p47 GTPase (Irgm1), or phagocyte oxidase (phox). These phenotypes suggest that ΔpstA1bacteria are sensitized to an IFN-γ-dependent immune mechanism(s) other than NOS2, Irgm1, or phox. In other species, the Pst system has a secondary role as a negative regulator of phosphate starvation-responsive gene expression through an interaction with a two-component signal transduction system. InM. tuberculosis, we found that ΔpstA1bacteria exhibited dysregulated gene expression during growth in phosphate-rich medium that was mediated by the two-component sensor kinase/response regulator system SenX3-RegX3. Remarkably, deletion of theregX3gene suppressed the replication and virulence defects of ΔpstA1bacteria in NOS2−/−mice, suggesting thatM. tuberculosisrequires the Pst system to negatively regulate activity of RegX3 in response to available phosphatein vivo. We therefore speculate that inorganic phosphate is readily available during replication in the lung and is an important signal controllingM. tuberculosisgene expression via the Pst-SenX3-RegX3 signal transduction system. Inability to sense this environmental signal, due to Pst deficiency, results in dysregulation of gene expression and sensitization of the bacteria to the host immune response.

scholarly journals YycH and YycI Interact To Regulate the Essential YycFG Two-Component System in Bacillus subtilis

2007 ◽  
Vol 189 (8) ◽  
pp. 3280-3289 ◽  
Author(s):  
Hendrik Szurmant ◽  
Michael A. Mohan ◽  
P. Michael Imus ◽  
James A. Hoch
Keyword(s):  
Signal Transduction ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Membrane Integrity ◽  
Direct Result ◽  
Signal Transduction System ◽  
Two Component System ◽  
Component System ◽  
Growth Defects ◽  
Two Component

ABSTRACT The YycFG two-component system is the only signal transduction system in Bacillus subtilis known to be essential for cell viability. This system is highly conserved in low-G+C gram-positive bacteria, regulating important processes such as cell wall homeostasis, cell membrane integrity, and cell division. Four other genes, yycHIJK, are organized within the same operon with yycF and yycG in B. subtilis. Recently, it was shown that the product of one of these genes, the YycH protein, regulated the activity of this signal transduction system, whereas no function could be assigned to the other genes. Results presented here show that YycI and YycH proteins interact to control the activity of the YycG kinase. Strains carrying individual in-frame deletion of the yycI and yycH coding sequences were constructed and showed identical phenotypes, namely a 10-fold-elevated expression of the YycF-dependent gene yocH, growth defects, as well as a cell wall defect. Cell wall and growth defects were a direct result of overregulation of the YycF regulon, since a strain overexpressing YycF showed phenotypes similar to those of yycH and yycI deletion strains. Both YycI and YycH proteins are localized outside the cytoplasm and attached to the membrane by an N-terminal transmembrane sequence. Bacterial two-hybrid data showed that the YycH, YycI, and the kinase YycG form a ternary complex. The data suggest that YycH and YycI control the activity of YycG in the periplasm and that this control is crucial in regulating important cellular processes.

The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2331-2343 ◽  
Author(s):  
Thierry Doan ◽  
Pascale Servant ◽  
Shigeo Tojo ◽  
Hirotake Yamaguchi ◽  
Guillaume Lerondel ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Malic Enzyme ◽  
Northern Analysis ◽  
Two Component System ◽  
Extracellular Medium ◽  
Component System ◽  
Two Component ◽  
Transcriptome Comparison ◽  
Physiological Tests

A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (k cat/K m 102 and 10 s−1 mM−1, respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.

scholarly journals Identification of a novel two-component system SenS/SenR modulating the production of the catalase-peroxidase CpeB and the haem-binding protein HbpS in Streptomyces reticuli

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3603-3614 ◽  
Author(s):  
Darío Ortiz de Orué Lucana ◽  
Peijian Zou ◽  
Marc Nierhaus ◽  
Hildgund Schrempf
Keyword(s):  
Peroxidase Activity ◽  
Response Regulator ◽  
Binding Protein ◽  
Streptomyces Avermitilis ◽  
Binding Motif ◽  
Two Component System ◽  
Component System ◽  
Regulatory Cascade ◽  
Two Component

The Gram-positive soil bacterium and cellulose degrader Streptomyces reticuli synthesizes the mycelium-associated enzyme CpeB, which displays haem-dependent catalase and peroxidase activity, as well as haem-independent manganese-peroxidase activity. The expression of the furS–cpeB operon depends on the redox regulator FurS and the presence of the haem-binding protein HbpS. Upstream of hbpS, the neighbouring senS and senR genes were identified. SenS is a sensor histidine kinase with five predicted N-terminally located transmembrane domains. SenR is the corresponding response regulator with a C-terminal DNA-binding motif. Comparative transcriptional and biochemical studies with a designed S. reticuli senS/senR chromosomal disruption mutant and a set of constructed Streptomyces lividans transformants showed that the presence of the novel two-component system SenS/SenR negatively modulates the expression of the furS–cpeB operon and the hbpS gene. The presence of SenS/SenR enhances considerably the resistance of S. reticuli to haemin and the redox-cycling compound plumbagin, suggesting that this system could participate directly or indirectly in the sensing of redox changes. Epitope-tagged HbpS (obtained from an Escherichia coli transformant) as well as the native S. reticuli HbpS interact in vitro specifically with the purified SenS fusion protein. On the basis of these findings, together with data deduced from the S. reticuli hbpS mutant strain, HbpS is suggested to act as an accessory protein that communicates with the sensor protein to modulate the corresponding regulatory cascade. Interestingly, close and distant homologues, respectively, of the SenS/SenR system are encoded within the Streptomyces coelicolor A3(2) and Streptomyces avermitilis genomes, but not within other known bacterial genomes. Hence the SenS/SenR system appears to be confined to streptomycetes.

scholarly journals CrdS and CrdA Comprise a Two-Component System That Is Cooperatively Regulated by the Che3 Chemosensory System in Myxococcus xanthus

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
Jonathan W. Willett ◽  
John R. Kirby
Keyword(s):  
Signal Transduction ◽  
Myxococcus Xanthus ◽  
Complex Signal ◽  
Central Processor ◽  
Two Component System ◽  
Component System ◽  
Chemosensory System ◽  
Content Type ◽  
Phosphorylation State ◽  
Two Component

ABSTRACTMyxococcus xanthusserves as a model organism for development and complex signal transduction. Regulation of developmental aggregation and sporulation is controlled, in part, by the Che3 chemosensory system. The Che3 pathway consists of homologs to two methyl-accepting chemotaxis proteins (MCPs), CheA, CheW, CheB, and CheR but not CheY. Instead, the output for Che3 is the NtrC homolog CrdA, which functions to regulate developmental gene expression. In this paper we have identified an additional kinase, CrdS, which directly regulates the phosphorylation state of CrdA. Both epistasis andin vitrophosphotransfer assays indicate that CrdS functions as part of the Che3 pathway and, in addition to CheA3, serves to regulate CrdA phosphorylation inM. xanthus. We provide kinetic data for CrdS autophosphorylation and demonstrate specificity for phosphotransfer from CrdS to CrdA. We further demonstrate that CheA3 destabilizes phosphorylated CrdA (CrdA~P), indicating that CheA3 likely acts as a phosphatase. Both CrdS and CheA3 control developmental progression by regulating the phosphorylation state of CrdA~P in the cell. These results support a model in which a classical two-component system and a chemosensory system act synergistically to control the activity of the response regulator CrdA.IMPORTANCEWhile phosphorylation-mediated signal transduction is well understood in prototypical chemotaxis and two-component systems (TCS), chemosensory regulation of alternative cellular functions (ACF) has not been clearly defined. The Che3 system inMyxococcus xanthusis a member of the ACF class of chemosensory systems and regulates development via the transcription factor CrdA (chemosensoryregulator ofdevelopment) (K. Wuichet and I. B. Zhulin, Sci. Signal. 3:ra50, 2010; J. R. Kirby and D. R. Zusman, Proc. Natl. Acad. Sci. U. S. A. 100:2008–2013, 2003). We have identified and characterized a homolog of NtrB, designated CrdS, capable of specifically phosphorylating the NtrC homolog CrdA inM. xanthus. Additionally, we demonstrate that the CrdSA two-component system is negatively regulated by CheA3, the central processor within the Che3 system ofM. xanthus. To our knowledge, this study provides the first example of an ACF chemosensory system regulating a prototypical two-component system and extends our understanding of complex regulation of developmental signaling pathways.

Sign in / Sign up

Export Citation Format

Share Document