The two-component response regulator LiaR regulates cell wall stress responses, pili expression and virulence in group B Streptococcus

Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a β-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a ΔprkA mutant to nearly wild-type levels. While a ΔprkA mutant is defective for PG synthesis during cell wall stress, a double ΔreoM ΔprkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a ΔprkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence.

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Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02461-16 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 19

Author(s):

Stephanie L. Kellogg ◽

Jaime L. Little ◽

Jessica S. Hoff ◽

Christopher J. Kristich

Keyword(s):

Cell Wall ◽

Enterococcus Faecium ◽

Wall Stress ◽

Signaling System ◽

Beta Lactam ◽

Two Component System ◽

Component System ◽

Content Type ◽

Cell Wall Stress ◽

Two Component

ABSTRACT Enterococci are serious opportunistic pathogens that are resistant to many cell wall-targeting antibiotics. The CroRS two-component signaling system responds to antibiotic-mediated cell wall stress and is critical for resistance to cell wall-targeting antibiotics in Enterococcus faecalis. Here, we identify and characterize an orthologous two-component system found in Enterococcus faecium that is functionally equivalent to the CroRS system of E. faecalis. Deletion of croRS in E. faecium resulted in marked susceptibility to cell wall-targeting agents including cephalosporins and bacitracin, as well as moderate susceptibility to ampicillin and vancomycin. As in E. faecalis, exposure to bacitracin and vancomycin stimulates signaling through the CroRS system in E. faecium. Moreover, the CroRS system is critical in E. faecium for enhanced beta-lactam resistance mediated by overexpression of Pbp5. Expression of a Pbp5 variant that confers enhanced beta-lactam resistance cannot overcome the requirement for CroRS function. Thus, the CroRS system is a conserved signaling system that responds to cell wall stress to promote intrinsic resistance to important cell wall-targeting antibiotics in clinically relevant enterococci.

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Loss of Bacterial Cell Pole Stabilization in Caulobacter crescentus Sensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics

10.1101/784066 ◽

2019 ◽

Author(s):

Simon-Ulysse Vallet ◽

Lykke Haastrup Hansen ◽

Freja Cecillie Bistrup ◽

Julien Bortoli Chapalay ◽

Marc Chambon ◽

...

Keyword(s):

Cell Wall ◽

Caulobacter Crescentus ◽

Cell Envelope ◽

Wall Stress ◽

Two Component System ◽

Component System ◽

Division Plane ◽

Cell Wall Stress ◽

Two Component ◽

Weak Points

AbstractRod-shaped bacteria frequently localise proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the role of such polar factors in responses to extracellular stimuli has been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal tolerance of cell envelope-directed antibiotics, including vancomycin that does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin tolerance. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive ΔtipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in ΔtipN cells relative to wild type under some, but not all, cell wall stress conditions that this strain was sensitised to, in particular cefixime and detergent exposure. Together, these results indicate that the ChvIG two-component system has been co-opted as a sensor of cell wall stress and that TipN can influence cell envelope stability and ChvIG-mediated signaling in addition to its roles in intracellular development.Author summaryMaintenance of an intact cell envelope is essential for free-living bacteria to survive harsh conditions they may encounter in their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and re-form parts of the cell envelope at the division plane in order to form new poles as the cells replicate and divide. We have found that TipN, a factor required for correct division and cell pole development in the rod-shaped bacterium, Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. We also identified an outer membrane receptor, ChvT, that was responsible for allowing vancomycin access to the cells and found that the two-component system that negatively regulates ChvT production was activated by various kinds of cell wall stress. Presence or absence of TipN influenced how active this system was in the presence of cefixime or of the membrane-disrupting detergent sodium deoxycholate. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilisation of these weak points of the cell envelope as well as its other roles inside the cell.

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The role of two-component system response regulatorBceRin antimicrobial resistance, virulence, biofilm formation, and stress response of group B Streptococcus

10.1101/340679 ◽

2018 ◽

Author(s):

Ying Yang ◽

Mingjing Luo ◽

Haokui ◽

Carmen Li ◽

Alison W. S. Luk ◽

...

Keyword(s):

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Response Regulator ◽

Group B Streptococcus ◽

Invasive Disease ◽

Wild Type ◽

Non Invasive ◽

Two Component ◽

Group B

AbstractThe hypervirulent Group B Streptococcus (Streptococcus agalactiae, GBS) serogroup III clonal cluster 17 has been associated with neonatal GBS invasive disease and meningits. Serogroup III, ST283 has recently been implicated in invasive disease among non-pregnant adults in Asia. These strains cluster with strains from freshwater fishes from aquaculture and a foodborne outbreak of sepsis, especially with septic arthritis, had been linked to such consumption in Singapore in 2015. Through comparative genome analyses of invasive and non-invasive strains of ST283, we identified a truncated response regulator gene in the non-invasive strain. This two component response gene, previously named a DNA binding regulator, is conserved among GBS strains and is a homologue ofBacillus subtilis BceR, the response regulator of the BceRSAB system. Loss of function of theBceRresponse gene in the invasive GBS strain demonstrated bacitracin susceptibility inΔBceRmutant with MICs of 256-fold and four-fold reduction in bacitracin and human cathelicin LL-37 compared to wild type and complementation strains. Upregulation ofdltAof wild type strain vsΔBceRmutant was demonstrated (p<0.0001), and was previously shown inStaphylococcus aureusto resist and repel cationic peptides through excess positive charges with D-alanylation of teichoic acids on the cell wall. In addition,ΔBceRmutant was less susceptible under oxidative stress under H2O2stress when compared to wild type strain (p<0.001) and inhibited biofilm formation (p<0.05 andp< 0.0001 for crystal violet staining and cfu counts). TheΔBceRmutant also showed reduced mortality as compared to wild type strain (p<0.01) in a murine infection model. Taken together,BceRSis involved in bacitracin and antimicrobial peptide resistance, survival under oxidative stress, biofilm formation and play an important role in the virulence of GBS.Author SummaryTwo-component systems (TCSs) play an important role in virulence in bacteria, and are involved in detecting environmental changes. AlthoughS. agalactiaewas reported to contain more predicted TCSs thanStreptococcus pneumoniae,few have been studied in detail. In this work, comparative genomic analysis of GBS invasive (hyper-virulent) and non-invasive serotype III-4 strains were performed to determine any gene differences that may account for severity of disease in humans.BceR-likeTCS was selected and suspected to be involved in virulence, and thusBceRwas deleted in a hyper-virulent GBS serotype III-4 strain. We demonstrated that thisBceR-likeTCS is involved in GBS virulence and induced proinflammatory host immune responses. Our study of TCSBceRmay guide further research into the role of other TCSs in GBS pathogenicity, and further explore therapeutic targets for GBS disease.

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Loss of Bacterial Cell Pole Stabilization in Caulobacter crescentus Sensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics

mBio ◽

10.1128/mbio.00538-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Simon-Ulysse Vallet ◽

Lykke Haastrup Hansen ◽

Freja Cecillie Bistrup ◽

Signe Aagaard Laursen ◽

Julien Bortoli Chapalay ◽

...

Keyword(s):

Cell Wall ◽

Caulobacter Crescentus ◽

Cell Envelope ◽

Wall Stress ◽

Two Component System ◽

Content Type ◽

Division Plane ◽

Cell Wall Stress ◽

Two Component ◽

Weak Points

ABSTRACT Rod-shaped bacteria frequently localize proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the roles of polar factors in responses to extracellular stimuli have been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal resistance of cell envelope-directed antibiotics, including vancomycin which does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin sensitivity. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive ΔtipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in ΔtipN cells relative to the wild type under some, but not all, cell wall stress conditions that this strain was sensitized to, in particular cefixime and detergent exposure. Together, these results indicate that TipN contributes to cell envelope stress resistance in addition to its roles in intracellular development, and its loss influences signaling through the ChvIG two-component system which has been co-opted as a sensor of cell wall stress in Caulobacter. IMPORTANCE Maintenance of an intact cell envelope is essential for free-living bacteria to protect themselves against their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and reform the cell envelope at the division plane as the cells divide. We have found that TipN, a factor required for correct division and cell pole development in Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilization of these weak points of the cell envelope as well as its other roles inside the cell.

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Epigallocatechin Gallate Induces Upregulation of the Two-Component VraSR System by Evoking a Cell Wall Stress Response in Staphylococcus aureus

Applied and Environmental Microbiology ◽

10.1128/aem.02253-12 ◽

2012 ◽

Vol 78 (22) ◽

pp. 7954-7959 ◽

Cited By ~ 10

Author(s):

Oren Levinger ◽

Tamar Bikels-Goshen ◽

Elad Landau ◽

Merav Fichman ◽

Roni Shapira

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Epigallocatechin Gallate ◽

Wall Stress ◽

Null Mutant ◽

Wild Type ◽

Content Type ◽

Cell Wall Stress ◽

Two Component ◽

A Cell

ABSTRACTWe previously found that a short exposure ofStaphylococcus aureusto subinhibitory (SI) doses of epigallocatechin gallate (EGCG) results in increased cell wall thickness, adaptation, and enhanced tolerance to cell-wall-targeted antibiotics. In this study, the response to EGCG ofsigBandvraSRtranscription factor mutants was characterized. We show that in contrast to the results observed for wild-type (WT) strains, anS. aureus315vraSRnull mutant exposed to SI doses of EGCG did not exhibit increased tolerance to EGCG and oxacillin. A diminished increase in tolerance to ampicillin (from 16-fold to 4-fold) and no change in the magnitude of resistance to vancomycin were observed. Preexposure to EGCG enhanced the tolerance of wild-type andsigBnull mutant cells to lysostaphin, but this enhancement was much weaker in thevraSRnull mutant. Marked upregulation (about 60-fold) ofvraRand upregulation of the peptidoglycan biosynthesis-associated genesmurA,murF, andpbp2(2-, 5-, and 6-fold, respectively) in response to SI doses of EGCG were determined by quantitative reverse transcription-PCR (qRT-PCR). EGCG also induced the promoter ofsas016(encoding a cell wall stress protein of unknown function which is not induced invraSRnull mutants) in a concentration-dependent manner, showing kinetics comparable to those of cell-wall-targeting antibiotics. Taken together, our results suggest that the two-component VraSR system is involved in modulating the cell response to SI doses of EGCG.

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PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses, cytosolic survival, and virulence in Listeria monocytogenes

10.1101/2021.08.20.457112 ◽

2021 ◽

Author(s):

Jessica L. Kelliher ◽

Caroline M. Grunenwald ◽

Rhiannon R. Abrahams ◽

McKenzie E. Daanen ◽

Cassandra I. Lew ◽

...

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Stress Responses ◽

Pathogenic Bacteria ◽

Critical Role ◽

Wall Stress ◽

Wild Type ◽

Comprehensive Overview ◽

Bacterial Physiology ◽

Cell Wall Stress

Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a β-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a ΔprkA mutant to nearly wild-type levels. While a ΔprkA mutant is defective for PG synthesis during cell wall stress, a double ΔreoM ΔprkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a ΔprkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence.

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Hph1p and Hph2p, Novel Components of Calcineurin-Mediated Stress Responses in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.3.3.695-704.2004 ◽

2004 ◽

Vol 3 (3) ◽

pp. 695-704 ◽

Cited By ~ 62

Author(s):

Victoria L Heath ◽

Sidney L. Shaw ◽

Sharmili Roy ◽

Martha S. Cyert

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Cell Wall ◽

Stress Responses ◽

Wall Stress ◽

Sequence Motif ◽

Alkaline Ph ◽

Cell Wall Stress ◽

High Concentrations

ABSTRACT Calcineurin is a Ca2+- and calmodulin-dependent protein phosphatase that plays a key role in animal and yeast physiology. In the yeast Saccharomyces cerevisiae, calcineurin is required for survival during several environmental stresses, including high concentrations of Na+, Li+, and Mn2+ ions and alkaline pH. One role of calcineurin under these conditions is to activate gene expression through its regulation of the Crz1p transcription factor. We have identified Hph1p as a novel substrate of calcineurin. HPH1 (YOR324C) and its homolog HPH2 (YAL028W) encode tail-anchored integral membrane proteins that interact with each other in the yeast two-hybrid assay and colocalize to the endoplasmic reticulum. Hph1p and Hph2p serve redundant roles in promoting growth under conditions of high salinity, alkaline pH, and cell wall stress. Calcineurin modifies the distribution of Hph1p within the endoplasmic reticulum and is required for full Hph1p activity in vivo. Furthermore, calcineurin directly dephosphorylates Hph1p and interacts with it through a sequence motif in Hph1p, PVIAVN. This motif is related to calcineurin docking sites in other substrates, such as NFAT and Crz1p, and is required for regulation of Hph1p by calcineurin. In contrast, Hph2p neither interacts with nor is dephosphorylated by calcineurin. Ca2+-induced Crz1p-mediated transcription is unaffected in hph1Δ hph2Δ mutants, and genetic analyses indicate that HPH1/HPH2 and CRZ1 act in distinct pathways downstream of calcineurin. Thus, Hph1p and Hph2p are components of a novel Ca2+- and calcineurin-regulated response required to promote growth under conditions of high Na+, alkaline pH, and cell wall stress.

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Functional Dissection of the CroRS Two-Component System Required for Resistance to Cell Wall Stressors in Enterococcus faecalis

Journal of Bacteriology ◽

10.1128/jb.00995-15 ◽

2016 ◽

Vol 198 (8) ◽

pp. 1326-1336 ◽

Cited By ~ 16

Author(s):

Stephanie L. Kellogg ◽

Christopher J. Kristich

Keyword(s):

Cell Wall ◽

Enterococcus Faecalis ◽

Histidine Kinase ◽

Response Regulator ◽

Wall Stress ◽

Opportunistic Pathogen ◽

Content Type ◽

Cell Wall Stress ◽

Cephalosporin Resistance ◽

Sense And Respond

ABSTRACTBacteria use two-component signal transduction systems (TCSs) to sense and respond to environmental changes via a conserved phosphorelay between a sensor histidine kinase and its cognate response regulator. The opportunistic pathogenEnterococcus faecalisutilizes a TCS comprised of the histidine kinase CroS and the response regulator CroR to mediate resistance to cell wall stresses such as cephalosporin antibiotics, but the molecular details by which CroRS promotes cephalosporin resistance have not been elucidated. Here, we analyzed mutants ofE. faecaliscarrying substitutions in CroR and CroS to demonstrate that phosphorylated CroR drives resistance to cephalosporins, and that CroS exhibits kinase and phosphatase activities to control the level of CroR phosphorylationin vivo. Deletion ofcroSin various lineages ofE. faecalisrevealed a CroS-independent mechanism for CroR phosphorylation and led to the identification of a noncognate histidine kinase capable of influencing CroR (encoded byOG1RF_12162; here calledcisS). Further analysis of this TCS network revealed that both systems respond to cell wall stress.IMPORTANCETCSs allow bacteria to sense and respond to many different environmental conditions. The opportunistic pathogenEnterococcus faecalisutilizes the CroRS TCS to mediate resistance to cell wall stresses, including clinically relevant antibiotics such as cephalosporins and glycopeptides. In this study, we use genetic and biochemical means to investigate the relationship between CroRS signaling and cephalosporin resistance inE. faecaliscells. Through this, we uncovered a signaling network formed between the CroRS TCS and a previously uncharacterized TCS that also responds to cell wall stress. This study provides mechanistic insights into CroRS signaling and cephalosporin resistance inE. faecalis.

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