scholarly journals The Pseudomonas aeruginosa PhoP-PhoQ Two-Component Regulatory System Is Induced upon Interaction with Epithelial Cells and Controls Cytotoxicity and Inflammation

2012 ◽  
Vol 80 (9) ◽  
pp. 3122-3131 ◽  
Author(s):  
Shaan L. Gellatly ◽  
Brittany Needham ◽  
Laurence Madera ◽  
M. Stephen Trent ◽  
Robert E. W. Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Lipid A ◽  
Regulatory System ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
Regulatory Systems ◽  
Two Component ◽  
Inflammatory Phenotype

ABSTRACTThe adaptation ofPseudomonas aeruginosato its environment, including the host, is tightly controlled by its network of regulatory systems. The two-component regulatory system PhoPQ has been shown to play a role in the virulence and polymyxin resistance ofP. aeruginosaas well as several other Gram-negative species. Dysregulation of this system has been demonstrated in clinical isolates, yet how it affects virulence ofP. aeruginosais unknown. To investigate this, an assay was used whereby bacteria were cocultured with human bronchial epithelial cells. The interaction of wild-type (WT) bacteria that had adhered to epithelial cells led to a large upregulation of the expression of theoprH-phoP-phoQoperon and its target, thearnlipopolysaccharide (LPS) modification operon, in a PhoQ-dependent manner, compared to cells in the supernatant that had failed to adhere. Relative to the wild type, aphoQmutant cocultured on epithelial cells produced less secreted protease and lipase and, like thephoQmutant,piv,lipH, andlasBmutants demonstrated reduced cytotoxicity toward epithelial cells. Mutation inphoQalso resulted in alterations to lipid A and to increased inflammatory LPS. These data indicate that mutation ofphoQresults in a phenotype that is similar to the less virulent but more inflammatory phenotype of clinical strains isolated from chronic-stage cystic fibrosis lung infections.

scholarly journals The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP

2016 ◽  
Vol 198 (6) ◽  
pp. 951-963 ◽  
Author(s):  
Manita Guragain ◽  
Michelle M. King ◽  
Kerry S. Williamson ◽  
Ailyn C. Pérez-Osorio ◽  
Tatsuya Akiyama ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Regulatory System ◽  
Dependent Manner ◽  
Content Type ◽  
Factor Production ◽  
Two Component ◽  
Intracellular Ca ◽  
Virulence Factor Production

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections,P. aeruginosaoften encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed thatP. aeruginosaresponds to externally added Ca2+through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+homeostasis are not known. Here, we characterized the genome-wide transcriptional response ofP. aeruginosato elevated [Ca2+] in both planktonic cultures and biofilms. Among the genes induced by CaCl2in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here calledcarSandcarR), while the closely related two-component regulatorsphoPQandpmrABwere repressed by CaCl2addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant ofcarRand performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2are the predicted periplasmic OB-fold protein, PA0320 (here calledcarO), and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here calledcarP). Mutations in bothcarOandcarPaffected Ca2+homeostasis, reducing the ability ofP. aeruginosato export excess Ca2+. In addition, a mutation incarPhad a pleotropic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+and responding through its regulatory targets that modulate Ca2+homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin.IMPORTANCEDuring infectious disease,Pseudomonas aeruginosaencounters environments with high calcium (Ca2+) concentrations, yet the cells maintain intracellular Ca2+at levels that are orders of magnitude less than that of the external environment. In addition, Ca2+signalsP. aeruginosato induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca2+homeostasis or how Ca2+acts as a signal. In this study, we identified a two-component regulatory system inP. aeruginosaPAO1, termed CarRS, that is induced at elevated Ca2+levels. CarRS modulates Ca2+signaling and Ca2+homeostasis through its regulatory targets, CarO and CarP. The results demonstrate thatP. aeruginosauses a two-component regulatory system to sense external Ca2+and relays that information for Ca2+-dependent cellular processes.

scholarly journals Impact of Two-Component Regulatory Systems PhoP-PhoQ and PmrA-PmrB on Colistin Pharmacodynamics in Pseudomonas aeruginosa

2012 ◽  
Vol 56 (6) ◽  
pp. 3453-3456 ◽  
Author(s):  
Neang S. Ly ◽  
Jenny Yang ◽  
Jurgen B. Bulitta ◽  
Brian T. Tsuji
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pharmacodynamic Model ◽  
Maximal Effect ◽  
Wild Type ◽  
Content Type ◽  
Regulatory Systems ◽  
Two Component ◽  
Subinhibitory Concentrations

ABSTRACTThein vitropharmacodynamics of colistin againstPseudomonas aeruginosaPAO1 wild-type and isogenic knockout strains ofphoPandpmrAwere evaluated. Colistin killing at subinhibitory concentrations was greater against thephoPandpmrAmutants than the wild type within the first 8 h: the concentration that results in 50% of maximal effect (EC50) of thepmrAmutant (0.413 mg/liter) was less than that of the wild type (0.718 mg/liter) (P< 0.05). Anin vitropharmacodynamic model simulating human colistin regimens displayed initial killing followed by regrowth in thephoPmutant and gradual regrowth in thepmrAmutant and wild type.

scholarly journals Polymyxin Resistance ofPseudomonas aeruginosa phoQMutants Is Dependent on Additional Two-Component Regulatory Systems

2013 ◽  
Vol 57 (5) ◽  
pp. 2204-2215 ◽  
Author(s):  
Alina D. Gutu ◽  
Nicole Sgambati ◽  
Pnina Strasbourger ◽  
Mark K. Brannon ◽  
Michael A. Jacobs ◽  
...  
Keyword(s):  
Lipid A ◽  
Regulatory System ◽  
Content Type ◽  
Regulatory Systems ◽  
Clinical Resistance ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
High Level

ABSTRACTPseudomonas aeruginosacan develop resistance to polymyxin as a consequence of mutations in the PhoPQ regulatory system, mediated by covalent lipid A modification. Transposon mutagenesis of a polymyxin-resistantphoQmutant defined 41 novel loci required for resistance, including two regulatory systems, ColRS and CprRS. Deletion of thecolRSgenes, individually or in tandem, abrogated the polymyxin resistance of a ΔphoQmutant, as did individual or tandem deletion ofcprRS. Individual deletion ofcolRorcolSin a ΔphoQmutant also suppressed 4-amino-l-arabinose addition to lipid A, consistent with the known role of this modification in polymyxin resistance. Surprisingly, tandem deletion ofcolRSorcprRSin the ΔphoQmutant or individual deletion ofcprRorcprSfailed to suppress 4-amino-l-arabinose addition to lipid A, indicating that this modification alone is not sufficient for PhoPQ-mediated polymyxin resistance inP. aeruginosa. Episomal expression ofcolRSorcprRSin tandem or ofcprRindividually complemented the Pm resistance phenotype in the ΔphoQmutant, while episomal expression ofcolR,colS, orcprSindividually did not. Highly polymyxin-resistantphoQmutants ofP. aeruginosaisolated from polymyxin-treated cystic fibrosis patients harbored mutant alleles ofcolRSandcprS; when expressed in a ΔphoQbackground, these mutant alleles enhanced polymyxin resistance. These results define ColRS and CprRS as two-component systems regulating polymyxin resistance inP. aeruginosa, indicate that addition of 4-amino-l-arabinose to lipid A is not the only PhoPQ-regulated biochemical mechanism required for resistance, and demonstrate thatcolRSandcprSmutations can contribute to high-level clinical resistance.

scholarly journals VraSR Two-Component Regulatory System Contributes tomprF-Mediated Decreased Susceptibility to Daptomycin inIn Vivo-Selected Clinical Strains of Methicillin-Resistant Staphylococcus aureus

2011 ◽  
Vol 56 (1) ◽  
pp. 92-102 ◽  
Author(s):  
Shrenik Mehta ◽  
Arabela X. Cuirolo ◽  
Konrad B. Plata ◽  
Sarah Riosa ◽  
Jared A. Silverman ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Regulatory System ◽  
Dependent Manner ◽  
Wild Type ◽  
Methicillin Resistant ◽  
Content Type ◽  
Clinical Strains ◽  
Two Component ◽  
The Impact

ABSTRACTDaptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistantStaphylococcus aureus(MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Ca-dependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients withS. aureusinfections after treatment with DAP. Different point mutations were found in themprFgene in DAP-resistant (DR) strains. Investigation of themprFL826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutatedmprFin DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested thatmprFwas not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory systemvraSR. Inactivation ofvraSRresulted in increased DAP susceptibility, while complementation ofvraSRmutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact ofvraSRandmprFmutations in the cell wall. Moreover, overexpression ofvraSRin DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations inmprF,vraSRcontributes to DAP resistance in the present group of clinical strains.

scholarly journals Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

2015 ◽  
Vol 83 (3) ◽  
pp. 1199-1209 ◽  
Author(s):  
Kivanc Bilecen ◽  
Jiunn C. N. Fong ◽  
Andrew Cheng ◽  
Christopher J. Jones ◽  
David Zamorano-Sánchez ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Lipid A ◽  
Response Regulator ◽  
Regulatory Region ◽  
Regulatory System ◽  
Polymyxin B ◽  
Content Type ◽  
Two Component ◽  
Antimicrobial Peptide Resistance

Two-component systems play important roles in the physiology of many bacterial pathogens.Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression ofvps(Vibriopolysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of thealmEFGgenes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of thealmEFGoperon. Similarly to acarRmutant, strains lackingalmE,almF, andalmGexhibited enhanced polymyxin B sensitivity. We also observed that strains lackingalmEor thealmEFGoperon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance inV. cholerae.

scholarly journals PmrAB, a Two-Component Regulatory System of Pseudomonas aeruginosa That Modulates Resistance to Cationic Antimicrobial Peptides and Addition of Aminoarabinose to Lipid A

2004 ◽  
Vol 186 (2) ◽  
pp. 575-579 ◽  
Author(s):  
Samuel M. Moskowitz ◽  
Robert K. Ernst ◽  
Samuel I. Miller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Peptides ◽  
Lipid A ◽  
Regulatory System ◽  
Signal Transduction System ◽  
Cationic Antimicrobial Peptides ◽  
Two Component ◽  
Serovar Typhimurium ◽  
Two Component Signal Transduction ◽  
Resistant Mutants

ABSTRACT Spontaneous polymyxin-resistant mutants of Pseudomonas aeruginosa were isolated. The mutations responsible for this phenotype were mapped to a two-component signal transduction system similar to PmrAB of Salmonella enterica serovar Typhimurium. Lipid A of these mutants contained aminoarabinose, an inducible modification that is associated with polymyxin resistance. Thus, P. aeruginosa possesses a mechanism that induces resistance to cationic antimicrobial peptides in response to environmental conditions.

scholarly journals PmrB Mutations Promote Polymyxin Resistance of Pseudomonas aeruginosa Isolated from Colistin-Treated Cystic Fibrosis Patients

2011 ◽  
Vol 56 (2) ◽  
pp. 1019-1030 ◽  
Author(s):  
Samuel M. Moskowitz ◽  
Mark K. Brannon ◽  
Nandini Dasgupta ◽  
Miyuki Pier ◽  
Nicole Sgambati ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Clinical Isolates ◽  
Gain Of Function ◽  
Content Type ◽  
Regulatory Systems ◽  
Repeated Passage ◽  
High Level

ABSTRACTPseudomonas aeruginosacan develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of colistin (polymyxin E) resistance in laboratory strains and clinical isolates of this organism (MICs of 8 to 64 mg/liter). To explore the role of PmrAB in high-level clinical polymyxin resistance,P. aeruginosaisolates from chronically colistin-treated cystic fibrosis patients, most with colistin MICs of >512 mg/liter, were analyzed. These cystic fibrosis isolates contained probable gain-of-functionpmrBalleles that conferred polymyxin resistance to strains with a wild-type orpmrABdeletion background. Double mutantpmrBalleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutantpmrBalleles induced transcription from the promoter of thearnBoperon and stimulated addition of 4-amino-l-arabinose to lipid A, consistent with the known role of this lipid A modification in polymyxin resistance. For some highly polymyxin-resistant clinical isolates, repeated passage without antibiotic selection pressure resulted in loss of resistance, suggesting that secondary suppressors occur at a relatively high frequency and account for the instability of this phenotype. These results indicate thatpmrBgain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains ofP. aeruginosa.

scholarly journals Bronchial Epithelial Tet2 Maintains Epithelial Integrity during Acute Pseudomonas aeruginosa Pneumonia

2020 ◽  
Vol 89 (1) ◽  
pp. e00603-20
Author(s):  
Wanhai Qin ◽  
Xanthe Brands ◽  
Cornelis van't Veer ◽  
Alex F. de Vos ◽  
Brendon P. Scicluna ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Bronchial Epithelial Cell ◽  
Mrna Levels ◽  
Wild Type ◽  
Bronchial Epithelial ◽  
Epithelial Integrity ◽  
Content Type ◽  
Protein Levels

ABSTRACTRespiratory epithelial cells are important for pulmonary innate immune responses during Pseudomonas aeruginosa infection. Tet methylcytosine dioxygenase 2 (Tet2) has been implicated in the regulation of host defense by myeloid and lymphoid cells, but whether Tet2 also contributes to epithelial responses during pneumonia is unknown. The aim of this study was to investigate the role of bronchial epithelial Tet2 in acute pneumonia caused by P. aeruginosa. To this end, we crossed mice with Tet2 flanked by two Lox-P sites (Tet2fl/fl mice) with mice expressing Cre recombinase under the bronchial epithelial cell-specific Cc10 promoter (Cc10Cre mice) to generate bronchial epithelial cell-specific Tet2-deficient (Tet2fl/fl Cc10Cre) mice. Six hours after infection with P. aeruginosa,Tet2fl/fl Cc10Cre and wild-type mice had similar bacterial loads in bronchoalveolar lavage fluid (BALF). At this time point, Tet2fl/fl Cc10Cre mice displayed reduced mRNA levels of the chemokines Cxcl1, Cxcl2, and Ccl20 in bronchial brushes. However, Cxcl1, Cxcl2, and Ccl20 protein levels and leukocyte recruitment in BALF were not different between groups. Tet2fl/fl Cc10Cre mice had increased protein levels in BALF after infection, indicating a disturbed epithelial barrier function, which was corroborated by reduced mRNA expression of tight junction protein 1 and occludin in bronchial brushes. Differences detected between Tet2fl/fl Cc10Cre and wild-type mice were no longer present at 24 h after infection. These results suggest that bronchial epithelial Tet2 contributes to maintaining epithelial integrity by enhancing intracellular connections between epithelial cells during the early phase of P. aeruginosa pneumonia.

scholarly journals The Efflux Pump MexXY/OprM Contributes to the Tolerance and Acquired Resistance of Pseudomonas aeruginosa to Colistin

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Hélène Puja ◽  
Arnaud Bolard ◽  
Aurélie Noguès ◽  
Patrick Plésiat ◽  
Katy Jeannot
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Efflux Pump ◽  
Acquired Resistance ◽  
Intrinsic Resistance ◽  
Drug Induced ◽  
Functional Link ◽  
Content Type ◽  
Regulatory Systems ◽  
Early Drug

ABSTRACT The intrinsic resistance of Pseudomonas aeruginosa to polymyxins in part relies on the addition of 4-amino-4-deoxy-l-arabinose (Ara4N) molecules to the lipid A of lipopolysaccharide (LPS), through induction of operon arnBCADTEF-ugd (arn) expression. As demonstrated previously, at least three two-component regulatory systems (PmrAB, ParRS, and CprRS) are able to upregulate this operon when bacteria are exposed to colistin. In the present study, gene deletion experiments with the bioluminescent strain PAO1::lux showed that ParRS is a key element in the tolerance of P. aeruginosa to this last-resort antibiotic (i.e., resistance to early drug killing). Other loci of the ParR regulon, such as those encoding the efflux proteins MexXY (mexXY), the polyamine biosynthetic pathway PA4773-PA4774-PA4775, and Ara4N LPS modification process (arnBCADTEF-ugd), also contribute to the bacterial tolerance in an intricate way with ParRS. Furthermore, we found that both stable upregulation of the arn operon and drug-induced ParRS-dependent overexpression of the mexXY genes accounted for the elevated resistance of pmrB mutants to colistin. Deletion of the mexXY genes in a constitutively activated ParR mutant of PAO1 was associated with significantly increased expression of the genes arnA, PA4773, and pmrA in the absence of colistin exposure, thereby highlighting a functional link between the MexXY/OprM pump, the PA4773-PA4774-PA4775 pathway, and Ara4N-based modification of LPS. The role played by MexXY/OprM in the adaptation of P. aeruginosa to polymyxins opens new perspectives for restoring the susceptibility of resistant mutants through the use of efflux inhibitors.

scholarly journals Two-Component Regulators ControlhilAExpression by ControllingfimZandhilEExpression within Salmonella enterica Serovar Typhimurium

2014 ◽  
Vol 83 (3) ◽  
pp. 978-985 ◽  
Author(s):  
M. Aaron Baxter ◽  
Bradley D. Jones
Keyword(s):  
Transcriptional Activator ◽  
Negative Regulator ◽  
Host Cells ◽  
Type I ◽  
Dependent Manner ◽  
Content Type ◽  
Regulatory Systems ◽  
Two Component ◽  
Serovar Typhimurium ◽  
Type I Fimbriae

Salmonellae initiate disease through the invasion of host cells within the intestine. This ability to invade requires the coordinated action of numerous genes, many of which are found withinSalmonellapathogenicity island 1 (SPI-1). The key to this process is the ability of the bacteria to respond to the environment, thereby upregulating the necessary genes under optimal conditions. Central to the control of SPI-1 is the transcriptional activatorhilA. Work has identified at least 10 different activators and 8 different repressors responsible for the control ofhilA. We have previously shown thathilEis aSalmonella-specific negative regulator that is able to represshilAexpression and invasion. Additionally,fimZ, a transcriptional activator responsible for the expression of type I fimbriae as well as flagellar genes, has also been implicated in this process.fimZis homologous to response regulators from other two-component regulatory systems, although a sensor for the system has not been identified. ThephoPQandphoBRregulons are both two-component systems that negatively affecthilAexpression, although the mechanism of action has not been determined. Our results show that PhoBR is capable of inducingfimZexpression, whereas PhoPQ does not affectfimZexpression but does upregulatehilEin an FimZ-dependent manner. Therefore, phosphate (sensed by PhoBR) and magnesium (sensed by PhoPQ) levels are important in controllinghilAexpression levels whenSalmonellais in the intestinal environment.

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