Reciprocal Regulation of Resistance-Nodulation-Division Efflux Systems and the Cpx Two-Component System in Vibrio cholerae

ABSTRACT The Cpx two-component regulatory system has been shown in Escherichia coli to alleviate stress caused by misfolded cell envelope proteins. The Vibrio cholerae Cpx system was previously found to respond to cues distinct from those in the E. coli system, suggesting that this system fulfills a different physiological role in the cholera pathogen. Here, we used microarrays to identify genes that were regulated by the V. cholerae Cpx system. Our observations suggest that the activation of the V. cholerae Cpx system does not induce expression of genes involved in the mitigation of stress generated by misfolded cell envelope proteins but promotes expression of genes involved in antimicrobial resistance. In particular, activation of the Cpx system induced expression of the genes encoding the VexAB and VexGH resistance-nodulation-division (RND) efflux systems and their cognate outer membrane pore protein TolC. The promoters for these loci contained putative CpxR consensus binding sites, and ectopic cpxR expression activated transcription from the promoters for the RND efflux systems. CpxR was not required for intrinsic antimicrobial resistance, but CpxR activation enhanced resistance to antimicrobial substrates of VexAB and VexGH. Mutations that inactivated VexAB or VexGH efflux activity resulted in the activation of the Cpx response, suggesting that vexAB and vexGH and the cpxP-cpxRA system are reciprocally regulated. We speculate that the reciprocal regulation of the V. cholerae RND efflux systems and the Cpx two-component system is mediated by the intracellular accumulation of an endogenously produced metabolic by-product that is normally extruded from the cell by the RND efflux systems.

[1]  T. Raivio,et al.  Mutations That Impact the Enteropathogenic Escherichia coli Cpx Envelope Stress Response Attenuate Virulence in Galleria mellonella , 2012, Infection and Immunity.

[2]  T. Raivio,et al.  Just scratching the surface: an expanding view of the Cpx envelope stress response. , 2012, FEMS microbiology letters.

[3]  T. Silhavy,et al.  Periplasmic stress and ECF sigma factors. , 2001, Annual review of microbiology.

[4]  L. Piddock.  Multidrug-resistance efflux pumps ? not just for resistance , 2006, Nature Reviews Microbiology.

[5]  M. Waldor,et al.  Genetic Analysis of Activation of the Vibrio cholerae Cpx Pathway , 2009, Journal of bacteriology.

[6]  V. L. Miller,et al.  Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Colin Hughes,et al.  Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export , 2000, Nature.

[8]  Koichiro Yamamoto,et al.  Culture Conditions for Stimulating Cholera Toxin Production by Vibrio cholerae O1 El Tor , 1986, Microbiology and immunology.

[9]  M. Waldor,et al.  Hfq is essential for Vibrio cholerae virulence and downregulates sigma expression. , 2004, Molecular microbiology.

[10]  J. Eswaran,et al.  Three's company: component structures bring a closer view of tripartite drug efflux pumps. , 2004, Current opinion in structural biology.

[11]  M. Waldor,et al.  The Vibrio cholerae ToxR-Regulated Porin OmpU Confers Resistance to Antimicrobial Peptides , 2004, Infection and Immunity.

[12]  V. L. Miller,et al.  Genetics of bacterial enterotoxins. , 1986, Annual review of microbiology.

[13]  S. Levy,et al.  Regulation of acrAB expression by cellular metabolites in Escherichia coli. , 2014, The Journal of antimicrobial chemotherapy.

[14]  G. Kovacikova,et al.  The Alternative Sigma Factor σE Plays an Important Role in Intestinal Survival and Virulence in Vibrio cholerae , 2002, Infection and Immunity.

[15]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[16]  T. Raivio,et al.  Three (and more) component regulatory systems – auxiliary regulators of bacterial histidine kinases , 2010, Molecular microbiology.

[17]  T. Silhavy,et al.  The sigma(E) and the Cpx signal transduction systems control the synthesis of periplasmic protein-folding enzymes in Escherichia coli. , 1997, Genes & development.

[18]  J. Mekalanos,et al.  Cholera toxin transcriptional activator ToxR is a transmembrane DNA binding protein , 1987, Cell.

[19]  Robert G. Martin,et al.  Reduction of Cellular Stress by TolC-Dependent Efflux Pumps in Escherichia coli Indicated by BaeSR and CpxARP Activation of spy in Efflux Mutants , 2012, Journal of bacteriology.

[20]  William R. Jacobs,et al.  Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice , 1999, Nature.

[21]  J. Martínez.  The role of natural environments in the evolution of resistance traits in pathogenic bacteria , 2009, Proceedings of the Royal Society B: Biological Sciences.

[22]  J. Eswaran,et al.  Structure and function of TolC: the bacterial exit duct for proteins and drugs. , 2004, Annual review of biochemistry.

[23]  T. Silhavy,et al.  The sigmaE and Cpx regulatory pathways: overlapping but distinct envelope stress responses. , 1999, Current opinion in microbiology.

[24]  D. Provenzano,et al.  Vibrio cholerae RND Family Efflux Systems Are Required for Antimicrobial Resistance, Optimal Virulence Factor Production, and Colonization of the Infant Mouse Small Intestine , 2008, Infection and Immunity.

[25]  Daniel L. Popkin,et al.  The Cpx Envelope Stress Response Is Controlled by Amplification and Feedback Inhibition , 1999, Journal of bacteriology.

[26]  M. Levine,et al.  Investigation of the Roles of Toxin-Coregulated Pili and Mannose-Sensitive Hemagglutinin Pili in the Pathogenesis of Vibrio cholerae O139 Infection , 1998, Infection and Immunity.

[27]  H. Zgurskaya,et al.  AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux Complex* , 2004, Journal of Biological Chemistry.

[28]  R. Sakazaki,et al.  [A NEW SELECTIVE ISOLATION MEDIUM FOR THE VIBRIO GROUP; ON A MODIFIED NAKANISHI'S MEDIUM (TCBS AGAR MEDIUM)]. , 1963, Nihon saikingaku zasshi. Japanese journal of bacteriology.

[29]  K. Klose,et al.  Characterization of the Role of the ToxR-Modulated Outer Membrane Porins OmpU and OmpT in Vibrio cholerae Virulence , 2001, Journal of bacteriology.

[30]  J. Pogliano,et al.  Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system. , 1997, Genes & development.

[31]  G. Segal,et al.  Identification of CpxR as a Positive Regulator of icm and dot Virulence Genes of Legionella pneumophila , 2003, Journal of bacteriology.

[32]  Maria Labandeira-Rey,et al.  Characterization of the CpxRA Regulon in Haemophilus ducreyi , 2010, Infection and Immunity.

[33]  R. Heermann,et al.  Purification, Reconstitution, and Characterization of the CpxRAP Envelope Stress System of Escherichia coli* , 2007, Journal of Biological Chemistry.

[34]  T. Silhavy,et al.  Transduction of envelope stress in Escherichia coli by the Cpx two-component system , 1997, Journal of bacteriology.

[35]  H. Goodrich-Blair,et al.  CpxRA Contributes to Xenorhabdus nematophila Virulence through Regulation of lrhA and Modulation of Insect Immunity , 2009, Applied and Environmental Microbiology.

[36]  J. Bina,et al.  The Cyclic Dipeptide Cyclo(Phe-Pro) Inhibits Cholera Toxin and Toxin-Coregulated Pilus Production in O1 El Tor Vibrio cholerae , 2010, Journal of bacteriology.

[37]  S. Faruque,et al.  ToxR regulon of Vibrio cholerae and its expression in vibrios shed by cholera patients , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[38]  C. Tacket,et al.  An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers , 2007, Proceedings of the National Academy of Sciences.

[39]  K. Poole,et al.  Influence of the MexAB-OprM Multidrug Efflux System on Quorum Sensing in Pseudomonas aeruginosa , 1998, Journal of bacteriology.

[40]  J. Mekalanos,et al.  Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[41]  S. Salzberg,et al.  DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae , 2000, Nature.

[42]  M H Saier,et al.  Phylogeny of multidrug transporters. , 2001, Seminars in cell & developmental biology.

[43]  Yunlong Liu,et al.  Activation of CpxRA in Haemophilus ducreyi Primarily Inhibits the Expression of Its Targets, Including Major Virulence Determinants , 2013, Journal of bacteriology.

[44]  M. Levine,et al.  Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans , 1988, The Journal of experimental medicine.

[45]  D. Nies,et al.  Efflux-mediated heavy metal resistance in prokaryotes. , 2003, FEMS microbiology reviews.

[46]  S. Imbeaud,et al.  Global Analysis of Extracytoplasmic Stress Signaling in Escherichia coli , 2009, PLoS genetics.

[47]  C. Prigent-Combaret,et al.  Complex Regulatory Network Controls Initial Adhesion and Biofilm Formation in Escherichia coli via Regulation of thecsgD Gene , 2001, Journal of bacteriology.

[48]  Susan M. Butler,et al.  Host-induced epidemic spread of the cholera bacterium , 2002, Nature.

[49]  J. Martínez,et al.  RND multidrug efflux pumps: what are they good for? , 2013, Front. Microbio..

[50]  K. Klose,et al.  Vibrio cholerae and cholera: out of the water and into the host. , 2002, FEMS microbiology reviews.

[51]  R. Taylor,et al.  Toxin-coregulated pilus, but not mannose-sensitive hemagglutinin, is required for colonization by Vibrio cholerae O1 El Tor biotype and O139 strains , 1996, Infection and immunity.

[52]  A. McGuire,et al.  Genome-wide Profiling of Promoter Recognition by the Two-component Response Regulator CpxR-P in Escherichia coli * , 2002, The Journal of Biological Chemistry.

[53]  A. Yamaguchi,et al.  Indole induces the expression of multidrug exporter genes in Escherichia coli , 2004, Molecular microbiology.

[54]  J. Bina,et al.  Vibrio cholerae vexH Encodes a Multiple Drug Efflux Pump That Contributes to the Production of Cholera Toxin and the Toxin Co-Regulated Pilus , 2012, PloS one.

[55]  A. Delcour,et al.  The Vibrio cholerae Porins OmpU and OmpT Have Distinct Channel Properties* , 2003, The Journal of Biological Chemistry.

[56]  H. Nikaido,et al.  Bypassing the periplasm: reconstitution of the AcrAB multidrug efflux pump of Escherichia coli. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[57]  C. van Delden,et al.  Overexpression of the MexEF-OprN Multidrug Efflux System Affects Cell-to-Cell Signaling in Pseudomonas aeruginosa , 2001, Journal of bacteriology.

[58]  J. Mekalanos,et al.  Characterization of the Vibrio cholerae vexAB and vexCD efflux systems , 2006, Archives of Microbiology.

[59]  T. Tsukihara,et al.  Crystal Structure of the Membrane Fusion Protein, MexA, of the Multidrug Transporter in Pseudomonas aeruginosa* , 2004, Journal of Biological Chemistry.

[60]  M. E. Bauer,et al.  Deletion of mtrC in Haemophilus ducreyi Increases Sensitivity to Human Antimicrobial Peptides and Activates the CpxRA Regulon , 2011, Infection and Immunity.

[61]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[62]  Vinod Nair,et al.  SQ109 Targets MmpL3, a Membrane Transporter of Trehalose Monomycolate Involved in Mycolic Acid Donation to the Cell Wall Core of Mycobacterium tuberculosis , 2012, Antimicrobial Agents and Chemotherapy.

[63]  T. Silhavy,et al.  CpxP, a Stress-Combative Member of the Cpx Regulon , 1998, Journal of bacteriology.

[64]  J. Bina,et al.  Vibrio cholerae ToxR Downregulates Virulence Factor Production in Response to Cyclo(Phe-Pro) , 2013, mBio.

[65]  V. Srinivasan,et al.  KpnEF, a New Member of the Klebsiella pneumoniae Cell Envelope Stress Response Regulon, Is an SMR-Type Efflux Pump Involved in Broad-Spectrum Antimicrobial Resistance , 2013, Antimicrobial Agents and Chemotherapy.

[66]  Rita Tamayo,et al.  Genes induced late in infection increase fitness of Vibrio cholerae after release into the environment. , 2007, Cell host & microbe.

[67]  P. Tulkens,et al.  Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy. , 2003, The Journal of antimicrobial chemotherapy.

[68]  M. Waldor,et al.  Hfq is essential for Vibrio cholerae virulence and downregulates σE expression , 2004 .

[69]  A. Alonso,et al.  Environmental selection of antibiotic resistance genes. , 2001, Environmental microbiology.

[70]  Robert G. Martin,et al.  An Excretory Function for the Escherichia coli Outer Membrane Pore TolC: Upregulation of marA and soxS Transcription and Rob Activity Due to Metabolites Accumulated in tolC Mutants , 2009, Journal of bacteriology.

[71]  B. Dhakal,et al.  The Cpx Stress Response System Potentiates the Fitness and Virulence of Uropathogenic Escherichia coli , 2013, Infection and Immunity.

[72]  K. Klose,et al.  Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae bile resistance, virulence factor expression, and intestinal colonization. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[73]  F. Baquero,et al.  Mechanisms of iron acquisition and bacterial virulence. , 1990, FEMS microbiology reviews.

[74]  T. Linn,et al.  Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ , 1990, Journal of bacteriology.

[75]  E. López-Solanilla,et al.  The role of several multidrug resistance systems in Erwinia chrysanthemi pathogenesis. , 2006, Molecular plant-microbe interactions : MPMI.

[76]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[77]  T. Tran,et al.  Toxic Waste Disposal in Escherichia coli , 2002, Journal of bacteriology.

[78]  M. Woodward,et al.  Role of the Two-Component Regulator CpxAR in the Virulence of Salmonella enterica Serotype Typhimurium , 2004, Infection and Immunity.