The Experts below are selected from a list of 10953 Experts worldwide ranked by ideXlab platform
Josep Casadesús - One of the best experts on this subject based on the ideXlab platform.
-
The cell division factor ZapB is required for Bile Resistance in Salmonella enterica
2019Co-Authors: Sara B. Hernández, Rocío Fernández-fernández, Elena Puerta-fernández, Verónica Urdaneta, Josep CasadesúsAbstract:ABSTRACT A gene annotated as yiiU in the genome of Salmonella enterica serovar Typhimurium encodes a protein homologous to E. coli ZapB, a non-essential cell division factor involved in Z-ring assembly. ZapB− null mutants of S. enterica are Bile-sensitive. The ZapB protein is degraded in the presence of sodium deoxycholate (DOC), and degradation appears to involve the Lon protease. The amount of zapB mRNA increases in the presence of a sublethal concentration of DOC. This increase is not caused by upregulation of zapB transcription but by increased stability of zapB mRNA. DOC-induced increase of the zapB transcript is suppressed by an hfq mutation, suggesting the involvement of a small regulatory RNA. We provide evidence that such sRNA is MicA. Increased stability of zapB mRNA in the presence of DOC may counter degradation of Bile-damaged ZapB, thus providing sufficient level of functional ZapB protein to permit Z-ring assembly in the presence of Bile. IMPORTANCE Bile salts have bactericidal activity as a consequence of membrane disruption, protein denaturation and DNA damage. However, intestinal bacteria are resistant to Bile. Envelope structures such as the lipopolysaccharide and the enterobacterial common antigen act as barriers that reduce intake of Bile salts. Remodelling of the outer membrane and the peptidoglycan, activation of efflux pumps, and upregulation of stress responses also contribute to Bile Resistance. This study adds the cell division factor ZapB (and presumably the Z-ring) to the list of cellular functions involved in Bile Resistance.
-
Adaptation of Salmonella enterica to Bile: essential role of AcrAB-mediated efflux.
Environmental microbiology, 2018Co-Authors: Verónica Urdaneta, Josep CasadesúsAbstract:Adaptation to Bile is the ability to endure the lethal effects of Bile salts after growth on sublethal concentrations. Surveys of adaptation to Bile in Salmonella enterica ser. Tyhimurium reveal that active efflux is essential for adaptation while other bacterial functions involved in Bile Resistance are not. Among S. enterica mutants lacking one or more efflux systems, only strains lacking AcrAB are unable to adapt, thus revealing an essential role for AcrAB. Transcription of the acrAB operon is upregulated in the presence of a sublethal concentration of sodium deoxycholate (DOC) while other efflux loci are either weakly upregulated or irresponsive. Upregulation of acrAB transcription is strong during exponential growth, and weak in stationary cultures. Single cell analysis of ethidium bromide accumulation indicates that DOC-induced AcrAB-mediated efflux occurs in both exponential and stationary cultures. Upregulation of acrAB expression may thus be crucial at early stages of adaptation, while sustained AcrAB activity may be sufficient to confer Bile Resistance in nondividing cells.
-
Increased Bile Resistance in Salmonella enterica mutants lacking Prc periplasmic protease
International microbiology : the official journal of the Spanish Society for Microbiology, 2013Co-Authors: Sara B. Hernández, Juan A. Ayala, Gadea Rico-pérez, Francisco García-del Portillo, Josep CasadesúsAbstract:Spanish Government; European Regional Fund (grants BIO2010-15023, BIO2010-18885, and BFU2009-09200); Consejeria de Innovacion, Ciencia y Empresa; Junta de Andalucia (grant CVI-5879); Spanish Ministry of Education and Culture
-
Adaptation and Preadaptation of Salmonella enterica to Bile
PLoS genetics, 2012Co-Authors: Sara B. Hernández, Ignacio Cota, Adrien Ducret, Laurent Aussel, Josep CasadesúsAbstract:Bile possesses antibacterial activity because Bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive Bile. Sublethal concentrations of the Bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of Bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to Bile is supported by the observation that RpoS− mutants are Bile-sensitive. While adaptation to Bile involves a response by the bacterial population, individual cells can become Bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of Bile yields Bile-resistant colonies at frequencies between 10−6 and 10−7 per cell and generation. Fluctuation analysis indicates that such colonies derive from Bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that Bile Resistance can be acquired by mutation. Full genome sequencing of Bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational Bile Resistance. However, selection on lethal concentrations of Bile also provides Bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering Bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations.
-
Identification of the Salmonella enterica damX Gene Product, an Inner Membrane Protein Involved in Bile Resistance
Journal of bacteriology, 2009Co-Authors: Javier López-garrido, Francisco García-del Portillo, Nancy Cheng, Fátima García-quintanilla, Josep CasadesúsAbstract:The damX gene product of Salmonella enterica serovar Typhimurium is a protein located in the inner membrane. DamX migrates as a 70-kDa protein in SDS-PAGE even though the predicted protein size is 46 kDa. Synthesis of DamX protein occurs in both exponential- and stationary-phase cultures. Disruption of damX causes severe sensitivity to Bile. Lack of the outer membrane protein AsmA suppresses Bile sensitivity in Salmonella damX mutants.
John S. Gunn - One of the best experts on this subject based on the ideXlab platform.
-
The interaction of Bile salts with pathogenic and nonpathogenic intestinal bacteria.
Food-Borne Microbes, 2014Co-Authors: Robert W. Crawford, John S. GunnAbstract:This chapter provides an overview of Bile composition and conjugation mediated by the normal flora and its aforementioned antimicrobial effects. It discusses known molecular mechanisms behind Bile Resistance and the role Bile has in altering the virulence of enteric pathogens. Fasting and malnourishment have been shown to decrease the amount of Bile in the intestine and, consequently, leave individuals vulnerable to bacterial pathogens. Similar to commensal enteric bacteria, pathogenic Listeria monocytogenes contains Bile salt hydrolases (BSH) genes thought to confer Bile Resistance and successful colonization and disease manifestation. Probiotics researchers suggest that subsequent limitations to enterohepatic circulation in the presence of unconjugated Bile acids cause enhanced fecal loss of Bile salts. The chapter talks about the effect of Bile on pathogenic bacteria. Adaptability to the harsh effects of Bile acids is a critical component of survival for gastrointestinal pathogens. Both conjugated and unconjugated Bile salts increased expression of the CmeABC efflux pump, while other antimicrobials, including chloramphenicol, ethidium bromide, and erythromycin, did not affect transcription of cmeABC. Surface plasmon resonance provided evidence that Bile salts were capable of inhibiting binding of CmeR to the cmeABC promoter, leading to increased pump expression and elevated Bile Resistance. Interestingly, the presence of Bile salts in culture media enhanced the Resistance of Campylobacter to multiple antibiotics, including cefotaxime, novobiocin, and fusidic acid. As long as the integrity of the normal microbial flora is maintained, compounds that are mentioned in the chapter could be manufactured to target known factors contributing to Bile Resistance.
-
Bile salt mediated induction of antimicrobial and Bile Resistance in salmonella typhimurium
Microbiology, 2004Co-Authors: Angela M Prouty, John S. Gunn, Igor E Brodsky, Stanley FalkowAbstract:By DNA microarray, the Salmonella typhimurium marRAB operon was identified as being Bile-activated. Transcriptional assays confirm that marRAB is activated in the presence of Bile and that this response is concentration-dependent. The Bile salt deoxycholate is alone able to activate transcription, while there was no response in the presence of other Bile salts tested or a non-ionic detergent. Deoxycholate is able to interact with MarR and interfere with its ability to bind to the mar operator. In addition, incubation of salmonellae in the presence of sublethal concentrations of Bile is able to enhance Resistance to chloramphenicol and Bile, by means of both mar-dependent and mar-independent pathways. To further characterize putative marRAB-regulated genes that may be important for the Resistance phenotype, acrAB, which encodes an efflux pump, was analysed. In S. typhimurium, acrAB is required for Bile Resistance, but while transcription of acrAB is activated by Bile, this activation is independent of marRAB, as well as Rob, RpoS or PhoP–PhoQ. These data suggest that Bile interacts with salmonellae to increase Resistance to Bile and other antimicrobials and that this can occur by marRAB- and acrAB-dependent pathways that function independently with respect to Bile activation.
-
Salmonella enterica Serovar Typhimurium Resistance to Bile: Identification and Characterization of the tolQRA Cluster
Journal of bacteriology, 2002Co-Authors: Angela M Prouty, Jennifer C. Van Velkinburgh, John S. GunnAbstract:Salmonella enterica serovar Typhimurium is resistant to the action of Bile salts, and Resistance to Bile is enhanced in strains in which the PhoP-PhoQ (PhoPQ) two-component regulatory system has been activated. To identify genes necessary for Bile Resistance, MudJ transposon mutagenesis was performed on a strain containing a phoP mutation that results in constitutive expression of PhoP-activated genes. After screening >10,000 mutants for the loss of growth on Luria-Bertani broth-Bile plates, 14 Bile-sensitive mutants were identified. Of these 14 mutants, 3 were found to retain the Bile sensitivity phenotype upon P22 transduction, to possess wild-type growth characteristics, and to contain a smooth lipopolysaccharide. Southern hybridization experiments showed that all three strains contained unique insertions. DNA sequencing of the transposon-chromosomal-DNA fusion junctions of these strains showed all to be linked to the putative Salmonella orf1-tolQRA operon, with insertions in tolQ, orf1, and a gene upstream of the orf1-tolQRA operon not previously associated with Tol function (orfX). Through the use of transcriptional fusions, none of the putative tol (or tol-associated) genes were shown to be regulated by PhoPQ, Bile, or the RcsC-RcsB two-component system; however, all of the genes (orfX, orf1, tolQRA) are predicted to be cotranscribed. This is the first identification of Salmonella serovar Typhimurium Tol homologs and the first demonstration of their role in Bile Resistance in this organism. In addition, the observed regulation, operon arrangement, and phenotypes associated with these tol genes demonstrate significant differences from their Escherichia coli homologs.
-
PhoP-PhoQ-Regulated Loci Are Required for Enhanced Bile Resistance in Salmonella spp.
Infection and immunity, 1999Co-Authors: Jennifer C. Van Velkinburgh, John S. GunnAbstract:As enteric pathogens, Salmonella spp. are resistant to the actions of Bile. Salmonella typhimurium and Salmonella typhi strains were examined to better define the Bile Resistance phenotype. The MICs of Bile for wild-type S. typhimurium and S. typhi were 18 and 12%, respectively, and pretreatment of log-phase S. typhimurium with 15% Bile dramatically increased Bile Resistance. Mutant strains of S. typhimurium and S. typhi lacking the virulence regulator PhoP-PhoQ were killed at significantly lower Bile concentrations than wild-type strains, while strains with constitutively active PhoP were able to survive prolonged incubation with Bile at concentrations of >60%. PhoP-PhoQ was shown to mediate Resistance specifically to the Bile components deoxycholate and conjugated forms of chenodeoxycholate, and the protective effect was not generalized to other membrane-active agents. Growth of both S. typhimurium and S. typhi in Bile and in deoxycholate resulted in the induction or repression of a number of proteins, many of which appeared identical to PhoP-PhoQ-activated or -repressed products. The PhoP-PhoQ regulon was not induced by Bile, nor did any of the 21 PhoP-activated or -repressed genes tested play a role in Bile Resistance. However, of the PhoP-activated or -repressed genes tested, two (prgC and prgH) were transcriptionally repressed by Bile in the medium independent of PhoP-PhoQ. These data suggest that salmonellae can sense and respond to Bile to increase Resistance and that this response likely includes proteins that are members of the PhoP regulon. These Bile- and PhoP-PhoQ-regulated products may play an important role in the survival of Salmonella spp. in the intestine or gallbladder.
Qijing Zhang - One of the best experts on this subject based on the ideXlab platform.
-
Bile salts modulate expression of the CmeABC multidrug efflux pump in Campylobacter jejuni.
Journal of bacteriology, 2005Co-Authors: Jun Lin, Cédric Cagliero, Baoqing Guo, Yi-wen Barton, Marie-christine Maurel, Sophie Payot, Qijing ZhangAbstract:CmeABC, a multidrug efflux pump, is involved in the Resistance of Campylobacter jejuni to a broad spectrum of antimicrobial agents and is essential for Campylobacter colonization in animal intestine by mediating Bile Resistance. Previously, we have shown that expression of this efflux pump is under the control of a transcriptional repressor named CmeR. Inactivation of CmeR or mutation in the cmeABC promoter (PcmeABC) region derepresses cmeABC, leading to overexpression of this efflux pump. However, it is unknown if the expression of cmeABC can be conditionally induced by the substrates it extrudes. In this study, we examined the expression of cmeABC in the presence of various antimicrobial compounds. Although the majority of the antimicrobials tested did not affect the expression of cmeABC, Bile salts drastically elevated the expression of this efflux operon. The induction was observed with both conjugated and unconjugated Bile salts and was in a dose- and time-dependent manner. Experiments using surface plasmon resonance demonstrated that Bile salts inhibited the binding of CmeR to PcmeABC, suggesting that Bile compounds are inducing ligands of CmeR. The interaction between Bile salts and CmeR likely triggers conformational changes in CmeR, resulting in reduced binding affinity of CmeR to PcmeABC. Bile did not affect the transcription of cmeR, indicating that altered expression of cmeR is not a factor in Bile-induced overexpression of cmeABC. In addition to the CmeR-dependent induction, some Bile salts (e.g., taurocholate) also activated the expression of cmeABC by a CmeR-independent pathway. Consistent with the elevated production of CmeABC, the presence of Bile salts in culture media resulted in increased Resistance of Campylobacter to multiple antimicrobials. These findings reveal a new mechanism that modulates the expression of cmeABC and further support the notion that Bile Resistance is a natural function of CmeABC.
-
The Campylobacter jejuni Response Regulator, CbrR, Modulates Sodium Deoxycholate Resistance and Chicken Colonization
Journal of bacteriology, 2005Co-Authors: Brian H. Raphael, Qijing Zhang, Sonia Pereira, Gary A. Flom, Julian M. Ketley, Michael E. KonkelAbstract:Two-component regulatory systems play a major role in the physiological response of bacteria to environmental stimuli. Such systems are composed of a sensor histidine kinase and a response regulator whose ultimate function is to affect the expression of target genes. Response regulator mutants of Campylobacter jejuni strain F38011 were screened for sensitivity to sodium deoxycholate. A mutation in Cj0643, which encodes a response regulator with no obvious cognate histidine kinase, resulted in an absence of growth on plates containing a subinhibitory concentration of sodium deoxcholate (1%, wt/vol). In broth cultures containing 0.05% (wt/vol) sodium deoxycholate, growth of the mutant was significantly inhibited compared to growth of the C. jejuni F38011 wild-type strain. Complementation of the C. jejuni cbrR mutant in trans restored growth in both broth and plate cultures supplemented with sodium deoxycholate. Based on the phenotype displayed by its mutation, we designated the gene corresponding to Cj0643 as cbrR (Campylobacter Bile Resistance regulator). While the MICs of a variety of Bile salts and other detergents for the C. jejuni cbrR mutant were lower, no difference was noted in its sensitivity to antibiotics or osmolarity. Finally, chicken colonization studies demonstrated that the C. jejuni cbrR mutant had a reduced ability to colonize compared to the wild-type strain. These data support previous findings that Bile Resistance contributes to colonization of chickens and establish that the response regulator, CbrR, modulates Resistance to Bile salts in C. jejuni.
-
critical role of multidrug efflux pump cmeabc in Bile Resistance and in vivo colonization of campylobacter jejuni
Infection and Immunity, 2003Co-Authors: Orhan Sahin, L.o. Michel, Qijing ZhangAbstract:CmeABC functions as a multidrug efflux pump contributing to the Resistance of Campylobacter to a broad range of antimicrobials. In this study, we examined the role of CmeABC in Bile Resistance and its contribution to the adaptation of Campylobacter jejuni in the intestinal tract of the chicken, a natural host and a major reservoir for Campylobacter. Inactivation of cmeABC drastically decreased the Resistance of Campylobacter to various Bile salts. Addition of choleate (2 mM) in culture medium impaired the in vitro growth of the cmeABC mutants but had no effect on the growth of the wild-type strain. Bile concentration varied in the duodenum, jejunum, and cecum of chicken intestine, and the inhibitory effect of the intestinal extracts on the in vitro growth of Campylobacter was well correlated with the total Bile concentration in the individual sections of chicken intestine. When inoculated into chickens, the wild-type strain colonized the birds as early as day 2 postinoculation with a density as high as 10 7 CFU/g of feces. In contrast, the cmeABC mutants failed to colonize any of the inoculated chickens throughout the study. The minimum infective dose for the cmeABC mutant was at least 2.6 10 4 -fold higher than that of the wild-type strain. Complementation of the cmeABC mutants with a wild-type cmeABC allele in trans fully restored the in vitro growth in Bile-containing media and the in vivo colonization to the levels of the wild-type strain. Immunoblotting analysis indicated that CmeABC is expressed and immunogenic in chickens experimentally infected with C. jejuni. Together, these findings provide compelling evidence that CmeABC, by mediating Resistance to Bile salts in the intestinal tract, is required for successful colonization of C. jejuni in chickens. Inhibition of CmeABC function may not only control antibiotic Resistance but also prevent the in vivo colonization of pathogenic Campylobacter. Bile is produced in the liver, stored in the gall bladder, and released into the small intestine for digestion of fats. Bile contains a group of detergent-like Bile salts which not only play
-
Critical role of multidrug efflux pump CmeABC in Bile Resistance and in vivo colonization of Campylobacter jejuni.
Infection and immunity, 2003Co-Authors: Jun Lin, Orhan Sahin, L.o. Michel, Qijing ZhangAbstract:ABSTRACT CmeABC functions as a multidrug efflux pump contributing to the Resistance of Campylobacter to a broad range of antimicrobials. In this study, we examined the role of CmeABC in Bile Resistance and its contribution to the adaptation of Campylobacter jejuni in the intestinal tract of the chicken, a natural host and a major reservoir for Campylobacter. Inactivation of cmeABC drastically decreased the Resistance of Campylobacter to various Bile salts. Addition of choleate (2 mM) in culture medium impaired the in vitro growth of the cmeABC mutants but had no effect on the growth of the wild-type strain. Bile concentration varied in the duodenum, jejunum, and cecum of chicken intestine, and the inhibitory effect of the intestinal extracts on the in vitro growth of Campylobacter was well correlated with the total Bile concentration in the individual sections of chicken intestine. When inoculated into chickens, the wild-type strain colonized the birds as early as day 2 postinoculation with a density as high as 107 CFU/g of feces. In contrast, the cmeABC mutants failed to colonize any of the inoculated chickens throughout the study. The minimum infective dose for the cmeABC mutant was at least 2.6 × 104-fold higher than that of the wild-type strain. Complementation of the cmeABC mutants with a wild-type cmeABC allele in trans fully restored the in vitro growth in Bile-containing media and the in vivo colonization to the levels of the wild-type strain. Immunoblotting analysis indicated that CmeABC is expressed and immunogenic in chickens experimentally infected with C. jejuni. Together, these findings provide compelling evidence that CmeABC, by mediating Resistance to Bile salts in the intestinal tract, is required for successful colonization of C. jejuni in chickens. Inhibition of CmeABC function may not only control antibiotic Resistance but also prevent the in vivo colonization of pathogenic Campylobacter.
-
outer membrane proteins key players for bacterial adaptation in host niches
Microbes and Infection, 2002Co-Authors: Jun Lin, Shouxiong Huang, Qijing ZhangAbstract:Outer membrane proteins (OMPs) of Gram-negative bacteria have diverse functions and are directly involved in the interaction with various environments encountered by pathogenic organisms. Thus, OMPs represent important virulence factors and play essential roles in bacterial adaptation to host niches, which are usually hostile to invading pathogens. Understanding the structure and functions of bacterial OMPs will facilitate the design of antimicrobial drugs and vaccines. In this paper, we will present a brief review on OMPs that contribute to bacterial adaptive responses including iron uptake, antimicrobial peptide Resistance, serum Resistance, and drug/Bile Resistance.
Ana I. Prieto - One of the best experts on this subject based on the ideXlab platform.
-
The GATC-Binding Protein SeqA Is Required for Bile Resistance and Virulence in Salmonella enterica Serovar Typhimurium
Journal of bacteriology, 2007Co-Authors: Ana I. Prieto, Francisco Ramos-morales, Francisco García-del Portillo, Marcello Jakomin, Ignacio Segura, M. Graciela Pucciarelli, Josep CasadesúsAbstract:Disruption of the seqA gene of Salmonella enterica serovar Typhimurium causes defects similar to those described in E. coli: filament formation, aberrant nucleoid segregation, induction of the SOS response, envelope instability, and increased sensitivity to membrane-damaging agents. Differences between SeqA− mutants of E. coli and S. enterica, however, are found. SeqA− mutants of S. enterica form normal colonies and do not exhibit alterations in phage plaquing morphology. Lack of SeqA causes attenuation of S. enterica virulence by the oral route but not by the intraperitoneal route, suggesting a virulence defect in the intestinal stage of infection. However, SeqA− mutants are fully proficient in the invasion of epithelial cells. We hypothesize that attenuation of SeqA− mutants by the oral route may be caused by Bile sensitivity, which in turn may be a consequence of envelope instability.
-
Repair of DNA damage induced by Bile salts in Salmonella enterica
Genetics, 2006Co-Authors: Ana I. Prieto, Francisco Ramos-morales, Josep CasadesúsAbstract:Exposure of Salmonella enterica to sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium glychocholate, sodium taurocholate, or sodium glycochenodeoxycholate induces the SOS response, indicating that the DNA-damaging activity of Bile resides in Bile salts. Bile increases the frequency of GC → AT transitions and induces the expression of genes belonging to the OxyR and SoxRS regulons, suggesting that Bile salts may cause oxidative DNA damage. S. enterica mutants lacking both exonuclease III (XthA) and endonuclease IV (Nfo) are Bile sensitive, indicating that S. enterica requires base excision repair (BER) to overcome DNA damage caused by Bile salts. Bile Resistance also requires DinB polymerase, suggesting the need of SOS-associated translesion DNA synthesis. Certain recombination functions are also required for Bile Resistance, and a key factor is the RecBCD enzyme. The extreme Bile sensitivity of RecB−, RecC−, and RecA− RecD− mutants provides evidence that Bile-induced damage may impair DNA replication.
-
Role for Salmonella enterica Enterobacterial Common Antigen in Bile Resistance and Virulence
Journal of bacteriology, 2003Co-Authors: Francisco Ramos-morales, Ana I. Prieto, Carmen R. Beuzón, David W. Holden, Josep CasadesúsAbstract:Passage through the digestive tract exposes Salmonella enterica to high concentrations of Bile salts, powerful detergents that disrupt biological membranes. Mutations in the wecD or wecA gene, both of which are involved in the synthesis of enterobacterial common antigen (ECA), render S. enterica serovar Typhimurium sensitive to the Bile salt deoxycholate. Competitive infectivity analysis of wecD and wecA mutants in the mouse model indicates that ECA is an important virulence factor for oral infection. In contrast, lack of ECA causes only a slight decrease in Salmonella virulence during intraperitoneal infection. A tentative interpretation is that ECA may contribute to Salmonella virulence by protecting the pathogen from Bile salts.
Karl E. Klose - One of the best experts on this subject based on the ideXlab platform.
-
Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae Bile Resistance, virulence factor expression, and intestinal colonization
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Daniele Provenzano, Karl E. KloseAbstract:The transmembrane transcriptional activators ToxR and TcpP modulate expression of Vibrio cholerae virulence factors by exerting control over toxT, which encodes the cytoplasmic transcriptional activator of the ctx, tcp, and acf virulence genes. However, ToxR, independently of TcpP and ToxT, activates and represses transcription of the genes encoding two outer-membrane porins, OmpU and OmpT. To determine the role of ToxR-dependent porin regulation in V. cholerae pathogenesis, the ToxR-activated ompU promoter was used to drive ompT transcription in a strain lacking OmpU. Likewise, the ToxR-repressed ompT promoter was used to drive ompU transcription in a strain lacking both ToxR and OmpT. This strategy allowed the generation of a toxR+ strain that expresses OmpT in place of OmpU, and a toxR− strain that expresses OmpU in place of OmpT. Growth rates in the presence of Bile salts and other anionic detergents were retarded for the toxR+ V. cholerae expressing OmpT in place of OmpU, but increased in toxR− V. cholerae expressing OmpU in place of OmpT. Additionally, the toxR+ V. cholerae expressing OmpT in place of OmpU expressed less cholera toxin and toxin-coregulated pilus, and this effect was shown to be caused by reduced toxT transcription in this strain. Finally, the toxR+ V. cholerae expressing OmpT in place of OmpU was ≈100-fold reduced in its ability to colonize the infant-mouse intestine. Our results indicate that ToxR-dependent modulation of the outer membrane porins OmpU and OmpT is critical for V. cholerae Bile Resistance, virulence factor expression, and intestinal colonization.
-
The Virulence Regulatory Protein ToxR Mediates Enhanced Bile Resistance in Vibrio cholerae and Other Pathogenic Vibrio Species
Infection and immunity, 2000Co-Authors: Daniele Provenzano, Darren A. Schuhmacher, Justin L. Barker, Karl E. KloseAbstract:The transmembrane regulatory protein ToxR is required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae, including cholera toxin (CT) and the toxin coregulated pilus (TCP). ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, which is the direct transcriptional activator of CT and TCP genes. However, ToxR, independent of ToxT, directly activates and represses transcription of the outer membrane porins OmpU and OmpT, respectively. The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic elements, while the toxR, ompU, and ompT genes are apparently in the ancestral Vibrio chromosome. The contribution of ToxR-dependent modulation of outer membrane porins to cholera pathogenesis has remained unknown. We demonstrate that ToxR mediates enhanced Bile Resistance in a ToxT-independent manner. In both classical and El Tor biotypes of V. cholerae, a toxR mutant strain has a reduced minimum bactericidal concentration (MBC) of Bile, the Bile component deoxycholate (DC), and the anionic detergent sodium dodecyl sulfate (SDS) compared to both wild-type and toxT mutant strains. Classical and El Tor toxR mutant strains also exhibit reduced growth rates at subinhibitory concentrations of DC and SDS. Growth of either V. cholerae biotype in subinhibitory concentrations of Bile or DC induces increased ToxR-dependent production of a major 38-kDa outer membrane protein, which was confirmed to be OmpU by Western blot. Measurement of transcription of a ompUp-lacZ fusion in both biotypes reveals stimulation (about two- to threefold) of ToxR-dependent ompU transcription by the presence of Bile or DC, suggesting that ToxR may respond to the presence of Bile. The toxR mutant strains of three additional human intestinal pathogenic Vibrio species, V. mimicus, V. fluvialis, and V. parahaemolyticus, display lower MBCs of Bile, DC, and SDS and have altered outer membrane protein profiles compared to the parental wild-type strains. Our results demonstrate a conserved role for ToxR in the modulation of outer membrane proteins and Bile Resistance of pathogenic Vibrio species and suggest that these ToxR-dependent outer membrane proteins may mediate enhanced Resistance to Bile. We speculate that ToxR-mediated Bile Resistance was an early step in the evolution of V. cholerae as an intestinal pathogen.