Klebsiella pneumoniae TolC contributes to antimicrobial resistance, exopolysaccharide production, and virulence.

Klebsiella pneumoniae is a Gram-negative bacterium that causes a variety of human diseases, ranging from pneumonia to urinary tract infections and invasive diseases. The emergence of K. pneumoniae strains that are resistant to multiple antibiotics has made treatment more complex and led to K. pneumoniae becoming a global health threat. Addressing this threat necessitates the development of new therapeutic strategies to combat this pathogen, including strategies to overcome antimicrobial resistance and therapeutics for novel targets such as antivirulence. Here, we investigated the function of TolC, an outer membrane protein essential for the function of tripartite transporters, in K. pneumoniae. Mutation of tolC rendered K. pneumoniae hypersensitive to multiple antibiotics. Moreover, the tolC mutation impaired capsule production and affected the expression of key capsule biosynthetic genes, indicating a regulatory role for TolC in capsule biosynthesis. Additionally, TolC was essential for growth under iron-limiting conditions, suggesting its involvement in iron acquisition. The tolC mutant exhibited increased adherence to human enterocytes and enhanced serum sensitivity. In the Galleria mellonella infection model, the tolC mutant displayed reduced virulence compared to the wild type. Our findings highlight the pleiotropic role of TolC in K. pneumoniae pathobiology, influencing antimicrobial resistance, capsule production, iron homeostasis, adherence to host cells, and virulence. Understanding the multifaceted role of TolC in K. pneumoniae may guide the development of new therapeutic strategies against this pathogen. .

Vibrio cholerae RND efflux systems: mediators of stress responses, colonization and pathogenesis.

Resistance Nodulation Division (RND) efflux systems are ubiquitous transporters in gram-negative bacteria that provide protection against antimicrobial agents and thereby enhance survival in virtually all environments these prokaryotes inhabit. Vibrio cholerae is a dual lifestyle enteric pathogen that spends much of its existence in aquatic environments. An unwitting encounter with a human host can lead to V. cholerae intestinal colonization by strains that encode cholera toxin and toxin co-regulated pilus virulence factors leading to potentially fatal cholera diarrhea and dissemination in the environment. Adaptive response mechanisms to host factors encountered by these pathogens are therefore critical both to engage survival mechanisms such as RND-mediated transporters and to induce timely expression of virulence factors. Sensing of cues encountered in the host may therefore activate more than protective responses such as efflux systems, but also be coordinated to initiate expression of virulence factors. This review summarizes recent advances that contribute towards the understanding of RND efflux physiological functions and how the transport systems interface with the regulation of virulence factor production in V. cholerae.

Bile Salts Promote ToxR Regulon Activation during Growth under Virulence-Inducing Conditions.

Cholera is an epidemic disease caused by the Gram-negative bacterium Vibrio cholerae. V. cholerae is found in aquatic ecosystems and infects people through the consumption of V. cholerae-contaminated food or water. Following ingestion, V. cholerae responds to host cues to activate the expression of critical virulence genes that are under the control of a hierarchical regulatory system called the ToxR regulon. The ToxR regulon is tightly regulated and is expressed in vitro only under special growth conditions referred to as AKI conditions. AKI conditions have been instrumental in elucidating V. cholerae virulence regulation, but the chemical cues within AKI medium that activate virulence gene expression are unknown. In this study, we fractionated AKI medium on a reverse-phase chromatography column (RPCC) and showed that the virulence-activating molecules were retained on the RPCC column and recovered in the eluate. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of the eluate revealed the presence of a known ToxR regulon activator, taurocholate, and other bile salts. The RPCC eluate activated the ToxR regulon when added to noninducing medium and promoted TcpP dimerization in a two-hybrid system, consistent with taurocholate being responsible for the virulence-inducing activity of AKI medium. Additional experiments using purified bile salts showed that the ToxR regulon was preferentially activated in response to primary bile acids. The results of this study shed light on the chemical cues involved in V. cholerae virulence activation and suggested that V. cholerae virulence genes are modulated in response to regionally specific bile acid species in the intestine.

Vibrio cholerae TolC Is Required for Expression of the ToxR Regulon.

Vibrio cholerae is a Gram-negative bacterium that causes the enteric disease cholera. V. cholerae colonization of the human intestine is dependent on the expression of both virulence genes and environmental adaptation genes involved in antimicrobial resistance. The expression of virulence genes, including the genes encoding the main virulence factors cholera toxin (CT) and the toxin-coregulated pilus (TCP), are coordinately regulated by the ToxR regulon. Tripartite transport systems belonging to the ATP binding cassette, major facilitator, and resistance-nodulation-division families are critical for V. cholerae pathogenesis. Transport systems belonging to these families contribute to myriad phenotypes, including protein secretion, antimicrobial resistance, and virulence. TolC plays a central role in bacterial physiology by functioning as the outer membrane pore protein for tripartite transport systems. Consistent with this, V. cholerae tolC was previously found to be required for MARTX toxin secretion and antimicrobial resistance. Here, we investigated the contribution of TolC to V. cholerae virulence. We documented that tolC was required for CT and TCP production in O1 El Tor V. cholerae. This phenotype was linked to repression of the critical ToxR regulon transcription factor aphA. Decreased aphA transcription correlated with increased expression of the LysR-family transcription factor leuO. Deletion of leuO restored aphA expression, and CT and TCP production, in a tolC mutant. The collective results document that tolC is required for ToxR regulon expression and further suggest that tolC participates in an efflux-dependent feedback circuit to regulate virulence gene expression.

ToxR Mediates the Antivirulence Activity of Phenyl-Arginine-ß-Naphthylamide To Attenuate Vibrio cholerae Virulence.

Multidrug efflux systems belonging to the resistance-nodulation-cell division (RND) family are ubiquitous in Gram-negative bacteria and are critical for antimicrobial resistance. This realization has led to efforts to develop efflux pump inhibitors (EPI) for use as adjuvants for antibiotic treatment of resistant organisms. However, the functions of RND transporters extend beyond antimicrobial resistance to include physiological functions that are critical for pathogenesis, suggesting that EPIs could also be used as antivirulence therapeutics. This was documented in the enteric pathogen Vibrio cholerae, in which EPIs were shown to attenuate the production of the critical virulence factors cholera toxin (CT) and the toxin-coregulated pilus (TCP). In this study, we investigated the antivirulence mechanism of action of the EPI phenyl-arginine-ß-naphthylamide (PAßN) on V. cholerae. Using bioassays, we documented that PAßN inhibited virulence factor production in three epidemic V. cholerae isolates. Transcriptional reporter studies and mutant analysis indicated that PAßN initiated a ToxR-dependent regulatory circuit to activate leuO expression and that LeuO repressed the expression of the critical virulence activator aphA to attenuate CT and TCP production. The antivirulence activity of PAßN was found to be dependent on the ToxR periplasmic sensing domain (PPD), suggesting that a feedback mechanism was involved in its activity. Collectively, the data indicated that PAßN inhibited V. cholerae virulence factor production by activating a ToxR-dependent metabolic feedback mechanism to repress the expression of the ToxR virulence regulon. This suggests that efflux pump inhibitors could be used as antivirulence therapeutics for the treatment of cholera and perhaps that of other Gram-negative pathogens.

Complete Genome Sequence of Klebsiella pneumoniae Strain ATCC 43816.

Klebsiella pneumoniae is a member of Enterobacteriaceae that causes a multitude of infections in compromised and healthy individuals. The rise of hypervirulent and multiple-drug-resistant K. pneumoniae strains has made this organism a global health threat. Here, we report the complete genome sequence of K. pneumoniae strain ATCC 43816.

Complete Genome Sequence of Vibrio cholerae O1 El Tor Strain C6706.

Vibrio cholerae is a global health threat and a model enteric pathogen that causes the human disease cholera. Here, we report the complete genome sequence of the seventh-pandemic V. cholerae O1 El Tor strain C6706.

Indole Inhibits ToxR Regulon Expression in Vibrio cholerae.

Indole is a degradation product of tryptophan that functions as a signaling molecule in many bacteria. This includes Vibrio cholerae, where indole was shown to regulate biofilm and type VI secretion in nontoxigenic environmental isolates. Indole is also produced by toxigenic V. cholerae strains in the human intestine, but its significance in the host is unknown. We investigated the effects of indole on toxigenic V. cholerae O1 El Tor during growth under virulence inducing conditions. The indole transcriptome was defined by RNA sequencing and showed widespread changes in the expression of genes involved in metabolism, biofilm production, and virulence factor production. In contrast, genes involved in type VI secretion were not affected by indole. We subsequently found that indole repressed genes involved in V. cholerae pathogenesis, including the ToxR virulence regulon. Consistent with this, indole inhibited cholera toxin and toxin-coregulated pilus production in a dose-dependent manner. The effects of indole on virulence factor production and biofilm were linked to ToxR and the ToxR-dependent regulator LeuO. The expression of leuO was increased by exogenous indole and linked to repression of the ToxR virulence regulon. This process was dependent on the ToxR periplasmic domain, suggesting that indole was a ToxR agonist. This conclusion was further supported by results showing that the ToxR periplasmic domain contributed to indole-mediated increased biofilm production. Collectively, our results suggest that indole may be a niche-specific cue that can function as a ToxR agonist to modulate virulence gene expression and biofilm production in V. cholerae.

The Vibrio cholerae RND efflux systems impact virulence factor production and adaptive responses via periplasmic sensor proteins.

Resistance-nodulation-division (RND) efflux systems are ubiquitous transporters in Gram-negative bacteria that are essential for antibiotic resistance. The RND efflux systems also contribute to diverse phenotypes independent of antimicrobial resistance, but the mechanism by which they affect most of these phenotypes is unclear. This is the case in Vibrio cholerae where the RND systems function in antimicrobial resistance and virulence factor production. Herein, we investigated the linkage between RND efflux and V. cholerae virulence. RNA sequencing revealed that the loss of RND efflux affected the activation state of periplasmic sensing systems including the virulence regulator ToxR. Activation of ToxR in an RND null mutant resulted in ToxR-dependent transcription of the LysR-family regulator leuO. Increased leuO transcription resulted in the repression of the ToxR virulence regulon and attenuated virulence factor production. Consistent with this, leuO deletion restored virulence factor production in an RND-null mutant, but not its ability to colonize infant mice; suggesting that RND efflux was epistatic to virulence factor production for colonization. The periplasmic sensing domain of ToxR was required for the induction of leuO transcription in the RND null mutant, suggesting that ToxR responded to metabolites that accumulated in the periplasm. Our results suggest that ToxR represses virulence factor production in response to metabolites that are normally effluxed from the cell by the RND transporters. We propose that impaired RND efflux results in periplasmic metabolite accumulation, which then activates periplasmic sensors including ToxR and two-component regulatory systems to initiate the expression of adaptive responses.

The Vibrio cholerae VexGH RND Efflux System Maintains Cellular Homeostasis by Effluxing Vibriobactin.

Resistance-nodulation-division (RND) superfamily efflux systems have been widely studied for their role in antibiotic resistance, but their native biological functions remain poorly understood. We previously showed that loss of RND-mediated efflux in Vibrio cholerae resulted in activation of the Cpx two-component regulatory system, which mediates adaptation to stress resulting from misfolded membrane proteins. Here, we investigated the mechanism linking RND-mediated efflux to the Cpx response. We performed transposon mutagenesis screening of RND-deficient V. cholerae to identify Cpx suppressors. Suppressor mutations mapped to genes involved in the biosynthesis of the catechol siderophore vibriobactin. We subsequently demonstrated that vibriobactin secretion is impaired in mutants lacking the VexGH RND efflux system and that impaired vibriobactin secretion is responsible for Cpx system activation, suggesting that VexGH secretes vibriobactin. This conclusion was bolstered by results showing that vexGH expression is induced by iron limitation and that vexH-deficient cells exhibit reduced fitness during growth under iron-limiting conditions. Our results support a model where VexGH contributes to cellular homeostasis by effluxing vibriobactin. In the absence of vexGH, retained vibriobactin appears to chelate iron from iron-rich components of the respiratory chain, with the deferrated proteins functioning to activate the Cpx response. Our collective results demonstrate that a native function of the V. cholerae VexGH RND efflux system is in vibriobactin secretion and that vibriobactin efflux is critical for maintenance of cellular homeostasis.IMPORTANCE RND efflux systems are ubiquitous Gram-negative transporters that play critical roles in antimicrobial resistance. In addition to antimicrobial resistance, RND transporters also affect the expression of diverse phenotypes, including virulence, cell metabolism, and stress responses. The latter observations suggest that RND transporters fulfill unknown physiological functions in the cell independently of their role in antimicrobial resistance. Vibrio cholerae is representative of many Gram-negative bacteria in encoding multiple RND transporters that are redundant in antimicrobial resistance and affect multiple phenotypes. Here we describe a novel function of the V. cholerae VexGH RND transporter in vibriobactin secretion. We show that vibriobactin production in VexGH-deficient cells impacts cell homeostasis, leading to activation of the Cpx stress response and reduced fitness under iron-limiting conditions. Our results highlight a native physiological function of an RND transporter and provide insight into the selective forces that maintain what was thought to be a redundant multidrug transporter.

Vibrio cholerae LeuO Links the ToxR Regulon to Expression of Lipid A Remodeling Genes.

Vibrio cholerae is an intestinal pathogen that causes the diarrheal disease cholera. Colonization of the intestine depends upon the expression of genes that allow V. cholerae to overcome host barriers, including low pH, bile acids, and the innate immune system. ToxR is a major contributor to this process. ToxR is a membrane-spanning transcription factor that coordinates gene expression in response to environmental cues. In previous work we showed that ToxR upregulated leuO expression in response to bile salts. LeuO is a LysR family transcription factor that contributes to acid tolerance, bile resistance, and biofilm formation in V. cholerae Here, we investigated the function of ToxR and LeuO in cationic antimicrobial peptide (CAMP) resistance. We report that ToxR and LeuO contribute to CAMP resistance by regulating carRS transcription. CarRS is a two-component regulatory system that positively regulates almEFG expression. AlmEFG confers CAMP resistance by glycinylation of lipid A. We found that the expression of carRS and almEFG and the polymyxin B MIC increased in mutants lacking toxRS or leuO Conversely, leuO overexpression decreased the polymyxin B MIC. Furthermore, we found that LeuO directly bound to the carRS promoter and that ToxR-dependent activation of leuO transcription regulated carRS transcription in response to bile salts. Our results suggest that LeuO functions downstream of ToxR to modulate carRS expression in response to environmental cues. This study extends the functional role of ToxR and LeuO in environmental adaptation to include cell surface remodeling and CAMP resistance.

The LysR-type regulator LeuO regulates the acid tolerance response in Vibrio cholerae.

Vibrio cholerae is a neutrophilic enteric pathogen that is extremely sensitive to acid. As V. cholerae passages through the host gastrointestinal tract it is exposed to a variety of environmental stresses including low pH and volatile fatty acids. Exposure to acidic environments induces expression of the V. cholerae acid tolerance response. A key component of the acid tolerance response is the cad system, which is encoded by cadC and the cadBA operon. CadB is a lysine/cadaverine antiporter and CadA is a lysine decarboxylase and these function together to counter low intracellular and extracellular pH. CadC is a membrane-associated transcription factor that activates cadBA expression in response to acidic conditions. Herein we investigated the role of the LysR-type transcriptional regulator LeuO in the V. cholerae acid tolerance response. Transcriptional reporter assays revealed that leuO expression repressed cadC transcription, indicating that LeuO was a cadC repressor. Consistent with this, leuO expression was inversely linked to lysine decarboxylase production and leuO overexpression resulted in increased sensitivity to organic acids. Overexpression of leuO in a cadA mutant potentiated killing by organic acids, suggesting that the function of leuO in the acid tolerance response extended beyond its regulation of the cad system. Collectively, these studies have identified a new physiological role for LeuO in V. cholerae acid tolerance.

Vibrio cholerae leuO Transcription Is Positively Regulated by ToxR and Contributes to Bile Resistance.

UNLABELLED: Vibrio cholerae is an aquatic organism and facultative human pathogen that colonizes the small intestine. In the small intestine, V. cholerae is exposed to a variety of antimicrobial compounds, including bile. V. cholerae resistance to bile is multifactorial and includes alterations in the membrane permeability barrier that are mediated by ToxR, a membrane-associated transcription factor. ToxR has also been shown to be required for activation of the LysR family transcription factor leuO in response to cyclic dipeptides. LeuO has been implicated in the regulation of multiple V. cholerae phenotypes, including biofilm production and virulence. In this study, we investigated the effects of bile on leuO expression. We show that leuO transcription increased in response to bile and bile salts but not in response to other detergents. The bile-dependent increase in leuO expression was dependent on ToxR, which was found to bind directly to the leuO promoter. The periplasmic domain of ToxR was required for basal leuO expression and for the bile-dependent induction of both leuO and ompU transcription. V. cholerae mutants that did not express leuO exhibited increased bile susceptibility, suggesting that LeuO contributes to bile resistance. Our collective results demonstrate that ToxR activates leuO expression in response to bile and that LeuO is a component of the ToxR-dependent responses that contribute to bile resistance. IMPORTANCE: The success of Vibrio cholerae as a human pathogen is dependent upon its ability to rapidly adapt to changes in its growth environment. Growth in the human gastrointestinal tract requires the expression of genes that provide resistance to host antimicrobial compounds, including bile. In this work, we show for the first time that the LysR family regulator LeuO mediates responses in V. cholerae that contribute to bile resistance.

Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels.

Construction of a tetracycline inducible expression vector and characterization of its use in Vibrio cholerae.

We report the construction of a tetracycline inducible expression vector that allows regulated gene expression in the enteric pathogen Vibrio cholerae. The expression vector, named pXB300, contains the tetracycline regulatory elements from Tn10, a multiple cloning site downstream of the tetA promoter and operator sequences, a ColE1 origin of replication, a ß-lactamase resistance gene for positive selection, and the hok/sok addiction system for selection in the absence of antibiotic. The function of the tetracycline expression system was demonstrated by cloning lacZ under control of the tetA promoter and quantifying ß-galactosidase expression in Escherichia coli and V. cholerae. The utility for pXB300 was documented by complementation of V. cholerae virulence mutants during growth under virulence inducing conditions. The results showed that pXB300 allowed high-level expression of recombinant genes with linear induction in response to the exogenous concentration of the inducer anhydrotetracycline. We further show that pXB300 was reliably maintained in V. cholerae during growth in the absence of antibiotic selection.

Reciprocal regulation of resistance-nodulation-division efflux systems and the Cpx two-component system in Vibrio cholerae.

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.

Cyclo(valine-valine) inhibits Vibrio cholerae virulence gene expression.

Vibrio cholerae has been shown to produce a cyclic dipeptide, cyclo(phenylalanine-proline) (cFP), that functions to repress virulence factor production. The objective of this study was to determine if heterologous cyclic dipeptides could repress V. cholerae virulence factor production. To that end, three synthetic cyclic dipeptides that differed in their side chains from cFP were assayed for virulence inhibitory activity in V. cholerae. The results revealed that cyclo(valine-valine) (cVV) inhibited virulence factor production by a ToxR-dependent process that resulted in the repression of the virulence regulator aphA. cVV-dependent repression of aphA was found to be independent of known aphA regulatory genes. The results demonstrated that V. cholerae was able to respond to exogenous cyclic dipeptides and implicated the hydrophobic amino acid side chains on both arms of the cyclo dipeptide scaffold as structural requirements for inhibitory activity. The results further suggest that cyclic dipeptides have potential as therapeutics for cholera treatment.

Vibrio cholerae ToxR downregulates virulence factor production in response to cyclo(Phe-Pro).

UNLABELLED: Vibrio cholerae is an aquatic organism that causes the severe acute diarrheal disease cholera. The ability of V. cholerae to cause disease is dependent upon the production of two critical virulence determinants, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The expression of the genes that encode for CT and TCP production is under the control of a hierarchical regulatory system called the ToxR regulon, which functions to activate virulence gene expression in response to in vivo stimuli. Cyclic dipeptides have been found to be produced by numerous bacteria, yet their biological function remains unknown. V. cholerae has been shown to produce cyclo(Phe-Pro). Previous studies in our laboratory demonstrated that cyclo(Phe-Pro) inhibited V. cholerae virulence factor production. For this study, we report on the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We have demonstrated that exogenous cyclo(Phe-Pro) activated the expression of leuO, a LysR-family regulator that had not been previously associated with V. cholerae virulence. Increased leuO expression repressed aphA transcription, which resulted in downregulation of the ToxR regulon and attenuated CT and TCP production. The cyclo(Phe-Pro)-dependent induction of leuO expression was found to be dependent upon the virulence regulator ToxR. Cyclo(Phe-Pro) did not affect toxR transcription or ToxR protein levels but appeared to enhance the ToxR-dependent transcription of leuO. These results have identified leuO as a new component of the ToxR regulon and demonstrate for the first time that ToxR is capable of downregulating virulence gene expression in response to an environmental cue. IMPORTANCE: The ToxR regulon has been a focus of cholera research for more than three decades. During this time, a model has emerged wherein ToxR functions to activate the expression of Vibrio cholerae virulence factors upon host entry. V. cholerae and other enteric bacteria produce cyclo(Phe-Pro), a cyclic dipeptide that we identified as an inhibitor of V. cholerae virulence factor production. This finding suggested that cyclo(Phe-Pro) was a negative effector of virulence factor production and represented a molecule that could potentially be exploited for therapeutic development. In this work, we investigated the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We found that cyclo(Phe-Pro) signaled through ToxR to activate the expression of leuO, a new virulence regulator that functioned to repress virulence factor production. Our results have identified a new arm of the ToxR regulon and suggest that ToxR may play a broader role in pathogenesis than previously known.