Publications by authors named "James E Bina"

35 Publications

Bile salts promote ToxR regulon activation during growth under virulence inducing conditions.

Infect Immun 2021 Sep 20:IAI0044121. Epub 2021 Sep 20.

University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, PA.

Cholera is an epidemic disease caused by the Gram-negative bacterium . is found in aquatic ecosystems and infects people through the consumption of contaminated food or water. Following ingestion, responds to host cues to activate the expression of critical virulence genes that are under control of a hierarchical regulatory system called the ToxR regulon. The ToxR regulon is tightly regulated and is only expressed under special growth conditions referred to as AKI conditions. AKI conditions have been instrumental in elucidating 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. 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 non-inducing media 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 ToxR regulon was preferentially activated in response to primary bile acids. The results of this study shed light on the chemical cues involved in virulence activation and suggested that virulence genes are modulated in response to regional-specific bile acid species in the intestine.
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http://dx.doi.org/10.1128/IAI.00441-21DOI Listing
September 2021

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

Infect Immun 2021 Sep 26;89(10):e0024221. Epub 2021 Jul 26.

University of Pittsburgh School of Medicinegrid.471408.e, Department of Microbiology and Molecular Genetics, Pittsburgh, Pennsylvania, USA.

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 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 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 . Decreased transcription correlated with increased expression of the LysR-family transcription factor . Deletion of restored expression, and CT and TCP production, in a mutant. The collective results document that is required for ToxR regulon expression and further suggest that participates in an efflux-dependent feedback circuit to regulate virulence gene expression.
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http://dx.doi.org/10.1128/IAI.00242-21DOI Listing
September 2021

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

Infect Immun 2021 06 16;89(7):e0014721. Epub 2021 Jun 16.

University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, Pennsylvania, USA.

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-β-naphthylamide (PAβN) on V. cholerae. Using bioassays, we documented that PAβN inhibited virulence factor production in three epidemic V. cholerae isolates. Transcriptional reporter studies and mutant analysis indicated that PAβN initiated a ToxR-dependent regulatory circuit to activate expression and that LeuO repressed the expression of the critical virulence activator to attenuate CT and TCP production. The antivirulence activity of PAβN 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 PAβN 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.
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http://dx.doi.org/10.1128/IAI.00147-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8208513PMC
June 2021

Complete Genome Sequence of Klebsiella pneumoniae Strain ATCC 43816.

Microbiol Resour Announc 2021 Feb 4;10(5). Epub 2021 Feb 4.

University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, Pennsylvania, USA

is a member of that causes a multitude of infections in compromised and healthy individuals. The rise of hypervirulent and multiple-drug-resistant strains has made this organism a global health threat. Here, we report the complete genome sequence of strain ATCC 43816.
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http://dx.doi.org/10.1128/MRA.01441-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862963PMC
February 2021

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

Microbiol Resour Announc 2021 Jan 21;10(3). Epub 2021 Jan 21.

University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, Pennsylvania, USA

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 O1 El Tor strain C6706.
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http://dx.doi.org/10.1128/MRA.01301-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8407767PMC
January 2021

Genome Sequence of Vibrio cholerae Strain RFB16, Isolated from North Park Lake in Allegheny County, Pennsylvania.

Microbiol Resour Announc 2020 Mar 5;9(10). Epub 2020 Mar 5.

University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, Pennsylvania, USA

is an aquatic organism and facultative human pathogen that typically resides in coastal areas and brackish water. Here, we report the complete genome sequence of strain RFB16, which was isolated from a freshwater lake in southwestern Pennsylvania.
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http://dx.doi.org/10.1128/MRA.00111-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171212PMC
March 2020

Indole Inhibits ToxR Regulon Expression in .

Infect Immun 2019 03 21;87(3). Epub 2019 Feb 21.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

Indole is a degradation product of tryptophan that functions as a signaling molecule in many bacteria. This includes , where indole was shown to regulate biofilm and type VI secretion in nontoxigenic environmental isolates. Indole is also produced by toxigenic strains in the human intestine, but its significance in the host is unknown. We investigated the effects of indole on toxigenic 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 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 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 .
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http://dx.doi.org/10.1128/IAI.00776-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386550PMC
March 2019

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

PLoS Pathog 2018 01 5;14(1):e1006804. Epub 2018 Jan 5.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1006804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773229PMC
January 2018

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

mBio 2017 05 16;8(3). Epub 2017 May 16.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

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 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 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 expression is induced by iron limitation and that -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 , 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 VexGH RND efflux system is in vibriobactin secretion and that vibriobactin efflux is critical for maintenance of cellular homeostasis. 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. 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 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.
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http://dx.doi.org/10.1128/mBio.00126-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5433094PMC
May 2017

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

Infect Immun 2016 Nov 17;84(11):3161-3171. Epub 2016 Oct 17.

University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, Pennsylvania, USA

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.
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http://dx.doi.org/10.1128/IAI.00445-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067749PMC
November 2016

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

Microbiology (Reading) 2015 Dec 29;161(12):2434-43. Epub 2015 Sep 29.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

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.
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http://dx.doi.org/10.1099/mic.0.000194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811655PMC
December 2015

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

J Bacteriol 2015 Nov 24;197(22):3499-510. Epub 2015 Aug 24.

University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, Pennsylvania, USA

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.
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http://dx.doi.org/10.1128/JB.00419-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621094PMC
November 2015

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

PLoS One 2015 19;10(2):e0117890. Epub 2015 Feb 19.

University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, Pennsylvania, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117890PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335029PMC
December 2015

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

Plasmid 2014 11 24;76:87-94. Epub 2014 Oct 24.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA. Electronic address:

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 β-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 β-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.
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http://dx.doi.org/10.1016/j.plasmid.2014.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467732PMC
November 2014

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

Infect Immun 2014 Jul 5;82(7):2980-91. Epub 2014 May 5.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

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.
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http://dx.doi.org/10.1128/IAI.00025-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097637PMC
July 2014

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

Microbiology (Reading) 2014 Jun 18;160(Pt 6):1054-1062. Epub 2014 Mar 18.

University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, PA 15219, USA.

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.
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http://dx.doi.org/10.1099/mic.0.077297-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4039241PMC
June 2014

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

mBio 2013 Aug 27;4(5):e00366-13. Epub 2013 Aug 27.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

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.
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http://dx.doi.org/10.1128/mBio.00366-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760244PMC
August 2013

Riboswitches for intracellular study of genes involved in Francisella pathogenesis.

mBio 2012 Nov 20;3(6). Epub 2012 Nov 20.

Department of Chemistry, Emory University, Atlanta, Georgia, USA.

Unlabelled: The study of many important intracellular bacterial pathogens requires an understanding of how specific virulence factors contribute to pathogenesis during the infection of host cells. This requires tools to dissect gene function, but unfortunately, there is a lack of such tools for research on many difficult-to-study, or understudied, intracellular pathogens. Riboswitches are RNA-based genetic control elements that directly modulate gene expression upon ligand binding. Here we report the application of theophylline-sensitive synthetic riboswitches to induce protein expression in the intracellular pathogen Francisella. We show that this system can be used to activate the bacterial expression of the reporter β-galactosidase during growth in rich medium. Furthermore, we applied this system to control the expression of green fluorescent protein during intracellular infection by the addition of theophylline directly to infected macrophages. Importantly, we could control the expression of a novel endogenous protein required for growth under nutrient-limiting conditions and replication in macrophages, FTN_0818. Riboswitch-mediated control of FTN_0818 rescued the growth of an FTN_0818 mutant in minimal medium and during macrophage infection. This is the first demonstration of the use of a synthetic riboswitch to control an endogenous gene required for a virulence trait in an intracellular bacterium. Since this system can be adapted to diverse bacteria, the ability to use riboswitches to regulate intracellular bacterial gene expression will likely facilitate the in-depth study of the virulence mechanisms of numerous difficult-to-study intracellular pathogens such as Ehrlichia chaffeensis, Anaplasma phagocytophilum, and Orientia tsutsugamushi, as well as future emerging pathogens.

Importance: Determining how specific bacterial genes contribute to virulence during the infection of host cells is critical to understanding how pathogens cause disease. This can be especially challenging with many difficult-to-study intracellular pathogens. Riboswitches are RNA-based genetic control elements that can be used to help dissect gene function, especially since they can be used in a broad range of bacteria. We demonstrate the utility of riboswitches, and for the first time show that riboswitches can be used to functionally control a bacterial gene that is critical to the ability of a pathogen to cause disease, during intracellular infection. Since this system can be adapted to diverse bacteria, riboswitches will likely facilitate the in-depth study of the virulence mechanisms of numerous difficult-to-study intracellular pathogens, as well as future emerging pathogens.
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http://dx.doi.org/10.1128/mBio.00253-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509410PMC
November 2012

Link between intraphagosomal biotin and rapid phagosomal escape in Francisella.

Proc Natl Acad Sci U S A 2012 Oct 15;109(44):18084-9. Epub 2012 Oct 15.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30329, USA.

Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15-60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. Although a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria in the phagosome are largely uncharacterized. We previously identified a Francisella protein, FTN_0818, as being essential for intracellular replication and involved in virulence in vivo. We now show that FTN_0818 is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP). Addition of biotin complemented the phagosomal escape defect of the FTN_0818 mutant, demonstrating that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome. Biotin also rescued the attenuation of the FTN_0818 mutant during infection in vitro and in vivo, highlighting the importance of this process. The key role of biotin in phagosomal escape implies biotin may be a limiting factor during infection. We demonstrate that a bacterial metabolite is required for phagosomal escape of an intracellular pathogen, providing insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens.
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http://dx.doi.org/10.1073/pnas.1206411109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497780PMC
October 2012

NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis.

Mol Microbiol 2012 Nov 11;86(3):611-27. Epub 2012 Sep 11.

Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University, Atlanta, GA, USA; Emory Vaccine Center, Emory University, Atlanta, GA, USA.

Modification of specific Gram-negative bacterial cell envelope components, such as capsule, O-antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram-negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria.
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http://dx.doi.org/10.1111/mmi.12004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841722PMC
November 2012

Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus.

PLoS One 2012 30;7(5):e38208. Epub 2012 May 30.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038208PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364225PMC
October 2012

Visualization of murine intranasal dosing efficiency using luminescent Francisella tularensis: effect of instillation volume and form of anesthesia.

PLoS One 2012 24;7(2):e31359. Epub 2012 Feb 24.

The University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.

Intranasal instillation is a widely used procedure for pneumonic delivery of drugs, vaccine candidates, or infectious agents into the respiratory tract of research mice. However, there is a paucity of published literature describing the efficiency of this delivery technique. In this report we have used the murine model of tularemia, with Francisella tularensis live vaccine strain (FTLVS) infection, to evaluate the efficiency of pneumonic delivery via intranasal dosing performed either with differing instillation volumes or different types of anesthesia. FTLVS was rendered luminescent via transformation with a reporter plasmid that constitutively expressed the Photorhabdus luminescens lux operon from a Francisella promoter. We then used an IVIS Spectrum whole animal imaging system to visualize FT dissemination at various time points following intranasal instillation. We found that instillation of FT in a dose volume of 10 µl routinely resulted in infection of the upper airways but failed to initiate infection of the pulmonary compartment. Efficient delivery of FT into the lungs via intranasal instillation required a dose volume of 50 µl or more. These studies also demonstrated that intranasal instillation was significantly more efficient for pneumonic delivery of FTLVS in mice that had been anesthetized with inhaled (isoflurane) vs. parenteral (ketamine/xylazine) anesthesia. The collective results underscore the need for researchers to consider both the dose volume and the anesthesia type when either performing pneumonic delivery via intranasal instillation, or when comparing studies that employed this technique.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031359PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286442PMC
August 2012

Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

PLoS One 2011 6;6(9):e24201. Epub 2011 Sep 6.

Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University, Atlanta, Georgia, United States of America.

Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024201PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167825PMC
February 2012

A galU mutant of Francisella tularensis is attenuated for virulence in a murine pulmonary model of tularemia.

BMC Microbiol 2011 Aug 5;11:179. Epub 2011 Aug 5.

Department of Microbiology, Immunology, and Biochemistry, The University of Tennessee Health Science Center, Memphis, 38163, USA.

Background: A number of studies have revealed that Francisella tularensis (FT) suppresses innate immune responses such as chemokine/cytokine production and neutrophil recruitment in the lungs following pulmonary infection via an unidentified mechanism. The ability of FT to evade early innate immune responses could be a very important virulence mechanism for this highly infectious bacterial pathogen.

Results: Here we describe the characterization of a galU mutant strain of FT live vaccine strain (LVS). We show that the galU mutant was highly attenuated in a murine model of tularemia and elicited more robust innate immune responses than the wild-type (WT) strain. These studies document that the kinetics of chemokine expression and neutrophil recruitment into the lungs of mice challenged with the galU mutant strain are significantly more rapid than observed with WT FT, despite the fact that there were no observed differences in TLR2 or TLR4 signaling or replication/dissemination kinetics during the early stages of infection. We also show that the galU mutant had a hypercytotoxic phenotype and more rapidly induced the production of IL-1β following infection either in vitro or in vivo, indicating that attenuation of the galU mutant strain may be due (in part) to more rapid activation of the inflammasome and/or earlier death of FT infected cells. Furthermore, we show that infection of mice with the galU mutant strain elicits protective immunity to subsequent challenge with WT FT.

Conclusions: Disruption of the galU gene of FTLVS has little (if any) effect on in vivo infectivity, replication, or dissemination characteristics, but is highly attenuating for virulence. The attenuated phenotype of this mutant strain of FT appears to be related to its increased ability to induce innate inflammatory responsiveness, resulting in more rapid recruitment of neutrophils to the lungs following pneumonic infection, and/or to its ability to kill infected cells in an accelerated fashion. These results have identified two potentially important virulence mechanisms used by FT. These findings could also have implications for design of a live attenuated vaccine strain of FT because sublethal infection of mice with the galU mutant strain of FTLVS promoted development of protective immunity to WT FTLVS.
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http://dx.doi.org/10.1186/1471-2180-11-179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173336PMC
August 2011

Construction of a bioluminescence reporter plasmid for Francisella tularensis.

Plasmid 2010 Nov 8;64(3):156-61. Epub 2010 Jul 8.

The University of Tennessee Health Science Center, Department of Molecular Sciences, Memphis, TN 38163, USA.

A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real-time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia research that is suitable for following F. tularensis growth in both in vitro and in vivo model systems.
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http://dx.doi.org/10.1016/j.plasmid.2010.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943566PMC
November 2010

The cyclic dipeptide cyclo(Phe-Pro) inhibits cholera toxin and toxin-coregulated pilus production in O1 El Tor Vibrio cholerae.

J Bacteriol 2010 Jul 7;192(14):3829-32. Epub 2010 May 7.

The University of Tennessee Health Sciences Center, Department of Molecular Sciences, 858 Madison Avenue, Memphis, TN 38163, USA.

Cyclo(Phe-Pro) is a cyclic dipeptide produced by multiple Vibrio species. In this work, we present evidence that cyclo(Phe-Pro) inhibits the production of the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) in O1 El Tor Vibrio cholerae strain N16961 during growth under virulence gene-inducing conditions. The cyclo(Phe-Pro) inhibition of CT and TCP production correlated with reduced transcription of the virulence regulator tcpPH and was alleviated by overexpression of tcpPH.
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http://dx.doi.org/10.1128/JB.00191-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2897329PMC
July 2010

Binding and activation of host plasminogen on the surface of Francisella tularensis.

BMC Microbiol 2010 Mar 12;10:76. Epub 2010 Mar 12.

Department of Molecular Sciences, The University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, Tennessee 38163, USA.

Background: Francisella tularensis (FT) is a gram-negative facultative intracellular coccobacillus and is the causal agent of a life-threatening zoonotic disease known as tularemia. Although FT preferentially infects phagocytic cells of the host, recent evidence suggests that a significant number of bacteria can be found extracellularly in the plasma fraction of the blood during active infection. This observation suggests that the interaction between FT and host plasma components may play an important role in survival and dissemination of the bacterium during the course of infection. Plasminogen (PLG) is a protein zymogen that is found in abundance in the blood of mammalian hosts. A number of both gram-positive and gram-negative bacterial pathogens have the ability to bind to PLG, giving them a survival advantage by increasing their ability to penetrate extracellular matrices and cross tissue barriers.

Results: We show that PLG binds to the surface of FT and that surface-bound PLG can be activated to plasmin in the presence of tissue PLG activator in vitro. In addition, using Far-Western blotting assays coupled with proteomic analyses of FT outer membrane preparations, we have identified several putative PLG-binding proteins of FT.

Conclusions: The ability of FT to acquire surface bound PLG that can be activated on its surface may be an important virulence mechanism that results in an increase in initial infectivity, survival, and/or dissemination of this bacterium in vivo.
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http://dx.doi.org/10.1186/1471-2180-10-76DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2848021PMC
March 2010

Effect of the efflux inhibitors 1-(1-naphthylmethyl)-piperazine and phenyl-arginine-beta-naphthylamide on antimicrobial susceptibility and virulence factor production in Vibrio cholerae.

J Antimicrob Chemother 2009 Jan 14;63(1):103-8. Epub 2008 Nov 14.

Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA.

Objectives: The aim of the study was to test the hypothesis that the efflux pump inhibitors (EPIs) 1-(1-naphthylmethyl)-piperazine (NMP) and phenyl-arginine-beta-naphthylamide (PAbetaN) can inhibit the Vibrio cholerae resistance-nodulation-division (RND) family efflux systems, and thereby render V. cholerae susceptible to antimicrobial agents and inhibit the production of the virulence factors cholera toxin (CT) and the toxin coregulated pilus (TCP).

Methods: The susceptibility of V. cholerae to antimicrobial compounds was determined in the presence or absence of NMP and PAbetaN. Transcriptional reporters were used to assess the effects of NMP and PAbetaN on the expression of the genes encoding the virulence factor regulators TcpP and ToxT, whereas CT and TCP production were determined by ELISA using GM1 ganglioside-coated microtitre plates and TcpA Western immunoblotting, respectively.

Results: NMP and PAbetaN potentiated antimicrobial compounds that were substrates for the V. cholerae RND efflux systems. PAbetaN exhibited complete inhibition of the RND efflux systems for Triton X-100 and deoxycholate, but partial inhibition of the efflux systems for cholate and erythromycin. NMP exhibited partial inhibition for all compounds tested except for SDS. The presence of NMP reduced the MIC of SDS to a level that was lower than that observed in an RND efflux-deficient strain, whereas the SDS MIC was unaffected by the presence of PAbetaN. Neither EPI potentiated polymyxin B, penicillin, ampicillin or chloramphenicol. Both NMP and PAbetaN inhibited the production of CT and the TCP and appeared to have additional virulence gene repressing activity independent of RND efflux inhibition.

Conclusions: RND efflux inhibitors represent potential novel therapeutics for the treatment of cholera.
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http://dx.doi.org/10.1093/jac/dkn466DOI Listing
January 2009

Vibrio cholerae RND family efflux systems are required for antimicrobial resistance, optimal virulence factor production, and colonization of the infant mouse small intestine.

Infect Immun 2008 Aug 19;76(8):3595-605. Epub 2008 May 19.

Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA.

Vibrio cholerae is a gram-negative human intestinal pathogen that causes the diarrheal disease cholera. Humans acquire cholera by ingesting V. cholerae-contaminated food or water. Upon ingestion, V. cholerae encounters several barriers to colonization, including bile acid toxicity and antimicrobial products of the innate immune system. In many gram-negative bacteria, resistance to the antimicrobial effects of these products is mediated by RND (resistance-nodulation-division) family efflux systems. In this study we tested the hypothesis that the V. cholerae RND efflux systems are required for antimicrobial resistance and virulence. The six V. cholerae genes encoding RND efflux pumps were deleted from the genome of the O1 El Tor strain N16961, resulting in the generation of 14 independent RND deletion mutants, including one RND-null strain. Determination of the antimicrobial susceptibilities of the mutants revealed that the RND efflux systems were responsible for resistance to multiple antimicrobial compounds, including bile acids, antimicrobial peptides, and antibiotics. VexB (VC0164) was found to be the RND efflux pump primarily responsible for the resistance of V. cholerae to multiple antimicrobial compounds in vitro. In contrast, VexD (VC1757) and VexK (VC1673) encoded efflux pumps with detergent-specific substrate specificities that were redundant with VexB. A strain lacking VexB, VexD, and VexK was attenuated in the infant mouse model, and its virulence factor production was unaffected. In contrast, a V. cholerae RND-null strain produced significantly less cholera toxin and fewer toxin-coregulated pili than the wild type and was unable to colonize the infant mouse. The decreased virulence factor production in the RND-null strain was linked to reduced transcription of tcpP and toxT. Our findings show that the V. cholerae RND efflux systems are required for antimicrobial resistance, optimal virulence factor production, and colonization of the infant mouse.
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http://dx.doi.org/10.1128/IAI.01620-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493215PMC
August 2008

The AcrAB RND efflux system from the live vaccine strain of Francisella tularensis is a multiple drug efflux system that is required for virulence in mice.

FEMS Microbiol Lett 2008 Feb 20;279(2):226-33. Epub 2007 Dec 20.

Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA.

The ability of bacterial pathogens to infect and cause disease is dependent upon their ability to resist antimicrobial components produced by their host, such as bile acids, fatty acids and other detergent-like molecules, and products of the innate immune system (e.g. cationic antimicrobial peptides). Bacterial resistance to the antimicrobial effects of such compounds is often mediated by active efflux systems belonging to the resistance-nodulation-division (RND) family of transporters. RND efflux systems have been implicated in antibiotic resistance and virulence extending their clinical relevance. In this report the hypothesis that the Francisella tularensis AcrAB RND efflux system contributes to antimicrobial resistance and pathogenesis has been tested. A null mutation was generated in the gene encoding the AcrB RND efflux pump protein of the live vaccine strain of F. tularensis. The resulting mutant exhibited increased sensitivity to multiple antibiotics and antimicrobial compounds. Murine challenge experiments revealed that the acrB mutant was attenuated. Collectively these results suggest that the F. tularensis AcrAB RND efflux system encodes a multiple drug efflux system that is important for virulence.
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http://dx.doi.org/10.1111/j.1574-6968.2007.01033.xDOI Listing
February 2008
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