scholarly journals ToxR mediates the antivirulence activity of phenyl-arginine-β-naphthylamide to attenuate Vibrio cholerae virulence.

2021 ◽  
Author(s):  
Yuding Weng ◽  
Thomas F. Bina ◽  
X. Renee Bina ◽  
James E. Bina
Keyword(s):  
Antimicrobial Resistance ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Efflux Pump ◽  
Feedback Mechanism ◽  
Gram Negative ◽  
Factor Production ◽  
Regulatory Circuit ◽  
Efflux Pump Inhibitors ◽  
Virulence Factor Production

Multidrug efflux systems belonging to the resistance-nodulation-cell division (RND) family are ubiquitous in Gram negative bacteria and 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 where 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 leuO expression and that LeuO repressed the expression of the critical virulence activator aphA to attenuate CT and TCP production. The antivirulence activity of PAβN was found to be dependent on the ToxR periplasmic sensing domain 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 other gram negative pathogens.

scholarly journals Vibrio cholerae RND Family Efflux Systems Are Required for Antimicrobial Resistance, Optimal Virulence Factor Production, and Colonization of the Infant Mouse Small Intestine

2008 ◽  
Vol 76 (8) ◽  
pp. 3595-3605 ◽  
Author(s):  
Xiaowen R. Bina ◽  
Daniele Provenzano ◽  
Nathalie Nguyen ◽  
James E. Bina
Keyword(s):  
Antimicrobial Resistance ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Efflux Pumps ◽  
Antimicrobial Compounds ◽  
Gram Negative ◽  
Infant Mouse ◽  
Factor Production ◽  
Virulence Factor Production ◽  
Null Strain

ABSTRACT 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.

scholarly journals Cyclo(valine–valine) inhibits Vibrio cholerae virulence gene expression

Microbiology ◽  
2014 ◽  
Vol 160 (6) ◽  
pp. 1054-1062 ◽  
Author(s):  
Amit Vikram ◽  
Vanessa M. Ante ◽  
X. Renee Bina ◽  
Qin Zhu ◽  
Xinyu Liu ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Inhibitory Activity ◽  
Virulence Gene ◽  
Side Chains ◽  
Cyclic Dipeptides ◽  
Virulence Gene Expression ◽  
Factor Production ◽  
Virulence Regulator ◽  
Virulence Factor Production

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.

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

2018 ◽  
Vol 14 (1) ◽  
pp. e1006804 ◽  
Author(s):  
X. Renee Bina ◽  
Mondraya F. Howard ◽  
Dawn L. Taylor-Mulneix ◽  
Vanessa M. Ante ◽  
Dillon E. Kunkle ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Adaptive Responses ◽  
Factor Production ◽  
Sensor Proteins ◽  
Virulence Factor Production

scholarly journals Regulation of Vibrio Polysaccharide Synthesis and Virulence Factor Production by CdgC, a GGDEF-EAL Domain Protein, in Vibrio cholerae

2007 ◽  
Vol 189 (3) ◽  
pp. 717-729 ◽  
Author(s):  
Bentley Lim ◽  
Sinem Beyhan ◽  
Fitnat H. Yildiz
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Stationary Phases ◽  
Cellular Processes ◽  
Factor Production ◽  
Expression Of Genes ◽  
Flagellar Biosynthesis ◽  
Polysaccharide Synthesis ◽  
Regulator Genes ◽  
Virulence Factor Production

ABSTRACT In Vibrio cholerae, the second messenger 3′,5′-cyclic diguanylic acid (c-di-GMP) regulates several cellular processes, such as formation of corrugated colony morphology, biofilm formation, motility, and virulence factor production. Both synthesis and degradation of c-di-GMP in the cell are modulated by proteins containing GGDEF and/or EAL domains, which function as a diguanylate cyclase and a phosphodiesterase, respectively. The expression of two genes, cdgC and mbaA, which encode proteins harboring both GGDEF and EAL domains is higher in the rugose phase variant of V. cholerae than in the smooth variant. In this study, we carried out gene expression analysis to determine the genes regulated by CdgC in the rugose and smooth phase variants of V. cholerae. We determined that CdgC regulates expression of genes required for V. cholerae polysaccharide synthesis and of the transcriptional regulator genes vpsR, vpsT, and hapR. CdgC also regulates expression of genes involved in extracellular protein secretion, flagellar biosynthesis, and virulence factor production. We then compared the genes regulated by CdgC and by MbaA, during both exponential and stationary phases of growth, to elucidate processes regulated by them. Identification of the regulons of CdgC and MbaA revealed that the regulons overlap, but the timing of regulation exerted by CdgC and MbaA is different, suggesting the interplay and complexity of the c-di-GMP signal transduction pathways operating in V. cholerae.

The major Vibrio cholerae autoinducer and its role in virulence factor production

Nature ◽  
2007 ◽  
Vol 450 (7171) ◽  
pp. 883-886 ◽  
Author(s):  
Douglas A. Higgins ◽  
Megan E. Pomianek ◽  
Christina M. Kraml ◽  
Ronald K. Taylor ◽  
Martin F. Semmelhack ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Factor Production ◽  
Virulence Factor Production

scholarly journals Effectiveness of Efflux Pump Inhibitors as Biofilm Disruptors and Resistance Breakers in Gram-negative (ESKAPEE) Bacteria

2019 ◽  
Author(s):  
Akif Reza ◽  
J. Mark Sutton ◽  
Khondaker Miraz Rahman
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Efflux Pump ◽  
Protein Structures ◽  
Underlying Mechanism ◽  
Resistance Mechanisms ◽  
Healthcare Provision ◽  
Gram Negative ◽  
Environmental Threats ◽  
Efflux Pump Inhibitors

Antibiotic resistance represents a significant threat to the modern healthcare provision. The ESKAPEE pathogens, in particular, have proven to be especially challenging to treat, due to their intrinsic and acquired ability to rapidly develop resistance mechanisms in response to environmental threats. The development of biofilm has been characterised as an essential contributing factor towards antimicrobial-resistance and tolerance. Several studies have implicated the involvement of efflux pumps in antibiotic resistance, both directly, via drug extrusion and indirectly, through the formation of biofilm. As a result, the underlying mechanism of these pumps has attracted considerable interest due to the potential of targeting these protein structures and developing novel adjunct therapies. Subsequent investigations have revealed the ability of efflux pump-inhibitors (EPIs) to block drug-extrusion and disrupt biofilm formation, thereby, potentiating antibiotics and reversing resistance of pathogen towards them. This review will discuss the potential of EPIs as a possible solution to antimicrobial resistance, examining different challenges to the design of these compounds, with an emphasis on Gram-negative ESKAPEE pathogens.

scholarly journals Vibrio cholerae ToxR Downregulates Virulence Factor Production in Response to Cyclo(Phe-Pro)

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
X. Renee Bina ◽  
Dawn L. Taylor ◽  
Amit Vikram ◽  
Vanessa M. Ante ◽  
James E. Bina
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Virulence Gene ◽  
Enteric Bacteria ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Factor Production ◽  
Virulence Regulator ◽  
Virulence Factor Production

ABSTRACTVibrio choleraeis an aquatic organism that causes the severe acute diarrheal disease cholera. The ability ofV. choleraeto 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 toin vivostimuli. Cyclic dipeptides have been found to be produced by numerous bacteria, yet their biological function remains unknown.V. choleraehas been shown to produce cyclo(Phe-Pro). Previous studies in our laboratory demonstrated that cyclo(Phe-Pro) inhibitedV. choleraevirulence 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 ofleuO, a LysR-family regulator that had not been previously associated withV. choleraevirulence. IncreasedleuOexpression repressedaphAtranscription, which resulted in downregulation of the ToxR regulon and attenuated CT and TCP production. The cyclo(Phe-Pro)-dependent induction ofleuOexpression was found to be dependent upon the virulence regulator ToxR. Cyclo(Phe-Pro) did not affecttoxRtranscription or ToxR protein levels but appeared to enhance the ToxR-dependent transcription ofleuO. These results have identifiedleuOas 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.IMPORTANCEThe 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 ofVibrio choleraevirulence factors upon host entry.V. choleraeand other enteric bacteria produce cyclo(Phe-Pro), a cyclic dipeptide that we identified as an inhibitor ofV. choleraevirulence 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 ofleuO, 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.

scholarly journals Vibrio cholerae OmpR Represses the ToxR Regulon in Response to Membrane Intercalating Agents That Are Prevalent in the Human Gastrointestinal Tract

2019 ◽  
Vol 88 (3) ◽  
Author(s):  
D. E. Kunkle ◽  
T. F. Bina ◽  
X. R. Bina ◽  
J. E. Bina
Keyword(s):  
Gastrointestinal Tract ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Diarrheal Disease ◽  
Multidrug Efflux ◽  
Content Type ◽  
Factor Production ◽  
Resistance Nodulation Division ◽  
Intercalating Agents ◽  
Virulence Factor Production

ABSTRACT Multidrug efflux systems belonging to the resistance-nodulation-division (RND) superfamily are ubiquitous in Gram-negative bacteria. RND efflux systems are often associated with multiple antimicrobial resistance and also contribute to the expression of diverse bacterial phenotypes including virulence, as documented in the intestinal pathogen Vibrio cholerae, the causative agent of the severe diarrheal disease cholera. Transcriptomic studies with RND efflux-negative V. cholerae suggested that RND-mediated efflux was required for homeostasis, as loss of RND efflux resulted in the activation of transcriptional regulators, including multiple environmental sensing systems. In this report, we investigated six RND efflux-responsive regulatory genes for contributions to V. cholerae virulence factor production. Our data showed that the V. cholerae gene VC2714, encoding a homolog of Escherichia coli OmpR, was a virulence repressor. The expression of ompR was elevated in an RND-null mutant, and ompR deletion partially restored virulence factor production in the RND-negative background. Virulence inhibitory activity in the RND-negative background resulted from OmpR repression of the key ToxR regulon virulence activator aphB, and ompR overexpression in wild-type cells also repressed virulence through aphB. We further show that ompR expression was not altered by changes in osmolarity but instead was induced by membrane-intercalating agents that are prevalent in the host gastrointestinal tract and which are substrates of the V. cholerae RND efflux systems. Our collective results indicate that V. cholerae ompR is an aphB repressor and regulates the expression of the ToxR virulence regulon in response to novel environmental cues.

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