Vibrio cholerae virulence regulator-coordinated evasion of host immunity

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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Structure and Function of the Vibrio cholerae Master Virulence Regulator ToxT

The FASEB Journal ◽

10.1096/fasebj.29.1_supplement.lb204 ◽

2015 ◽

Vol 29 (S1) ◽

Author(s):

Justin Cruite ◽

Gabriela Kovacikova ◽

Karen Skorupski ◽

Ronald Taylor ◽

F. Jon Kull

Keyword(s):

Vibrio Cholerae ◽

Structure And Function ◽

And Function ◽

Virulence Regulator

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Calcium Enhances Bile Salt-Dependent Virulence Activation in Vibrio cholerae

Infection and Immunity ◽

10.1128/iai.00707-16 ◽

2016 ◽

Vol 85 (1) ◽

Cited By ~ 7

Author(s):

Amanda J. Hay ◽

Menghua Yang ◽

Xiaoyun Xia ◽

Zhi Liu ◽

Justin Hammons ◽

...

Keyword(s):

Bile Salt ◽

Vibrio Cholerae ◽

Bile Salts ◽

Diarrheal Disease ◽

Content Type ◽

Inner Membrane Protein ◽

Virulence Regulator ◽

Host Signals

ABSTRACT Vibrio cholerae is the causative bacteria of the diarrheal disease cholera, but it also persists in aquatic environments, where it displays an expression profile that is distinct from that during infection. Upon entry into the host, a tightly regulated circuit coordinates the induction of two major virulence factors: cholera toxin and a toxin-coregulated pilus (TCP). It has been shown that a set of bile salts, including taurocholate, serve as host signals to activate V. cholerae virulence through inducing the activity of the transmembrane virulence regulator TcpP. In this study, we investigated the role of calcium, an abundant mental ion in the gut, in the regulation of virulence. We show that whereas Ca2+ alone does not affect virulence, Ca2+ enhances bile salt-dependent virulence activation for V. cholerae. The induction of TCP by murine intestinal contents is counteracted when Ca2+ is depleted by the high-affinity calcium chelator EGTA, suggesting that the calcium present in the gut is a relevant signal for V. cholerae virulence induction in vivo. We further show that Ca2+ enhances virulence by promoting bile salt-induced TcpP-TcpP interaction. Moreover, fluorescence recovery after photobleaching (FRAP) analysis demonstrated that exposure to bile salts and Ca2+ together decreases the recovery rate for fluorescently labeled TcpP, but not for another inner membrane protein (TatA). Together, these data support a model in which physiological levels of Ca2+ may result in altered bile salt-induced TcpP protein movement and activity, ultimately leading to an increased expression of virulence.

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The Fatty Acid Regulator FadR Influences the Expression of the Virulence Cascade in the El Tor Biotype of Vibrio cholerae by Modulating the Levels of ToxT via Two Different Mechanisms

Journal of Bacteriology ◽

10.1128/jb.00762-16 ◽

2017 ◽

Vol 199 (7) ◽

Cited By ~ 8

Author(s):

Gabriela Kovacikova ◽

Wei Lin ◽

Ronald K. Taylor ◽

Karen Skorupski

Keyword(s):

Gene Expression ◽

Vibrio Cholerae ◽

Virulence Gene ◽

Enteric Bacteria ◽

Posttranslational Regulation ◽

Content Type ◽

Virulence Gene Expression ◽

Expression Of Genes ◽

El Tor ◽

Virulence Regulator

ABSTRACT FadR is a master regulator of fatty acid (FA) metabolism that coordinates the pathways of FA degradation and biosynthesis in enteric bacteria. We show here that a ΔfadR mutation in the El Tor biotype of Vibrio cholerae prevents the expression of the virulence cascade by influencing both the transcription and the posttranslational regulation of the master virulence regulator ToxT. FadR is a transcriptional regulator that represses the expression of genes involved in FA degradation, activates the expression of genes involved in unsaturated FA (UFA) biosynthesis, and also activates the expression of two operons involved in saturated FA (SFA) biosynthesis. Since FadR does not bind directly to the toxT promoter, we determined whether the regulation of any of its target genes indirectly influenced ToxT. This was accomplished by individually inserting a double point mutation into the FadR-binding site in the promoter of each target gene, thereby preventing their activation or repression. Although preventing FadR-mediated activation of fabA, which encodes the enzyme that carries out the first step in UFA biosynthesis, did not significantly influence either the transcription or the translation of ToxT, it reduced its levels and prevented virulence gene expression. In the mutant strain unable to carry out FadR-mediated activation of fabA, expressing fabA ectopically restored the levels of ToxT and virulence gene expression. Taken together, the results presented here indicate that V. cholerae FadR influences the virulence cascade in the El Tor biotype by modulating the levels of ToxT via two different mechanisms. IMPORTANCE Fatty acids (FAs) play important roles in membrane lipid homeostasis and energy metabolism in all organisms. In Vibrio cholerae, the causative agent of the acute intestinal disease cholera, they also influence virulence by binding into an N-terminal pocket of the master virulence regulator, ToxT, and modulating its activity. FadR is a transcription factor that coordinately controls the pathways of FA degradation and biosynthesis in enteric bacteria. This study identifies a new link between FA metabolism and virulence in the El Tor biotype by showing that FadR influences both the transcription and posttranslational regulation of the master virulence regulator ToxT by two distinct mechanisms.

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A new class of inhibitors of the AraC family virulence regulator Vibrio cholerae ToxT

Scientific Reports ◽

10.1038/srep45011 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 9

Author(s):

Anne K. Woodbrey ◽

Evans O. Onyango ◽

Maria Pellegrini ◽

Gabriela Kovacikova ◽

Ronald K. Taylor ◽

...

Keyword(s):

Vibrio Cholerae ◽

New Class ◽

Virulence Regulator ◽

Arac Family

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Faculty Opinions recommendation of Vibrio cholerae anaerobic induction of virulence gene expression is controlled by thiol-based switches of virulence regulator AphB.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717967920.793467554 ◽

2012 ◽

Author(s):

Victor DiRita ◽

Rebecca Anthouard

Keyword(s):

Gene Expression ◽

Vibrio Cholerae ◽

Virulence Gene ◽

Virulence Gene Expression ◽

Anaerobic Induction ◽

Virulence Regulator

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

mBio ◽

10.1128/mbio.00366-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 33

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.

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Identification of Residues Critical for the Function of the Vibrio cholerae Virulence Regulator ToxT by Scanning Alanine Mutagenesis

Journal of Molecular Biology ◽

10.1016/j.jmb.2007.01.061 ◽

2007 ◽

Vol 367 (5) ◽

pp. 1413-1430 ◽

Cited By ~ 31

Author(s):

Brandon M. Childers ◽

Gregor G. Weber ◽

Michael G. Prouty ◽

Melissa M. Castaneda ◽

Fen Peng ◽

...

Keyword(s):

Vibrio Cholerae ◽

Virulence Regulator

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The Cyclic Dipeptide Cyclo(Phe-Pro) Inhibits Cholera Toxin and Toxin-Coregulated Pilus Production in O1 El Tor Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.00191-10 ◽

2010 ◽

Vol 192 (14) ◽

pp. 3829-3832 ◽

Cited By ~ 21

Author(s):

Xiaowen R. Bina ◽

James E. Bina

Keyword(s):

Cholera Toxin ◽

Vibrio Cholerae ◽

Virulence Factors ◽

Virulence Gene ◽

Cyclic Dipeptide ◽

Present Evidence ◽

Toxin Coregulated Pilus ◽

El Tor ◽

Virulence Regulator

ABSTRACT 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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The Virulence Transcriptional Activator AphA Enhances Biofilm Formation by Vibrio cholerae by Activating Expression of the Biofilm Regulator VpsT

Infection and Immunity ◽

10.1128/iai.00429-09 ◽

2009 ◽

Vol 78 (2) ◽

pp. 697-703 ◽

Cited By ~ 47

Author(s):

Menghua Yang ◽

Erin M. Frey ◽

Zhi Liu ◽

Rima Bishar ◽

Jun Zhu

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Virulence Genes ◽

Diarrheal Disease ◽

Genomic Library ◽

Gene Products ◽

Regulatory Pathway ◽

Transcriptional Fusion ◽

E Coli ◽

Virulence Regulator

ABSTRACT Vibrio cholerae is the agent of the severe diarrheal disease cholera, and it perpetuates in aquatic reservoirs when not in the host. Within the host's intestines, the bacteria execute a complex regulatory pathway culminating with the production of virulence factors that allow colonization and cause disease. The ability of V. cholerae to form biofilms is thought to aid its persistence in the aquatic environment and passage through the gastric acid barrier of the stomach. The transcriptional activators VpsR and VpsT are part of the biofilm formation-regulatory network. In this study, we screened a V. cholerae genomic library in Escherichia coli cells containing a P vpsT -luxCDBAE transcriptional fusion reporter and found that a plasmid clone containing the aphA gene activates the expression of vpsT in E. coli. AphA is a master virulence regulator in V. cholerae that is required to activate the expression of tcpP, whose gene products in turn activate all virulence genes including those responsible for the synthesis of the toxin-coregulated pilus (TCP) and cholera toxin through the activation of toxT. AphA has a direct effect on the vpsT promoter, as gel shift experiments demonstrated that AphA binds to the vpsT promoter region. Furthermore, V. cholerae aphA mutants exhibit significantly lower levels of vpsT expression as well as reduced biofilm formation. AphA thus links the expression of virulence and biofilm synthesis genes.

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