scholarly journals Mechanism for Inhibition of Vibrio cholerae ToxT Activity by the Unsaturated Fatty Acid Components of Bile

2015 ◽  
Vol 197 (10) ◽  
pp. 1716-1725 ◽  
Author(s):  
Sarah C. Plecha ◽  
Jeffrey H. Withey
Keyword(s):  
Gene Expression ◽  
Fatty Acid ◽  
Linoleic Acid ◽  
Dna Binding ◽  
Vibrio Cholerae ◽  
Unsaturated Fatty Acid ◽  
Binding Sites ◽  
Virulence Gene ◽  
Content Type ◽  
Virulence Gene Expression

ABSTRACTThe Gram-negative curved bacillusVibrio choleraecauses the severe diarrheal illness cholera. During host infection, a complex regulatory cascade results in production of ToxT, a DNA-binding protein that activates the transcription of major virulence genes that encode cholera toxin (CT) and toxin-coregulated pilus (TCP). Previous studies have shown that bile and its unsaturated fatty acid (UFA) components reduce virulence gene expression and therefore are likely important signals upon entering the host. However, the mechanism for the bile-mediated reduction of TCP and CT expression has not been clearly defined. There are two likely hypotheses to explain this reduction: (i) UFAs decrease DNA binding by ToxT, or (ii) UFAs decrease dimerization of ToxT. The work presented here elucidates that bile or UFAs directly affect DNA binding by ToxT. UFAs, specifically linoleic acid, can enterV. choleraewhen added exogenously and are present in the cytoplasm, where they can then interact with ToxT. Electrophoretic mobility shift assays (EMSAs) with ToxT and various virulence promoters in the presence or absence of UFAs showed a direct reduction in ToxT binding to DNA, even in promoters with only one ToxT binding site. Virstatin, a synthetic ToxT inhibitor, was previously shown to reduce ToxT dimerization. Here we show that virstatin affects DNA binding only at ToxT promoters with two binding sites, unlike linoleic acid, which affects ToxT binding promoters having either one or two ToxT binding sites. This suggests a mechanism in which UFAs, unlike virstatin, do not affect dimerization but affect monomeric ToxT binding to DNA.IMPORTANCEVibrio choleraemust produce the major virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) to cause cholera. CT and TCP production depends on ToxT, the major virulence transcription activator. ToxT activity is negatively regulated by unsaturated fatty acids (UFAs) present in the lumen of the upper small intestine. This study investigated the mechanism for inhibition of ToxT activity by UFAs and found that UFAs directly reduce specific ToxT binding to DNA at virulence promoters and subsequently reduce virulence gene expression. UFAs inhibit ToxT monomers from binding DNA. This differs from the inhibitory mechanism of a synthetic ToxT inhibitor, virstatin, which inhibits ToxT dimerization. Understanding the mechanisms for inhibition of virulence could lead to better cholera therapeutics.

scholarly journals A Genome-Wide Screen Reveals that the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Modulates Virulence Gene Expression

2015 ◽  
Vol 83 (9) ◽  
pp. 3381-3395 ◽  
Author(s):  
Qiyao Wang ◽  
Yves A. Millet ◽  
Michael C. Chao ◽  
Jumpei Sasabe ◽  
Brigid M. Davis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Insertion Site ◽  
Virulence Gene ◽  
Loss Of Function ◽  
Phosphotransferase System ◽  
Content Type ◽  
Virulence Gene Expression ◽  
A Genome

Diverse environmental stimuli and a complex network of regulatory factors are known to modulate expression ofVibrio cholerae's principal virulence factors. However, there is relatively little known about how metabolic factors impinge upon the pathogen's well-characterized cascade of transcription factors that induce expression of cholera toxin and the toxin-coregulated pilus (TCP). Here, we used a transposon insertion site (TIS) sequencing-based strategy to identify new factors required for expression oftcpA, which encodes the major subunit of TCP, the organism's chief intestinal colonization factor. Besides identifying most of the genes known to modulatetcpAexpression, the screen yieldedptsIandptsH, which encode the enzyme I (EI) and Hpr components of theV. choleraephosphoenolpyruvate phosphotransferase system (PTS). In addition to reduced expression of TcpA, strains lacking EI, Hpr, or the associated EIIAGlcprotein produced less cholera toxin (CT) and had a diminished capacity to colonize the infant mouse intestine. The PTS modulates virulence gene expression by regulating expression oftcpPHandaphAB, which themselves control expression oftoxT, the central activator of virulence gene expression. One mechanism by which PTS promotes virulence gene expression appears to be by modulating the amounts of intracellular cyclic AMP (cAMP). Our findings reveal that theV. choleraePTS is an additional modulator of the ToxT regulon and demonstrate the potency of loss-of-function TIS sequencing screens for defining regulatory networks.

scholarly journals Interactions of the Metalloregulatory Protein SloR from Streptococcus mutans with Its Metal Ion Effectors and DNA Binding Site

2015 ◽  
Vol 197 (22) ◽  
pp. 3601-3615 ◽  
Author(s):  
Grace Spatafora ◽  
John Corbett ◽  
Louis Cornacchione ◽  
William Daly ◽  
Diego Galan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Real Time ◽  
Streptococcus Mutans ◽  
Metal Ion ◽  
Virulence Gene ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Metal Ion Homeostasis ◽  
Binding Interface

ABSTRACTStreptococcus mutansis the causative agent of dental caries, a significant concern for human health, and therefore an attractive target for therapeutics development. Previous work in our laboratory has identified a homodimeric, manganese-dependent repressor protein, SloR, as an important regulator of cariogenesis and has used site-directed mutagenesis to map functions to specific regions of the protein. Here we extend those studies to better understand the structural interaction between SloR and its operator and its effector metal ions. The results of DNase I assays indicate that SloR protects a 42-bp region of DNA that overlaps thesloABCpromoter on theS. mutansUA159 chromosome, while electrophoretic mobility shift and solution binding assays indicate that each of two SloR dimers binds to this region. Real-time semiquantitative reverse transcriptase PCR (real-time semi-qRT-PCR) experiments were used to determine the individual base pairs that contribute to SloR-DNA binding specificity. Solution studies indicate that Mn2+is better than Zn2+at specifically activating SloR to bind DNA, and yet the 2.8-Å resolved crystal structure of SloR bound to Zn2+provides insight into the means by which selective activation by Mn2+may be achieved and into how SloR may form specific interactions with its operator. Taken together, these experimental observations are significant because they can inform rational drug design aimed at alleviating and/or preventingS. mutans-induced caries formation.IMPORTANCEThis report focuses on investigating the SloR protein as a regulator of essential metal ion transport and virulence gene expression in the oral pathogenStreptococcus mutansand on revealing the details of SloR binding to its metal ion effectors and binding to DNA that together facilitate this expression. We used molecular and biochemical approaches to characterize the interaction of SloR with Mn2+and with its SloR recognition element to gain a clearer picture of the regulatory networks that optimize SloR-mediated metal ion homeostasis and virulence gene expression inS. mutans. These experiments can have a significant impact on caries treatment and/or prevention by revealing theS. mutansSloR-DNA binding interface as an appropriate target for the development of novel therapeutic interventions.

scholarly journals 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

2017 ◽  
Vol 199 (7) ◽  
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.

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 Crystal Structure of the Vibrio cholerae Quorum-Sensing Regulatory Protein HapR

2007 ◽  
Vol 189 (15) ◽  
pp. 5683-5691 ◽  
Author(s):  
Rukman S. De Silva ◽  
Gabriela Kovacikova ◽  
Wei Lin ◽  
Ronald K. Taylor ◽  
Karen Skorupski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Crystal Structure ◽  
Dna Binding ◽  
Quorum Sensing ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Regulatory Protein ◽  
Virulence Gene ◽  
Binding Motif ◽  
Virulence Gene Expression

ABSTRACT Quorum sensing in Vibrio cholerae involves signaling between two-component sensor protein kinases and the response regulator LuxO to control the expression of the master regulator HapR. HapR, in turn, plays a central role in regulating a number of important processes, such as virulence gene expression and biofilm formation. We have determined the crystal structure of HapR to 2.2-Å resolution. Its structure reveals a dimeric, two-domain molecule with an all-helical structure that is strongly conserved with members of the TetR family of transcriptional regulators. The N-terminal DNA-binding domain contains a helix-turn-helix DNA-binding motif and alteration of certain residues in this domain completely abolishes the ability of HapR to bind to DNA, alleviating repression of both virulence gene expression and biofilm formation. The C-terminal dimerization domain contains a unique solvent accessible tunnel connected to an amphipathic cavity, which by analogy with other TetR regulators, may serve as a binding pocket for an as-yet-unidentified ligand.

scholarly journals Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae

2015 ◽  
Vol 198 (3) ◽  
pp. 498-509 ◽  
Author(s):  
Sarah J. Morgan ◽  
Emily L. French ◽  
Joshua J. Thomson ◽  
Craig P. Seaborn ◽  
Christian A. Shively ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Vibrio Cholerae ◽  
Disulfide Bond ◽  
Virulence Gene ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Intramolecular Disulfide Bond ◽  
Periplasmic Domain ◽  
The Stability

ABSTRACTTcpP and ToxR coordinately regulate transcription oftoxT, the master regulator of numerous virulence factors inVibrio cholerae. TcpP and ToxR are membrane-localized transcription factors, each with a periplasmic domain containing two cysteines. In ToxR, these cysteines form an intramolecular disulfide bond and a cysteine-to-serine substitution affects activity. We determined that the two periplasmic cysteines of TcpP also form an intramolecular disulfide bond. Disruption of this intramolecular disulfide bond by mutation of either cysteine resulted in formation of intermolecular disulfide bonds. Furthermore, disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP. While the decreased stability of TcpP-C207S resulted in a nearly complete loss oftoxTactivation and cholera toxin (CT) production, the second cysteine mutant, TcpP-C218S, was partially resistant to proteolytic degradation and maintained ∼50%toxTactivation capacity. TcpP-C218S was also TcpH independent, since deletion oftcpHdid not affect the stability of TcpP-C218S, whereas wild-type TcpP was degraded in the absence of TcpH. Finally, TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, suggesting that the single periplasmic cysteine in TcpH may assist with disulfide bond formation in TcpP by interacting with the periplasmic cysteines of TcpP. Consistent with this finding, a TcpH-C114S mutant was unable to stabilize TcpP and was itself unstable. Our findings demonstrate a periplasmic disulfide bond in TcpP is critical for TcpP stability and virulence gene expression.IMPORTANCETheVibrio choleraetranscription factor TcpP, in conjunction with ToxR, regulates transcription oftoxT, the master regulator of numerous virulence factors inVibrio cholerae. TcpP is a membrane-localized transcription factor with a periplasmic domain containing two cysteines. We determined that the two periplasmic cysteines of TcpP form an intramolecular disulfide bond and disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP and reduced virulence gene expression. Normally TcpH, another membrane-localized periplasmic protein, protects TcpP from degradation. However, we found that TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, indicating that the periplasmic cysteines of TcpP are required for functional interaction with TcpH and that this interaction is required for both TcpP and TcpH stability.

scholarly journals Sequence Variations within PrfA DNA Binding Sites and Effects on Listeria monocytogenes Virulence Gene Expression

2000 ◽  
Vol 182 (3) ◽  
pp. 837-841 ◽  
Author(s):  
James R. Williams ◽  
Chetna Thayyullathil ◽  
Nancy E. Freitag
Keyword(s):  
Gene Expression ◽  
Listeria Monocytogenes ◽  
Dna Binding ◽  
Binding Sites ◽  
Virulence Genes ◽  
Regulation Of Gene Expression ◽  
Virulence Gene ◽  
Virulence Gene Expression ◽  
Dna Binding Sites ◽  
Sequence Variations

ABSTRACT Reporter gene fusions were used to investigate the contributions of PrfA DNA binding sites to Listeria monocytogenes virulence gene expression. Our results suggest that the DNA sequence of PrfA binding sites determines the levels of expression of certain virulence genes, such as hly and mpl. Other virulence genes, such as actA and plcB, may depend upon additional factors for full regulation of gene expression.

scholarly journals Nutritional Regulation of the Sae Two-Component System by CodY inStaphylococcus aureus

2018 ◽  
Vol 200 (8) ◽  
Author(s):  
Kevin D. Mlynek ◽  
William E. Sause ◽  
Derek E. Moormeier ◽  
Marat R. Sadykov ◽  
Kurt R. Hill ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Factors ◽  
Virulence Gene ◽  
Nutrient Depletion ◽  
Global Regulator ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Two Component

ABSTRACTStaphylococcus aureussubverts innate defenses during infection in part by killing host immune cells to exacerbate disease. This human pathogen intercepts host cues and activates a transcriptional response via theS. aureusexoprotein expression (SaeR/SaeS [SaeR/S]) two-component system to secrete virulence factors critical for pathogenesis. We recently showed that the transcriptional repressor CodY adjusts nuclease (nuc) gene expression via SaeR/S, but the mechanism remained unknown. Here, we identified two CodY binding motifs upstream of thesaeP1 promoter, which suggested direct regulation by this global regulator. We show that CodY shares a binding site with the positive activator SaeR and that alleviating direct CodY repression at this site is sufficient to abrogate stochastic expression, suggesting that CodY repressessaeexpression by blocking SaeR binding. Epistasis experiments support a model that CodY also controlssaeindirectly through Agr and Rot-mediated repression of thesaeP1 promoter. We also demonstrate that CodY repression ofsaerestrains production of secreted cytotoxins that kill human neutrophils. We conclude that CodY plays a previously unrecognized role in controlling virulence gene expression via SaeR/S and suggest a mechanism by which CodY acts as a master regulator of pathogenesis by tying nutrient availability to virulence gene expression.IMPORTANCEBacterial mechanisms that mediate the switch from a commensal to pathogenic lifestyle are among the biggest unanswered questions in infectious disease research. Since the expression of most virulence genes is often correlated with nutrient depletion, this implies that virulence is a response to the lack of nourishment in host tissues and that pathogens likeS. aureusproduce virulence factors in order to gain access to nutrients in the host. Here, we show that specific nutrient depletion signals appear to be funneled to the SaeR/S system through the global regulator CodY. Our findings reveal a strategy by whichS. aureusdelays the production of immune evasion and immune-cell-killing proteins until key nutrients are depleted.

scholarly journals Pathovar Transcriptomes

mSystems ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Amy Platenkamp ◽  
Jay L. Mellies
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Regulatory Networks ◽  
Virulence Gene ◽  
Model System ◽  
Genomic Sequences ◽  
Bacterial Strains ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Link Type

ABSTRACT Archetypal pathogenic bacterial strains are often used to elucidate regulatory networks of an entire pathovar, which encompasses multiple lineages and phylogroups. With enteropathogenic Escherichia coli (EPEC) as a model system, Hazen and colleagues (mSystems 6:e00024-17, 2017, https://doi.org/10.1128/mSystems.00024-17 ) used 9 isolates representing 8 lineages and 3 phylogroups to find that isolates with similar genomic sequences exhibit similarities in global transcriptomes under conditions of growth in medium that induces virulence gene expression, and they found variation among individual isolates. Archetypal pathogenic bacterial strains are often used to elucidate regulatory networks of an entire pathovar, which encompasses multiple lineages and phylogroups. With enteropathogenic Escherichia coli (EPEC) as a model system, Hazen and colleagues (mSystems 6:e00024-17, 2017, https://doi.org/10.1128/mSystems.00024-17 ) used 9 isolates representing 8 lineages and 3 phylogroups to find that isolates with similar genomic sequences exhibit similarities in global transcriptomes under conditions of growth in medium that induces virulence gene expression. They also found variation among individual isolates. Their work illustrates the importance of moving beyond observing regulatory phenomena of a limited number of regulons in a few archetypal strains, with the possibility of correlating clinical symptoms to key transcriptional pathways across lineages and phylogroups.

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