scholarly journals Vibrio cholerae H-NS Silences Virulence Gene Expression at Multiple Steps in the ToxR Regulatory Cascade

2000 ◽  
Vol 182 (15) ◽  
pp. 4295-4303 ◽  
Author(s):  
Melinda B. Nye ◽  
James D. Pfau ◽  
Karen Skorupski ◽  
Ronald K. Taylor
Keyword(s):  
Gene Expression ◽  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Transcriptional Start Site ◽  
Regulatory Protein ◽  
Virulence Gene ◽  
Virulence Gene Expression ◽  
Activator Proteins ◽  
Regulatory Cascade ◽  
Genes Encoding

ABSTRACT H-NS is an abundant nucleoid-associated protein involved in the maintenance of chromosomal architecture in bacteria. H-NS also has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. In this study we demonstrate a role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon ofVibrio cholerae. Deletion of hns resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin-coregulated pilus, and the ToxT virulence gene regulatory protein. For the cholera toxin- and ToxT-encoding genes, elevated expression in an hns mutant was found to occur in the absence of the cognate activator proteins, suggesting that H-NS functions directly at these promoters to decrease gene expression. Deletion analysis of the region upstream of toxT suggests that an extensive region located far upstream of the transcriptional start site is required for complete H-NS-mediated repression of gene expression. These data indicate that H-NS negatively influences multiple levels of gene expression within the V. choleraevirulence cascade and raise the possibility that the transcriptional activator proteins in the ToxR regulon function to counteract the repressive effects of H-NS at the various promoters as well as to recruit RNA polymerase.

scholarly journals Bicarbonate Induces Vibrio cholerae Virulence Gene Expression by Enhancing ToxT Activity

2009 ◽  
Vol 77 (9) ◽  
pp. 4111-4120 ◽  
Author(s):  
Basel H. Abuaita ◽  
Jeffrey H. Withey
Keyword(s):  
Gene Expression ◽  
Carbonic Anhydrase ◽  
Vibrio Cholerae ◽  
Regulatory Protein ◽  
Virulence Gene ◽  
Chemical Stimulus ◽  
High Concentration ◽  
Virulence Gene Expression ◽  
Regulatory Cascade ◽  
El Tor

ABSTRACT Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera, a severe diarrheal illness. The two biotypes of V. cholerae O1 capable of causing cholera, classical and El Tor, require different in vitro growth conditions for induction of virulence gene expression. Growth under the inducing conditions or infection of a host initiates a complex regulatory cascade that results in production of ToxT, a regulatory protein that directly activates transcription of the genes encoding cholera toxin (CT), toxin-coregulated pilus (TCP), and other virulence genes. Previous studies have shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype. However, the mechanism for bicarbonate-mediated CT induction has not been defined. In this study, we demonstrate that bicarbonate stimulates virulence gene expression by enhancing ToxT activity. Both the classical and El Tor biotypes produce inactive ToxT protein when they are cultured statically in the absence of bicarbonate. Addition of bicarbonate to the culture medium does not affect ToxT production but causes a significant increase in CT and TCP expression in both biotypes. Ethoxyzolamide, a potent carbonic anhydrase inhibitor, inhibits bicarbonate-mediated virulence induction, suggesting that conversion of CO2 into bicarbonate by carbonic anhydrase plays a role in virulence induction. Thus, bicarbonate is the first positive effector for ToxT activity to be identified. Given that bicarbonate is present at high concentration in the upper small intestine where V. cholerae colonizes, bicarbonate is likely an important chemical stimulus that V. cholerae senses and that induces virulence during the natural course of infection.

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 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 Analysis of an Autoregulatory Loop Controlling ToxT, Cholera Toxin, and Toxin-Coregulated Pilus Production inVibrio cholerae

1999 ◽  
Vol 181 (8) ◽  
pp. 2584-2592 ◽  
Author(s):  
Rosa R. Yu ◽  
Victor J. DiRita
Keyword(s):  
Cholera Toxin ◽  
Virulence Gene ◽  
Primer Extension ◽  
The Other ◽  
Wild Type ◽  
Virulence Gene Expression ◽  
Regulatory Cascade ◽  
Genes Encoding ◽  
Toxin Coregulated Pilus ◽  
Extension Analysis

ABSTRACT Coordinate expression of many virulence genes in the human pathogenVibrio cholerae is controlled by the ToxR, TcpP, and ToxT proteins. These proteins function in a regulatory cascade in which ToxR and TcpP, two inner membrane proteins, are required to activatetoxT and ToxT is the direct activator of virulence gene expression. ToxT-activated genes include those whose products are required for the biogenesis of cholera toxin (CTX) and the toxin-coregulated pilus, the major subunit of which is TcpA. This work examined control of toxT transcription. We tested a model whereby activation of toxT by ToxR and TcpP is required to prime an autoregulatory loop in which ToxT-dependent transcription of the tcpA promoter reads through a proposed terminator between the tcpF and toxT genes to result in continued ToxT production. Primer extension analysis of RNA from wild-type classical strain O395 showed that there are two products encoding toxT, one of which is longer than the other by 105 bp. Deletion of the toxT promoter (toxTΔpro ) resulted in the abolishment oftoxT transcription, as predicted. Deletion of thetcpA promoter (tcpAΔpro ) had no effect on subsequent detection of the smaller toxT primer extension product, but the larger toxT product was not detected, indicating that this product may be the result of transcription from the tcpA promoter and not of initiation directly upstream of toxT. Neither mutant strain produced detectable TcpA, but the CTX levels of the strains were different. ThetoxTΔpro strain produced little detectable CTX, while the tcpAΔpro strain produced CTX levels intermediate between those of the wild-type andtoxTΔpro strains. Dependence oftoxT transcription on TcpP and TcpH was confirmed by analyzing RNAs from strains carrying deletions in the genes encoding these regulators. The tcpP defect resulted in undetectabletoxT transcription, whereas the tcpH mutation led to a diminishing of toxT RNA but not complete abolishment. Taken together, these results suggest thattoxT transcription is dependent on two different promoters; one is directly upstream and is activated in part by TcpP and TcpH, and the other is much further upstream and is activated by ToxT.

scholarly journals Integration Host Factor Positively Regulates Virulence Gene Expression in Vibrio cholerae

2008 ◽  
Vol 190 (13) ◽  
pp. 4736-4748 ◽  
Author(s):  
Emily Stonehouse ◽  
Gabriela Kovacikova ◽  
Ronald K. Taylor ◽  
Karen Skorupski
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Virulence Gene ◽  
Toxin Production ◽  
Dnase I ◽  
Integration Host Factor ◽  
Host Factor ◽  
Mobility Shift ◽  
Virulence Gene Expression ◽  
Dnase I Footprinting

ABSTRACT Virulence gene expression in Vibrio cholerae is dependent upon a complex transcriptional cascade that is influenced by both specific and global regulators in response to environmental stimuli. Here, we report that the global regulator integration host factor (IHF) positively affects virulence gene expression in V. cholerae. Inactivation of ihfA and ihfB, the genes encoding the IHF subunits, decreased the expression levels of the two main virulence factors tcpA and ctx and prevented toxin-coregulated pilus and cholera toxin production. IHF was found to directly bind to and bend the tcpA promoter region at an IHF consensus site centered at position −162 by using gel mobility shift assays and DNase I footprinting experiments. Deletion or mutation of the tcpA IHF consensus site resulted in the loss of IHF binding and additionally disrupted the binding of the repressor H-NS. DNase I footprinting revealed that H-NS protection overlaps with both the IHF and the ToxT binding sites at the tcpA promoter. In addition, disruption of ihfA in an hns or toxT mutant background had no effect on tcpA expression. These results suggest that IHF may function at the tcpA promoter to alleviate H-NS repression.

scholarly journals Interplay between the QseC and QseE Bacterial Adrenergic Sensor Kinases in Salmonella enterica Serovar Typhimurium Pathogenesis

2012 ◽  
Vol 80 (12) ◽  
pp. 4344-4353 ◽  
Author(s):  
Cristiano G. Moreira ◽  
Vanessa Sperandio
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Salmonella Enterica ◽  
Double Mutant ◽  
Virulence Gene ◽  
Virulence Gene Expression ◽  
Regulatory Cascade ◽  
Serovar Typhimurium ◽  
Sensor Kinases

ABSTRACTThe bacterial adrenergic sensor kinases QseC and QseE respond to epinephrine and/or norepinephrine to initiate a complex phosphorelay regulatory cascade that modulates virulence gene expression in several pathogens. We have previously shown that QseC activates virulence gene expression inSalmonella entericaserovar Typhimurium. Here we report the role of QseE inS. Typhimurium pathogenesis as well as the interplay between these two histidine sensor kinases in gene regulation. AnS. TyphimuriumqseEmutant is hampered in the invasion of epithelial cells and intramacrophage replication. The ΔqseCstrain is highly attenuated for intramacrophage survival but has only a minor defect in invasion. However, the ΔqseECstrain has only a slight attenuation in invasion, mirroring the ΔqseCstrain, and has an intermediary intramacrophage replication defect in comparison to the ΔqseEand ΔqseCstrains. The expressions of thesipAandsopBgenes, involved in the invasion of epithelial cells, are activated by epinephrine via QseE. The expression levels of these genes are still decreased in the ΔqseECdouble mutant, albeit to a lesser extent, congruent with the invasion phenotype of this mutant. The expression level of thesifAgene, important for intramacrophage replication, is decreased in theqseEmutant and the ΔqseECdouble mutant grownin vitro. However, as previously reported by us, the epinephrine-dependent activation of this gene occurs via QseC. In the systemic model ofS. Typhimurium infection of BALB/c mice, theqseCandqseEmutants are highly attenuated, while the double mutant has an intermediary phenotype. Altogether, these data suggest that both adrenergic sensors play an important role in modulating several aspects ofS. Typhimurium pathogenesis.

scholarly journals TolC Affects Virulence Gene Expression in Vibrio cholerae

2011 ◽  
Vol 193 (20) ◽  
pp. 5850-5852 ◽  
Author(s):  
Y. Minato ◽  
R. L. Siefken ◽  
C. C. Hase
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Virulence Gene ◽  
Virulence Gene Expression
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