scholarly journals Enhanced Interaction of Vibrio cholerae Virulence Regulators TcpP and ToxR under Oxygen-Limiting Conditions

2014 ◽  
Vol 82 (4) ◽  
pp. 1676-1682 ◽  
Author(s):  
Fenxia Fan ◽  
Zhi Liu ◽  
Nusrat Jabeen ◽  
L. Dillon Birdwell ◽  
Jun Zhu ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Virulence Gene ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Low Oxygen ◽  
Content Type ◽  
Multiple Components ◽  
Effect Of Oxygen ◽  
Protein Disulfide

ABSTRACTVibrio choleraeis the causative agent of the diarrheal disease cholera. The ability ofV. choleraeto colonize and cause disease requires the intricately regulated expression of a number of virulence factors during infection. One of the signals sensed byV. choleraeis the presence of oxygen-limiting conditions in the gut. It has been shown that the virulence activator AphB plays a key role in sensing low oxygen concentrations and inducing the transcription of another key virulence activator, TcpP. In this study, we used a bacterial two-hybrid system to further examine the effect of oxygen on different virulence regulators. We found that anoxic conditions enhanced the interaction between TcpP and ToxR, identified as the first positive regulator ofV. choleraevirulence genes. We further demonstrated that the TcpP-ToxR interaction was dependent on the primary periplasmic protein disulfide formation enzyme DsbA and cysteine residues in the periplasmic domains of both ToxR and TcpP. Furthermore, we showed that inV. cholerae, an interaction between TcpP and ToxR is important for virulence gene induction. Under anaerobic growth conditions, we detected ToxR-TcpP heterodimers, which were abolished in the presence of the reducing agent dithiothreitol. Our results suggest thatV. choleraemay sense intestinal anoxic signals by multiple components to activate virulence.

scholarly journals A Small Unstructured Region in Vibrio cholerae ToxT Mediates the Response to Positive and Negative Effectors and ToxT Proteolysis

2014 ◽  
Vol 197 (3) ◽  
pp. 654-668 ◽  
Author(s):  
Joshua J. Thomson ◽  
Sarah C. Plecha ◽  
Jeffrey H. Withey
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factors ◽  
Unsaturated Fatty Acids ◽  
Diarrheal Disease ◽  
Virulence Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
Terminal Domain ◽  
Virulence Gene Regulation ◽  
Host Signals

Vibrio choleraeis the causative agent of the severe diarrheal disease cholera. The production of the virulence factors that are required for human disease is controlled by a complex network of transcriptional and posttranscriptional regulators. ToxT is the transcription regulator that directly controls the production of the two major virulence factors, toxin-coregulated pilus (TCP) and cholera toxin (CT). The solved crystal structure of ToxT revealed an unstructured region in the N-terminal domain between residues 100 and 110. This region and the surrounding amino acids have been previously implicated in ToxT proteolysis, resistance to inhibition by negative effectors, and ToxT dimerization. To better characterize this region, site-directed mutagenesis was performed to assess the effects on ToxT proteolysis and bile sensitivity. This analysis identified specific mutations within this unstructured region that prevent ToxT proteolysis and other mutations that reduce inhibition by bile and unsaturated fatty acids. In addition, we found that mutations that affect the sensitivity of ToxT to bile also affect the sensitivity of ToxT to its positive effector, bicarbonate. These results suggest that a small unstructured region in the ToxT N-terminal domain is involved in multiple aspects of virulence gene regulation and response to human host signals.

scholarly journals Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae

2015 ◽  
Vol 84 (2) ◽  
pp. 511-523 ◽  
Author(s):  
Eric D. Peng ◽  
Elizabeth E. Wyckoff ◽  
Alexandra R. Mey ◽  
Carolyn R. Fisher ◽  
Shelley M. Payne
Keyword(s):  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Iron Acquisition ◽  
Growth Conditions ◽  
Growth Defect ◽  
Null Mutant ◽  
Content Type ◽  
Infant Mouse ◽  
Iron Transporter

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, thrives in both marine environments and the human host. To do so, it must encode the tools necessary to acquire essential nutrients, including iron, under these vastly different conditions. A number ofV. choleraeiron acquisition systems have been identified; however, the precise role of each system is not fully understood. To test the roles of individual systems, we generated a series of mutants in which only one of the four systems that support iron acquisition on unsupplemented LB agar, Feo, Fbp, Vct, and Vib, remains functional. Analysis of these mutants under different growth conditions showed that these systems are not redundant. The strain carrying only the ferrous iron transporter Feo grew well at acidic, but not alkaline, pH, whereas the ferric iron transporter Fbp promoted better growth at alkaline than at acidic pH. A strain defective in all four systems (null mutant) had a severe growth defect under aerobic conditions but accumulated iron and grew as well as the wild type in the absence of oxygen, suggesting the presence of an additional, unidentified iron transporter inV. cholerae. In support of this, the null mutant was only moderately attenuated in an infant mouse model of infection. While the null mutant used heme as an iron sourcein vitro, we demonstrate that heme is not available toV. choleraein the infant mouse intestine.

scholarly journals Host Intestinal Signal-Promoted Biofilm Dispersal Induces Vibrio cholerae Colonization

2014 ◽  
Vol 83 (1) ◽  
pp. 317-323 ◽  
Author(s):  
Amanda J. Hay ◽  
Jun Zhu
Keyword(s):  
Bile Salt ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Virulence Gene ◽  
Human Infection ◽  
Content Type ◽  
Abiotic Degradation ◽  
Contaminated Food ◽  
Free Media ◽  
Biofilm Matrix

Vibrio choleraecauses human infection through ingestion of contaminated food and water, leading to the devastating diarrheal disease cholera.V. choleraeforms matrix-encased aggregates, known as biofilms, in the native aquatic environment. While the formation ofV. choleraebiofilms has been well studied, little is known about the dispersal from biofilms, particularly upon entry into the host. In this study, we found that the exposure of mature biofilms to physiologic levels of the bile salt taurocholate, a host signal for the virulence gene induction ofV. cholerae, induces an increase in the number of detached cells with a concomitant decrease in biofilm mass. Scanning electron microscopy micrographs of biofilms exposed to taurocholate revealed an altered, perhaps degraded, appearance of the biofilm matrix. The inhibition of protein synthesis did not alter rates of detachment, suggesting thatV. choleraeundergoes a passive dispersal. Cell-free media from taurocholate-exposed biofilms contains a larger amount of free polysaccharide, suggesting an abiotic degradation of biofilm matrix by taurocholate. Furthermore, we found thatV. choleraeis only able to induce virulence in response to taurocholate after exit from the biofilm. Thus, we propose a model in whichV. choleraeingested as a biofilm has coopted the host-derived bile salt signal to detach from the biofilm and go on to activate virulence.

scholarly journals EnterotoxigenicE. colivirulence gene regulation in human infections

2018 ◽  
Vol 115 (38) ◽  
pp. E8968-E8976 ◽  
Author(s):  
Alexander A. Crofts ◽  
Simone M. Giovanetti ◽  
Erica J. Rubin ◽  
Frédéric M. Poly ◽  
Ramiro L. Gutiérrez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Factor ◽  
Virulence Gene ◽  
Human Infection ◽  
Anaerobic Growth ◽  
Infection Model ◽  
Low Oxygen ◽  
Virulence Gene Expression ◽  
Factor Expression

EnterotoxigenicEscherichia coli(ETEC) is a global diarrheal pathogen that utilizes adhesins and secreted enterotoxins to cause disease in mammalian hosts. Decades of research on virulence factor regulation in ETEC has revealed a variety of environmental factors that influence gene expression, including bile, pH, bicarbonate, osmolarity, and glucose. However, other hallmarks of the intestinal tract, such as low oxygen availability, have not been examined. Further, determining how ETEC integrates these signals in the complex host environment is challenging. To address this, we characterized ETEC’s response to the human host using samples from a controlled human infection model. We found ETEC senses environmental oxygen to globally influence virulence factor expression via the oxygen-sensitive transcriptional regulator fumarate and nitrate reduction (FNR) regulator. In vitro anaerobic growth replicates the in vivo virulence factor expression profile, and deletion offnrin ETEC strain H10407 results in a significant increase in expression of all classical virulence factors, including the colonization factor antigen I (CFA/I) adhesin operon and both heat-stable and heat-labile enterotoxins. These data depict a model of ETEC infection where FNR activity can globally influence virulence gene expression, and therefore proximity to the oxygenated zone bordering intestinal epithelial cells likely influences ETEC virulence gene expression in vivo. Outside of the host, ETEC biofilms are associated with seasonal ETEC epidemics, and we find FNR is a regulator of biofilm production. Together these data suggest FNR-dependent oxygen sensing in ETEC has implications for human infection inside and outside of the host.

scholarly journals The Cpx System Regulates Virulence Gene Expression in Vibrio cholerae

2015 ◽  
Vol 83 (6) ◽  
pp. 2396-2408 ◽  
Author(s):  
Nicole Acosta ◽  
Stefan Pukatzki ◽  
Tracy L. Raivio
Keyword(s):  
Vibrio Cholerae ◽  
Response Regulator ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Receptor Protein ◽  
Virulence Determinants ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Envelope Stress ◽  
El Tor

Bacteria possess signal transduction pathways capable of sensing and responding to a wide variety of signals. The Cpx envelope stress response, composed of the sensor histidine kinase CpxA and the response regulator CpxR, senses and mediates adaptation to insults to the bacterial envelope. The Cpx response has been implicated in the regulation of a number of envelope-localized virulence determinants across bacterial species. Here, we show that activation of the Cpx pathway inVibrio choleraeEl Tor strain C6706 leads to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT) and the toxin-coregulated pilus (TCP). Our results indicate that this occurs through the repression of production of the ToxT regulator and an additional upstream transcription factor, TcpP. The effect of the Cpx response on CT and TCP expression is mostly abrogated in a cyclic AMP receptor protein (CRP) mutant, although expression of thecrpgene is unaltered. Since TcpP production is controlled by CRP, our data suggest a model whereby the Cpx response affects CRP function, which leads to diminished TcpP, ToxT, CT, and TCP production.

scholarly journals Structural basis for virulence regulation in Vibrio cholerae by unsaturated fatty acid components of bile

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Justin T. Cruite ◽  
Gabriela Kovacikova ◽  
Kenzie A. Clark ◽  
Anne K. Woodbrey ◽  
Karen Skorupski ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Structural Model ◽  
Unsaturated Fatty Acids ◽  
Diarrheal Disease ◽  
Bacterial Pathogen ◽  
Virulence Gene ◽  
Structural Basis ◽  
Active Conformation ◽  
Structural Mechanism ◽  
Virulence Gene Regulation

AbstractThe AraC/XylS-family transcriptional regulator ToxT is the master virulence activator of Vibrio cholerae, the gram-negative bacterial pathogen that causes the diarrheal disease cholera. Unsaturated fatty acids (UFAs) found in bile inhibit the activity of ToxT. Crystal structures of inhibited ToxT bound to UFA or synthetic inhibitors have been reported, but no structure of ToxT in an active conformation had been determined. Here we present the 2.5 Å structure of ToxT without an inhibitor. The structure suggests release of UFA or inhibitor leads to an increase in flexibility, allowing ToxT to adopt an active conformation that is able to dimerize and bind DNA. Small-angle X-ray scattering was used to validate a structural model of an open ToxT dimer bound to the cholera toxin promoter. The results presented here provide a detailed structural mechanism for virulence gene regulation in V. cholerae by the UFA components of bile and other synthetic ToxT inhibitors.

scholarly journals Neutrophils Are Essential for Containment of Vibrio cholerae to the Intestine during the Proinflammatory Phase of Infection

2012 ◽  
Vol 80 (8) ◽  
pp. 2905-2913 ◽  
Author(s):  
Jessica Queen ◽  
Karla J. Fullner Satchell
Keyword(s):  
Immune Response ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Innate Immune ◽  
Infection Model ◽  
Proinflammatory Response ◽  
Necrosis Factor Alpha ◽  
Live Attenuated Vaccine ◽  
Content Type

ABSTRACTCholera is classically considered a noninflammatory diarrheal disease, in comparison to invasive enteric organisms, although there is a low-level proinflammatory response during early infection withVibrio choleraeand a strong proinflammatory reaction to live attenuated vaccine strains. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host defense to infection. Nontoxigenic El Tor O1V. choleraeinfection is characterized by the upregulation of interleukin-6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha in the intestine, indicating an acute innate immune response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to decreased survival of mice. The role of neutrophils in protection of the host is to limit the infection to the intestine and control bacterial spread to extraintestinal organs. In the absence of neutrophils, the infection spread to the spleen and led to increased systemic levels of IL-1β and tumor necrosis factor alpha, suggesting the decreased survival in neutropenic mice is due to systemic shock. Neutrophils were found not to contribute to either clearance of colonizing bacteria or to alter the local immune response. However, when genes for secreted accessory toxins were deleted, the colonizing bacteria were cleared from the intestine, and this clearance is dependent upon neutrophils. Thus, the requirement for accessory toxins in virulence is negated in neutropenic mice, which is consistent with a role of accessory toxins in the evasion of innate immune cells in the intestine. Overall, these data support that neutrophils impact disease progression and suggest that neutrophil effectiveness can be manipulated through the deletion of accessory toxins.

scholarly journals Integration of Cyclic di-GMP and Quorum Sensing in the Control ofvpsTandaphAin Vibrio cholerae

2011 ◽  
Vol 193 (22) ◽  
pp. 6331-6341 ◽  
Author(s):  
Disha Srivastava ◽  
Rebecca C. Harris ◽  
Christopher M. Waters
Keyword(s):  
Quorum Sensing ◽  
Binding Site ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Virulence Gene ◽  
Transcriptional Activator ◽  
Transcriptional Regulators ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Signaling Systems

Vibrio choleraetransitions between aquatic environmental reservoirs and infection in the gastrointestinal tracts of human hosts. The second-messenger molecule cyclic di-GMP (c-di-GMP) and quorum sensing (QS) are important signaling systems that enableV. choleraeto alternate between these distinct environments by controlling biofilm formation and virulence factor expression. Here we identify a conserved regulatory mechanism inV. choleraethat integrates c-di-GMP and QS to control the expression of two transcriptional regulators:aphA, an activator of virulence gene expression and an important regulator of the quorum-sensing pathway, andvpsT, a transcriptional activator that induces biofilm formation. Surprisingly,aphAexpression was induced by c-di-GMP. Activation of bothaphAandvpsTby c-di-GMP requires the transcriptional activator VpsR, which binds to c-di-GMP. The VpsR binding site at each of these promoters overlaps with the binding site of HapR, the master QS regulator at high cell densities. Our results suggest thatV. choleraecombines information conveyed by QS and c-di-GMP to appropriately respond and adapt to divergent environments by modulating the expression of key transcriptional regulators.

scholarly journals Type III Secretion Is Essential for the Rapidly Fatal Diarrheal Disease Caused by Non-O1, Non-O139 Vibrio cholerae

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Ok S. Shin ◽  
Vincent C. Tam ◽  
Masato Suzuki ◽  
Jennifer M. Ritchie ◽  
Roderick T. Bronson ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Intestinal Epithelium ◽  
Type Iii Secretion ◽  
Diarrheal Disease ◽  
Secretion Systems ◽  
Content Type ◽  
Type Iii ◽  
Severe Diarrhea ◽  
Toxin Coregulated Pilus

ABSTRACTCholera is a severe diarrheal disease typically caused by O1 serogroup strains ofVibrio cholerae. The pathogenicity of all pandemicV. choleraeO1 strains relies on two critical virulence factors: cholera toxin, a potent enterotoxin, and toxin coregulated pilus (TCP), an intestinal colonization factor. However, certain non-O1, non-O139V. choleraestrains, such as AM-19226, do not produce cholera toxin or TCP, yet they still cause severe diarrhea. The molecular basis for the pathogenicity of non-O1, non-O139V. choleraehas not been extensively characterized, but many of these strains encode related type III secretion systems (TTSSs). Here, we used infant rabbits to assess the contribution of the TTSS to non-O1, non-O139V. choleraepathogenicity. We found that all animals infected with wild-type AM-19226 developed severe diarrhea even more rapidly than rabbits infected withV. choleraeO1. UnlikeV. choleraeO1 strains, which do not damage the intestinal epithelium in rabbits or humans, AM-19226 caused marked disruptions of the epithelial surface in the rabbit small intestine. TTSS proved to be essential for AM-19226 virulence in infant rabbits; an AM-19226 derivative deficient for TTSS did not elicit diarrhea, colonize the intestine, or induce pathological changes in the intestine. Deletion of either one of the two previously identified or two newly identified AM-19226 TTSS effectors reduced but did not eliminate AM-19226 pathogenicity, suggesting that at least four effectors contribute to this strain’s virulence. In aggregate, our results suggest that the TTSS-dependent virulence in non-O1, non-O139V. choleraerepresents a new type of diarrheagenic mechanism.IMPORTANCECholera, which is caused byVibrio cholerae, is an important cause of diarrheal disease in many developing countries. The mechanisms of virulence of nonpandemic strains that can cause a diarrheal illness are poorly understood. AM-19226, like several other pathogenic, nonpandemicV. choleraestrains, carries genes that encode a type III secretion system (TTSS), but not cholera toxin (CT) or toxin coregulated pilus (TCP). In this study, we used infant rabbits to study AM-19226 virulence. Infant rabbits orally inoculated with this strain rapidly developed a fatal diarrheal disease, which was accompanied by marked disruptions of the intestinal epithelium. This strain’s TTSS proved essential for its pathogenicity, and there was no diarrhea, intestinal pathology, or colonization in rabbits infected with a TTSS mutant. The effector proteins translocated by the TTSS all appear to contribute to AM-19226 virulence. Thus, our study provides insight intoin vivomechanisms by which a novel TTSS contributes to diarrheal disease caused by nonpandemic strains ofV. cholerae.

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.

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