scholarly journals H-NS and ToxT inversely control cholera toxin production by binding to overlapping DNA sequences

2021 ◽  
Author(s):  
Jennifer B. Stone ◽  
Jeffrey H. Withey
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Binding Sites ◽  
Current Model ◽  
Toxin Production ◽  
Negative Regulator ◽  
Watery Diarrhea ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Heptad Repeats

Vibrio cholerae infects human hosts following ingestion of contaminated food or water, resulting in the severe diarrheal disease cholera. The watery diarrhea that is characteristic of the disease is directly caused by production of cholera toxin (CT). A complex regulatory cascade controls production of CT and other virulence factors. However, ultimately a single protein, ToxT, directly binds to virulence gene promoters and activates their transcription. Previously, we identified two ToxT binding sites, or toxboxes, within the cholera toxin promoter (P ctxAB ). The toxboxes overlap with the two promoter-proximal GATTTTT heptad repeats found within P ctxAB in classical biotype V. cholerae strain O395. These heptad repeats were previously found to be within a large DNA region bound by H-NS, a global transcriptional repressor present in Gram-negative bacteria. The current model for control of P ctxAB transcription proposed complete H-NS displacement from the DNA by ToxT, followed by direct activation by ToxT-RNAP contacts. The goal of this study was to determine more precisely where H-NS binds to P ctxAB and test the hypothesis that ToxT completely displaces H-NS from the P ctxAB promoter before activating transcription. Results suggest that H-NS binds only to the region of P ctxAB encompassing the heptad repeats and ToxT only displaces H-NS from its most promoter proximal binding sites, calling for a revision of the current model involving H-NS and ToxT at P ctxAB . Importance H-NS is a global negative regulator of transcription in Gram negative bacteria, particularly in horizontally acquired genetic islands. Previous work in Vibrio cholerae suggested that H-NS represses transcription of cholera toxin genes by binding to a large region upstream of its promoter, and that virulence activator ToxT derepresses transcription by removing H-NS from the promoter. Here, new data support a revised model in which ToxT only displaces H-NS bound to the most promoter proximal DNA sites that overlap the ToxT binding sites, leaving the upstream sites occupied by H-NS. This introduces a higher resolution mechanism for antirepression of H-NS in control of cholera toxin production.

Cholera Toxin Production Induced upon Anaerobic Respiration is Suppressed by Glucose Fermentation in Vibrio cholerae

2016 ◽  
Vol 26 (3) ◽  
pp. 627-636 ◽  
Author(s):  
Young Taek Oh ◽  
Kang-Mu Lee ◽  
Wasimul Bari ◽  
Hwa Young Kim ◽  
Hye Jin Kim ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Anaerobic Respiration ◽  
Toxin Production ◽  
Glucose Fermentation

scholarly journals Expression of the p20 Gene from Bacillus thuringiensis H-14 Increases Cry11A Toxin Production and Enhances Mosquito-Larvicidal Activity in Recombinant Gram-Negative Bacteria

2001 ◽  
Vol 67 (7) ◽  
pp. 3010-3015 ◽  
Author(s):  
Y. Xu ◽  
M. Nagai ◽  
M. Bagdasarian ◽  
T. W. Smith ◽  
E. D. Walker
Keyword(s):  
Bacillus Thuringiensis ◽  
Toxin Production ◽  
Gram Negative Bacteria ◽  
Western Blots ◽  
Toxic Activity ◽  
Gram Negative ◽  
E Coli ◽  
Recombinant Cells ◽  
C Terminus ◽  
Insoluble Form

ABSTRACT Experimental analyses with recombinant Escherichia coliand Pseudomonas putida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing both proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 104 to 105cells/ml. Western blots revealed a higher production of Cry11A when thep20 gene was coexpressed. Cry11A was detected primarily in insoluble form in recombinant cells. Cry11A was not detected inP. putida when P20 was not coproduced, and these recombinants were not toxic to larvae, whereas P. putidarecombinants producing both proteins were toxic at concentrations similar to those for E. coli. A coelution experiment was conducted, in which a p20 gene construct producing the P20 protein with an extension of six histidines on the C terminus was mixed with the Cry11A protein. The results showed that Cry11A bound to the P20(His6) on a nickel chelating column, whereas Cry11A produced without the P20(His6) protein was washed through the column, thus indicating that Cry11A and P20 physically interact. Thus, P20 protein either stabilizes Cry11A or helps it attain the folding important for its toxic activity.

scholarly journals Inhibition of cholera toxin production by thiols in Vibrio cholerae.

1986 ◽  
Vol 53 (3) ◽  
pp. 700-701 ◽  
Author(s):  
T Shimamura ◽  
S Watanabe ◽  
S Sasaki
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Toxin Production

scholarly journals Characterization of theVibrio choleraePhage Shock Protein Response

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Cara M. DeAngelis ◽  
Dhrubajyoti Nag ◽  
Jeffrey H. Withey ◽  
Jyl S. Matson
Keyword(s):  
Stress Response ◽  
Vibrio Cholerae ◽  
Membrane Damage ◽  
Regulatory Elements ◽  
Life Cycles ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Genetic Components ◽  
Stress Response Pathway

ABSTRACTThe phage shock protein (Psp) system is a stress response pathway that senses and responds to inner membrane damage. The genetic components of the Psp system are present in several clinically relevant Gram-negative bacteria, includingVibrio cholerae. However, most of the current knowledge about the Psp response stems fromin vitrostudies inEscherichia coliandYersinia enterocolitica. In fact, the Psp response inV. choleraehas remained completely uncharacterized. In this study, we demonstrate thatV. choleraedoes have a functional Psp response system. We found that overexpression of GspD (EpsD), the type II secretion system secretin, induces the Psp response, whereas otherV. choleraesecretins do not. In addition, we have identified several environmental conditions that induce this stress response. Our studies on the genetic regulation and induction of the Psp system inV. choleraesuggest that the key regulatory elements are conserved with those of other Gram-negative bacteria. While apspnull strain is fully capable of colonizing the infant mouse intestine, it exhibits a colonization defect in a zebrafish model, indicating that this response may be important for disease transmission in the environment. Overall, these studies provide an initial understanding of a stress response pathway that has not been previously investigated inV. cholerae.IMPORTANCEVibrio choleraeleads a dual life cycle, as it can exist in the aquatic environment and colonize the human small intestine. In both life cycles,V. choleraeencounters a variety of stressful conditions, including fluctuating pH and temperature and exposure to other agents that may negatively affect cell envelope homeostasis. The phage shock protein (Psp) response is required to sense and respond to such insults in other bacteria but has remained unstudied inV. cholerae. Interestingly, the Psp system has protein homologs, principally, PspA, in a number of bacterial clades as well as in archaea and plants. Therefore, our findings not only fill a gap in knowledge about an unstudied extracytoplasmic stress response inV. cholerae, but also may have far-reaching implications.

scholarly journals Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

2016 ◽  
Vol 113 (34) ◽  
pp. E5034-E5043 ◽  
Author(s):  
Wanatchaporn Arunmanee ◽  
Monisha Pathania ◽  
Alexandra S. Solovyova ◽  
Anton P. Le Brun ◽  
Helen Ridley ◽  
...  
Keyword(s):  
Binding Sites ◽  
Calcium Ion ◽  
Gram Negative Bacteria ◽  
Core Oligosaccharide ◽  
Gram Negative ◽  
X Ray ◽  
Cross Links ◽  
Phosphate Groups ◽  
Positively Charged ◽  
Lps Binding

The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin–LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin–LPS interactions and a bridging calcium ion.

scholarly journals Activation of the Vibrio cholerae SOS Response Is Not Required for Intestinal Cholera Toxin Production or Colonization

2006 ◽  
Vol 74 (2) ◽  
pp. 927-930 ◽  
Author(s):  
Mariam Quinones ◽  
Brigid M. Davis ◽  
Matthew K. Waldor
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Sos Response ◽  
Toxin Production ◽  
Mrna Levels ◽  
Suckling Mouse ◽  
Potent Inducer ◽  
Genes Encoding ◽  
Mouse Intestine ◽  
Ctx Prophage

ABSTRACT Cholera toxin, one of the main virulence factors of Vibrio cholerae, is encoded in the genome of CTXφ, a V. cholerae-specific lysogenic filamentous bacteriophage. Although the genes encoding cholera toxin, ctxAB, are known to have their own promoter, the toxin genes can also be transcribed from an upstream CTXφ promoter, PrstA . The V. cholerae SOS response to DNA damage induces the CTX prophage by stimulating gene expression initiating from PrstA . Here, we investigated whether ctxA mRNA levels increase along with the levels of the transcripts for the other CTXφ genes following stimulation of the V. cholerae SOS response. Treatment of V. cholerae with the SOS-inducing agent mitomycin C increased the level of ctxA mRNA approximately sevenfold, apparently by augmenting the activity of PrstA . However, using suckling mice as a model host, we found that intraintestinal ctxA transcription does not depend on PrstA . In fact, the suckling mouse intestine does not appear to be a potent inducer of the V. cholerae SOS response. Furthermore, alleviation of LexA-mediated repression of the V. cholerae SOS regulon was not required for V. cholerae growth in the suckling mouse intestine. Our observations suggest that pathogenicity of V. cholerae does not depend on its SOS response.

scholarly journals Back to the Future: Studying Cholera Pathogenesis Using Infant Rabbits

mBio ◽  
2010 ◽  
Vol 1 (1) ◽  
Author(s):  
Jennifer M. Ritchie ◽  
Haopeng Rui ◽  
Roderick T. Bronson ◽  
Matthew K. Waldor
Keyword(s):  
Animal Model ◽  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Close Association ◽  
Disease Model ◽  
Rabbit Model ◽  
Secretory Diarrhea ◽  
Watery Diarrhea ◽  
Content Type

ABSTRACTCholera is a severe diarrheal disease, caused byVibrio cholerae, for which there has been no reproducible, nonsurgical animal model. Here, we report that orogastric inoculation ofV. choleraeinto 3-day-old rabbits pretreated with cimetidine led to lethal, watery diarrhea in virtually all rabbits. The appearance and chemical composition of the rabbit diarrheal fluid were comparable to those of the “rice-water stool” produced by cholera patients. As in humans,V. choleraemutants that do not produce cholera toxin (CT) and toxin-coregulated pilus (TCP) did not induce cholera-like disease in rabbits. CT induced extensive exocytosis of mucin from intestinal goblet cells, and wild-typeV. choleraewas predominantly found in close association with mucin. Large aggregates of mucin-embeddedV. choleraewere observed, both attached to the epithelium and floating within the diarrheal fluid. These findings suggest that CT-dependent mucin secretion significantly influencesV. cholerae’s association with the host intestine and its exit from the intestinal tract. Our model should facilitate identification and analyses of factors that may governV. choleraeinfection, survival, and transmission, such as mucin. In addition, our results using nontoxigenicV. choleraesuggest that infant rabbits will be useful for study of the reactogenicity of live attenuated-V. choleraevaccines.IMPORTANCECholera remains a significant threat to populations in developing nations. Currently, there is no reproducible, nonsurgical animal model of cholera, the secretory diarrheal disease caused byVibrio cholerae. We found that oral infection of infant rabbits withV. choleraeled to lethal, watery diarrhea in most rabbits. Using this disease model, we discovered a new role for cholera toxin (CT) during infection. This toxin not only caused secretory diarrhea but also profoundly influenced howV. choleraeassociates with the intestine and how the pathogen exits from the host. Rabbits inoculated withV. choleraethat does not produce CT developed mild diarrhea, suggesting that this model may prove useful for generating improved live attenuated-V. choleraevaccine candidates. Overall, our findings suggest that the infant rabbit model will enable pursuit of several new avenues for research on cholera pathogenesis, as well as serve as a vehicle for testing new therapeutics.

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