Cholera Toxin Production Research Articles

A Novel Marker for the Species-Specific Detection and Quantitation of Vibrio cholerae by Targeting an Outer Membrane Lipoprotein lolB Gene

Vibrio cholerae O1 and O139 are the major serotypes associated with illness, and some V. cholera non-O1 and non-O139 isolates produce cholera toxin. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the species-specific detection and quantitation of V. cholera using a primer pair based on an outer membrane lipoprotein lolB gene for the amplification of a 195 bp DNA fragment. The qPCR primer set for the accurate diagnosis of V. cholera was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.

Activation of Cholera Toxin Production by Anaerobic Respiration of Trimethylamine N-oxide in Vibrio cholerae

Vibrio cholerae is a gram-negative bacterium that causes cholera. Although the pathogenesis caused by this deadly pathogen takes place in the intestine, commonly thought to be anaerobic, anaerobiosis-induced virulence regulations are not fully elucidated. Anerobic growth of the V. cholerae strain, N16961, was promoted when trimethylamine N-oxide (TMAO) was used as an alternative electron acceptor. Strikingly, cholera toxin (CT) production was markedly induced during anaerobic TMAO respiration. N16961 mutants unable to metabolize TMAO were incapable of producing CT, suggesting a mechanistic link between anaerobic TMAO respiration and CT production. TMAO reductase is transported to the periplasm via the twin arginine transport (TAT) system. A similar defect in both anaerobic TMAO respiration and CT production was also observed in a N16961 TAT mutant. In contrast, the abilities to grow on TMAO and to produce CT were not affected in a mutant of the general secretion pathway. This suggests that V. cholerae may utilize the TAT system to secrete CT during TMAO respiration. During anaerobic growth with TMAO, N16961 cells exhibit green fluorescence when stained with 2',7'-dichlorofluorescein diacetate, a specific dye for reactive oxygen species (ROS). Furthermore, CT production was decreased in the presence of an ROS scavenger suggesting a positive role of ROS in regulating CT production. When TMAO was co-administered to infant mice infected with N16961, the mice exhibited more severe pathogenic symptoms. Together, our results reveal a novel anaerobic growth condition that stimulates V. cholerae to produce its major virulence factor.

Functional Characterization of the Stringent Response Regulatory GenedksAof Vibrio cholerae and Its Role in Modulation of Virulence Phenotypes

In bacteria, nutrient deprivation evokes the stringent response, which is mediated by the small intracellular signaling molecule ppGpp. In Gram negatives, the RelA enzyme synthesizes and SpoT hydrolyzes ppGpp, although the latter protein also has weak synthetase activity. DksA, a recently identified RNA polymerase binding transcription factor, acts as a coregulator along with ppGpp for controlling the stringent response. Recently, we have shown that three genes, relA, spoT, and relV, govern cellular levels of ppGpp during various starvation stresses in the Gram-negative cholera pathogen Vibrio cholerae. Here we report functional characterization of the dksA gene of V. cholerae (dksA(Vc)), coding for the protein DksA(Vc). Extensive genetic analyses of the ΔdksA(Vc) mutants suggest that DksA(Vc) is an important component involved in the stringent response in V. cholerae. Further analysis of mutants revealed that DksA(Vc) positively regulates various virulence-related processes, namely, motility, expression of the major secretory protease, called hemagglutinin protease (HAP), and production of cholera toxin (CT), under in vitro conditions. We found that DksA(Vc) upregulates expression of the sigma factor FliA (σ(28)), a critical regulator of motility in V. cholerae. Altogether, it appears that apart from stringent-response regulation, DksA(Vc) also has important roles in fine regulation of virulence-related phenotypes of V. cholerae.

Mlp24 (McpX) of Vibrio cholerae Implicated in Pathogenicity Functions as a Chemoreceptor for Multiple Amino Acids

The chemotaxis of Vibrio cholerae, the causative agent of cholera, has been implicated in pathogenicity. The bacterium has more than 40 genes for methyl-accepting chemotaxis protein (MCP)-like proteins (MLPs). In this study, we found that glycine and at least 18 L-amino acids, including serine, arginine, asparagine, and proline, serve as attractants to the classical biotype strain O395N1. Based on the sequence comparison with Vibrio parahaemolyticus, we speculated that at least 17 MLPs of V. cholerae may mediate chemotactic responses. Among them, Mlp24 (previously named McpX) is required for the production of cholera toxin upon mouse infection. mlp24 deletion strains of both classical and El Tor biotypes showed defects in taxis toward several amino acids, which were complemented by the expression of Mlp24. These amino acids enhanced methylation of Mlp24. Serine, arginine, asparagine, and proline were shown to bind directly to the periplasmic fragment of Mlp24. The structural information of its closest homolog, Mlp37, predicts that Mlp24 has two potential ligand-binding pockets per subunit, the membrane distal of which was suggested, by mutational analyses, to be involved in sensing of amino acids. These results suggest that Mlp24 is a chemoreceptor for multiple amino acids, including serine, arginine, and asparagine, which were previously shown to stimulate the expression of several virulence factors, implying that taxis toward a set of amino acids plays critical roles in pathogenicity of V. cholerae.

Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus.

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.

Phenotypic and Genetic Analysis of Altered Variants of <i>Vibrio cholerae</i> Biovar El Tor

Determined is cholera toxin production by altered Vibrio cholerae O1 eltor strains, isolated on the territory of Russia in 1993-2010, which contain classical type ctxB gene. Studied are their genetic characteristics related to the production of this virulence factor. Determined is that altered eltor variants yield 10 to 20 times more cholera toxin than typical eltor strains. Changes in promotor region of ctxAB operon are not detected but elucidated is that 90 % of studied altered strains contain two copies of CTXϕ prophage in their genome whereas typical strains carry one copy of CTXϕ prophage. Despite the higher copy number of CTXϕ prophage, the identified differences in production of cholera toxin can be associated mostly with a change of transcriptional activity of regulatory genes. That causes the active production of cholera toxin by the altered variants of Vibrio cholerae eltor in non typical environment.

TolC Affects Virulence Gene Expression in Vibrio cholerae

A Vibrio cholerae tolC mutant showed increased toxT expression in M9 medium, but not in the presence of four amino acids that induce cholera toxin production, and in LB with high osmolarity but not high pH or temperature. TolC did not affect expression of other regulatory genes in the ToxR regulon.

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

ABSTRACTCholera is a severe diarrheal disease typically caused by O1 serogroup strains of Vibrio cholerae. The pathogenicity of all pandemic V. cholerae O1 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-O139 V. cholerae strains, 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-O139 V. cholerae has 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-O139 V. cholerae pathogenicity. We found that all animals infected with wild-type AM-19226 developed severe diarrhea even more rapidly than rabbits infected with V. cholerae O1. Unlike V. cholerae O1 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-O139 V. cholerae represents a new type of diarrheagenic mechanism.

Binding efficiencies of carbohydrate ligands with different genotypes of cholera toxin B: molecular modeling, dynamics and docking simulation studies

Vibrio cholerae produces cholera toxin (CT) that consists of two subunits, A and B, and is encoded by a filamentous phage CTXΦ. The A subunit carries enzymatic activity that ribosylates ADP, whereas the B subunit binds to monosialoganglioside (GM1) receptor in epithelial cells. Molecular analysis of toxigenic V. cholerae strains indicated the presence of multiple ctxB genotypes. In this study, we employed a comparative modeling approach to define the structural features of all known variants of ctxB found in O139 serogroup V. cholerae. Modeling, molecular dynamics and docking simulations studies suggested subtle variations in the binding ability of ctxB variants to carbohydrate ligands of GM1 (galactose, sialic acid and N-acetyl galactosamine). These findings throw light on the molecular efficiencies of pathogenic isolates of V. cholerae harboring natural variants of ctxB in causing the disease, thus suggesting the need to consider ctxB variations when designing vaccines against cholera.

Characterization of Vibrio cholerae O139 belonging to multiple ribotypes and isolated from diarrhoeal patients in Kerala, southern India

Twenty-four Vibrio cholerae O139 strains isolated from Kerala, southern India were characterized by PCR, CTX typing and ribotyping; all of which, except three strains, carried the core of the CTX genetic element, colonization-toxin co-regulated pilus, the adherence outer membrane protein, haemolysin, central regulatory protein encoded toxR, SXT genetic element, and produced cholera toxin and biofilm. Results of RFLP analysis revealed twenty-one of the O139 strains possess two copies of CTXΦ and pre-CTXФ always preceded by tandemly arranged RS1 element; one had two copies of pre-CTXΦ and two a single copy of pre-CTXΦ. Nucleotide sequencing detected classical ctxB in CTXETΦ and CTXCalcФ with additional change at 28th amino acid position of CTXCalcФ. Ribotype analysis revealed the presence of multiple ribotypes, including B-I and B-II, and new ribotypes designated B-VIIIa, B-VIIIb and B-IX, not reported earlier among V. cholerae O139 strains. These observations thus indicate that genetic recombination or mutations had occurred in conserved rrn operon and variations in CTXΦ may have implications on the evolution of the organism.

Two Different Diagnosis Methods For The detection of cholera toxin production From Vibrio cholerae isolated from different areas in Iraq

Fifty isolates of Vibrio cholerae obtained from different areas in Iraq from patients with acute secretory diarrhea were diagnosed serologically in central public health laboratory(CPHL), Two different methods were used for detection cholera toxin production ,the first based on the degradation of Nicotin Amide Adinine Dinucleotide( NAD) by Cholera toxin (CT). and the second was by detection CT gene by polymerase chain reaction (PCR).All Vibrio cholerae were cultured in alkaline peptone water, out of 50 isolates 46 (92%) were positive when we used NAD for the detection of cholera toxin(CT).Whereas all isolates gave positive results by PCR. From these data we conclude that the degradation of NAD by CT is simple and can be carried out in small laboratories, it is also easy to perform, and gives reproducible results, while PCR provides a more sensitive and specific assay for rapid diagnosis of cholera than currently available methods.

Treatment of cholera-like diarrhoea with oral rehydration

Cholera diarrhoea remains a major global health problem that has caused seven pandemics. The pathogenesis of cholera is attributable to the production of cholera toxin by the causative pathogen, Vibrio cholerae. The toxin causes increased production of cyclic adenosine monophosphate and this results in massive water and electrolyte secretion into the intestinal lumen. These changes manifest clinically as the painless defecation of voluminous stools that resemble 'rice water', leading to severe dehydration. The cornerstone in the management of cholera diarrhoea is the use of oral rehydration solutions (ORS) to replace the water and electrolytes lost as stools. The World Health Organization recommends the use of ORS of 'reduced osmolarity' for the treatment of acute non-cholera diarrhoea and the use of rice-based ORS for the management of cholera diarrhoea. Although several attempts have been made to improve ORS, studies to evaluate some of the modifications, which include the addition of amylase-resistant starch, the use of amino acids (such as glycine, alanine and glutamine) as sodium cotransporters, and zinc-supplemented ORS, are still needed.

Vibrio cholerae O1 Avirulent Strains - Producers of Protective O1 Antigen: Obtaining and Peculiarities

Among 15 Vibrio cholerae El Tor O1 virulent strains of Inaba and Ogawa serovars grown in sterile river water, 6 strains were found to lose spontaneously the core region of CTXφ prophage including structural genes ( ctxAB ) of cholera toxin. It was determined that the obtained ctxA - mutants did not produce cholera toxin and did not cause specific choleragenic reaction in the intra-intestinally inoculated suckling rabbits suggesting that they belonged to the III d pathogenicity group. Analysis of the population structure of two selected non-toxigenic strains M569A and 5/65A revealed clones with high level of O1 antigen of Inaba and Ogawa serovars production. The obtained avirulent strains can be used as producers of above-noted protective antigens both in production of chemical cholera vaccine and in constructing of diagnostic immunoenzyme test-systems.

Back to the Future: Studying Cholera Pathogenesis Using Infant Rabbits

ABSTRACTCholera is a severe diarrheal disease, caused by Vibrio cholerae, for which there has been no reproducible, nonsurgical animal model. Here, we report that orogastric inoculation of V. cholerae into 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. cholerae mutants 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-type V. cholerae was predominantly found in close association with mucin. Large aggregates of mucin-embedded V. cholerae were observed, both attached to the epithelium and floating within the diarrheal fluid. These findings suggest that CT-dependent mucin secretion significantly influences V. 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 govern V. cholerae infection, survival, and transmission, such as mucin. In addition, our results using nontoxigenic V. cholerae suggest that infant rabbits will be useful for study of the reactogenicity of live attenuated-V. cholerae vaccines.

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

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.

In Vitro Production of Cholera Toxin by Vibrio cholerae El Tor Strains from Human Beings and the Environment

In Vitro Production of Cholera Toxin by Vibrio cholerae El Tor Strains from Human Beings and the Environment

Capsaicin, a potential inhibitor of cholera toxin production inVibrio cholerae

The use of natural compounds as inhibitory agents for virulence factor production is a new approach to overcome increased antimicrobial resistance in pathogenic bacteria. In this study, we examined whether red chilli (Capsicum annuum) contains any such compound(s) that can repress the cholera toxin (CT) production in Vibrio cholerae. We found that the methanol extract of red chilli could inhibit CT production in recently emerged V. cholerae O1 El Tor variant strains without affecting their viability. Interestingly, capsaicin, a well-studied active component of red chilli, also drastically inhibited CT production in V. cholerae strains belonging to various serogroups including variants. Real-time quantitative reverse transcription-PCR assay revealed that capsaicin effectively repressed the transcription of ctxA, tcpA and toxT genes, but not of toxR and toxS genes. On the contrary, capsaicin significantly enhanced the transcription of the hns gene, the product of which is known to regulate negatively the transcription of ctxAB, tcpA and toxT genes. These results suggest that capsaicin might act as a potent repressor for CT production possibly by enhancing the transcription of hns.

Construction and characterization of rtxA and rtxC mutants of auxotrophic O139 Vibrio cholerae

Vibrio cholerae is a Gram-negative bacterium that causes diarrheal disease. V. cholerae O1 and O139 serogroups are toxigenic and are known to cause epidemic cholera. These serogroups produce cholera toxin and other accessory toxins such as accessory cholera enterotoxin, zonula occludens toxin, and multifunctional, autoprocessing repeat in toxin (MARTX). In the present study, we incorporated mutated rtxA and rtxC genes that encode MARTX toxin into the existing aminolevulinic acid (ALA) auxotrophic vaccine candidate VCUSM2 of V. cholerae O139 serogroup. The rtxC mutant was named VCUSM9 and the rtxC/ rtxA mutant was named VCUSM10. VCUSM9 and VCUSM10 were able to colonize intestinal cells well, compared with the parent vaccine strain, and produced no fluid accumulation in a rabbit ileal loop model. Cell rounding and western blotting assays indicated that mutation of the rtxC gene alone (VCUSM9 strain) did not abolish MARTX toxicity. However mutation of both the rtxA and rtxC genes (VCUSM10) completely abolished MARTX toxicity. Thus we have produced a new, less reactogenic, auxotrophic rtxC/ rtxA mutated vaccine candidate against O139 V. cholerae.

Levels of the Secreted Vibrio cholerae Attachment Factor GbpA Are Modulated by Quorum-Sensing-Induced Proteolysis

Vibrio cholerae is the etiologic agent of cholera in humans. Intestinal colonization occurs in a stepwise fashion, initiating with attachment to the small intestinal epithelium. This attachment is followed by expression of the toxin-coregulated pilus, microcolony formation, and cholera toxin (CT) production. We have recently characterized a secreted attachment factor, GlcNAc binding protein A (GbpA), which functions in attachment to environmental chitin sources as well as to intestinal substrates. Studies have been initiated to define the regulatory network involved in GbpA induction. At low cell density, GbpA was detected in the culture supernatant of all wild-type (WT) strains examined. In contrast, at high cell density, GbpA was undetectable in strains that produce HapR, the central regulator of the cell density-dependent quorum-sensing system of V. cholerae. HapR represses the expression of genes encoding regulators involved in V. cholerae virulence and activates the expression of genes encoding the secreted proteases HapA and PrtV. We show here that GbpA is degraded by HapA and PrtV in a time-dependent fashion. Consistent with this, Delta hapA Delta prtV strains attach to chitin beads more efficiently than either the WT or a Delta hapA Delta prtV Delta gbpA strain. These results suggest a model in which GbpA levels fluctuate in concert with the bacterial production of proteases in response to quorum-sensing signals. This could provide a mechanism for GbpA-mediated attachment to, and detachment from, surfaces in response to environmental cues.

Detection of Clones with Different Expression of Virulence and Persistence Factors in Natural Populations of Cholera Vibrio and Their Phenotypic Analysis

Analysis of the population structure of 41 Vibrio cholerae biovar eltor natural strains permitted to detect 12 strains forming typical transparent and atypical opaque colonies. Among the later were identified 3 strains which morphology variability correlated with altered production of exopolysaccharide, cholera toxin, hemolysine, soluble hemagglutinin/protease and altered mobility. One of the external signals causing coordinating switching of production of the indicated pathogenicity and persistence factors is alkaline (pH 9,0) reaction of the medium.