Cholera Toxin Gene Research Articles

Genetics of type IIa heat-labile enterotoxin of Escherichia coli: operon fusions, nucleotide sequence, and hybridization studies.

Operon fusions for the Escherichia coli heat-labile enterotoxin type IIa (LT-IIa) operon were isolated and characterized. The LT-IIa genes are organized in a transcriptional unit similar to those of cholera toxin (CT) and the closely related E. coli heat-labile toxin type I (LT-I, with subtypes LTh-I and LTp-I). The nucleotide sequence of the LT-IIa genes was determined and compared with the sequences of LTh-I and CT. The A subunit gene of LT-IIa was found to be 57% homologous with the A subunit gene of LTh-I and 55% homologous with the A gene of CT. Most of the homology derived from the region of the A gene which encodes the A1 fragment. The B gene of LT-IIa was not homologous with the B gene of LTh-I or CT. DNA probes containing various portions of the LT-IIa genes and adjacent sequences were used for hybridization studies with restriction endonuclease fragments of DNA from a collection of LT-II-producing strains. These studies showed that a probe containing much of the A subunit gene hybridized well to DNA from the various strains, but a probe for the B subunit gene did not.

Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.

Effect of a recA mutation on cholera toxin gene amplification and deletion events.

The cholera toxin operon (ctxAB) is located on a 7-kilobase pair variable genetic element which undergoes genetic duplication and amplification events in Vibrio cholerae. Amplification of the ctx genetic element was investigated by substituting the resident ctx loci of two V. cholerae strains with a DNA fragment encoding resistance to kanamycin. Although these strains were not normally resistant to greater than 150 micrograms of kanamycin per ml, spontaneous derivatives could be obtained that grew well on 3 mg of kanamycin per ml. Southern blot analysis of these highly resistant isolates demonstrated that the ctx element was amplified approximately 20-fold. This amplification process was completely inhibited in the absence of a functional recA gene. The V. cholerae RecA protein, therefore, is essential for cholera toxin gene amplification. Spontaneous deletions of the ctx structural genes were observed in both recA+ and recA- V. cholerae strains, although such deletions occurred at a 21-fold-lower frequency in the latter case. Structural analysis of these ctx amplification and deletion events supports a model for their formation that involves unequal crossing over between repetitive sequences located upstream and downstream of the ctx operon.

Molecular epidemiology of non-O1 Vibrio cholerae and Vibrio mimicus in the U.S. Gulf Coast region.

Ten toxigenic Vibrio cholerae non-O1 and V. mimicus strains isolated from clinical and environmental sources in the U.S. Gulf Coast region were examined for genetic relatedness. Restriction digest patterns of chromosomal DNA and Southern blot analysis with a cholera toxin gene probe revealed that the strains exhibited greater genetic divergence than the highly conserved V. cholerae O1 strains isolated from clinical and sewage samples in this region.

Non-O1 Vibrio cholerae in Thailand: homology with cloned cholera toxin genes.

We examined 281 non-O1 Vibrio cholerae isolates from Thailand for homology with genes coding for cholera toxin. Five isolates from environmental sources were homologous with the cholera toxin gene probe and produced both the A and B subunits of cholera toxin.

Mechanism of toxin secretion by Vibrio cholerae investigated in strains harboring plasmids that encode heat-labile enterotoxins of Escherichia coli.

A genetically engineered Vibrio cholerae strain from which the cholera toxin genes had previously been deleted was used as a host in which to study the expression and secretion of related toxins and their subunits. Recombinant plasmids encoding heat-labile enterotoxins (LTs) from Escherichia coli of human and porcine origin were expressed in the V. cholerae host, and this resulted in the secretion of the LTs into the extracellular milieu. The secreted LTs were isolated and it was found that the A subunits of human and porcine LT were "unnicked" polypeptides, which indicates that nicking is not obligatory for toxin secretion. V. cholerae strains were also constructed that harbored plasmids encoding either the A or the B subunits of human LT (A+B-, or A-B+). Approximately 90% of the B subunits were secreted from the A-B+ strain, while all of the A subunits expressed by the A+B- strain remained cell associated. This implies that strains synthesizing both subunits assemble the A and B subunits prior to their secretion. We propose that the entry of the toxin into the secretory step of the export pathway is mediated by a secretory apparatus that recognizes structural domains within the B subunit of LT.

Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR.

We have cloned a positive regulatory gene ( toxR ) from Vibrio cholerae that controls cholera toxin transcription. This was done by first constructing a genetic fusion consisting of the lacZ gene fused to the promoter of the cholera toxin operon ctxAB . This operon fusion was used to screen a V. cholerae genomic library for genes that could activate the ctx promoter in Escherichia coli. This method allowed the identification of a gene, toxR , that increases ctx expression by more than 100-fold. Complementation analysis indicated that certain hypotoxinogenic mutants of V. cholerae 569B probably have mutations in the toxR gene. Southern blot analysis suggests that all V. cholerae, including nontoxinogenic strains, have the toxR gene. Moreover, nontoxinogenic strains not only lack the structural genes for cholera toxin but also sequences associated with the larger 7-kilobase ctx genetic element.

Evolution and structure of two ADP-ribosylation enterotoxins, Escherichia coli heat-labile toxin and cholera toxin

Nucleotide sequence comparisons of the heat-labile enterotoxin (LTh) genes of E. coli pathogenic for humans with cholera toxin (CT) genes suggest that the two toxin genes have evolved from a common ancestry by a series of single selective base changes, while conserving the catalytic fragment A1 (ADP-ribose transferase). Based on the local hydrophilicity profiles of LTh and CT peptides, a transmembrane segment appears to be present in A1 in both toxins.

A Recombinant Live Oral Cholera Vaccine

A pathogenic strain of Vibrio cholerae was attenuated by deletion of DNA sequences encoding the A1 subunit of the cholera enterotoxin. A restriction endonuclease fragment encoding the A1, but not the A2 or B sequences was deleted in vitro from cloned cholera toxin genes. The mutation was then recombined into the chromosome of V.cholerae Ogawa 395, a pathogenic strain that confers complete immunity to subsequent infection following an initial clinical infection. The resulting strain, which produces the immunogenic but nontoxic B subunit of cholera toxin and is unaltered in other critical antigens, represents a promising candidate for an attenuated live oral cholera vaccine.

Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development.

Nucleotide sequence and deletion analysis have been used to identify the regulatory and coding sequences comprising the cholera toxin operon (ctx). Incorporation of defined in vitro-generated ctx deletion mutations into Vibrio cholerae by in vivo genetic recombination produced strains which have practical value in cholera vaccine development.

Molecular epidemiology of Vibrio cholerae in the U.S. Gulf Coast.

Enterotoxigenic strains of Vibrio cholerae O-1, biotype El Tor, isolated from a case of cholera in Texas in 1973, an outbreak of cholera in Louisiana in 1978, and Louisiana sewage samples in 1980 and 1981 were analyzed for their genetic similarities. Chromosomal DNA was isolated from each strain, digested with restriction endonuclease, and analyzed by the Southern blot technique. A radioactive probe consisting of Escherichia coli heat-labile enterotoxin DNA detected cholera toxin gene sequences in these strains and demonstrated that the toxin gene sequence, if not the entire chromosomal DNA, is identical in these strains and distinctly different from other strains of V. cholerae isolated throughout the world. In addition, two strains of enterotoxigenic V. cholerae non-O-1 isolated from clinical cases, were analyzed and found to possess cholera toxin genes which differed in the DNA sequence from the V. cholerae O-1 strains. We concluded that a single strain of enterotoxigenic V. cholerae O-1 is resident in the U.S. Gulf Coast and that a second reservoir of cholera toxin genes exists in V. cholerae non-O-1 strains in Louisiana.

Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12.

Hybridization probes derived from the A and B subunit genes of the heat-labile enterotoxin (LT) of Escherichia coli were used to analyze DNA from Vibrio cholera strain 569B for cholera toxin gene sequences. Southern blot analysis indicated that the cholera toxin A and B subunit genes were each duplicated in the strain. One of the two toxin subunit gene pairs was cloned as a 5.1-kilobase DNA insert in plasmid pBR322. E. coli cells carrying the recombinant plasmid pJM17 were shown to produce cholera toxin, which was found to be largely cell associated. Protein chemical analysis indicated that the toxin was in its unnicked form and required additional proteolytic processing by trypsin to exhibit full toxicity in tissue culture. The alteration in E. coli of the secretion and proteolytic processing of cholera toxin parallels that previously observed for LT. An in vitro generated insertion mutation in the A subunit gene on pJM17 was shown to abolish production of the A chain but still allow production of the B chain. These observations, together with restriction mapping data, have demonstrated that the cholera toxin and LT genes are very similar in their genetic organization.

Influence of Some Physicochemical Stresses on the Survival of Vibrio cholerae O1 at Non-Culturable State

Survival study of toxigenic Vibrio cholerae O1 Classical Inaba and El Tor Ogawa biotypes was carried out in artificial microcosm without any nutrients under stressed physiochemical conditions. The organisms were found to undergo viable but non-culturable (VBNC) state, which was detected by polymerase chain reaction (PCR) technique using primers that flank a specific fragment of the cholera toxin gene (ctx). The findings of the study showed that about 89% of samples gave positive result although the organisms were in nonculturable state. Depending upon the conditions the Classical biotype of vibrios had undergone non-culturable state within 2-15 days and 55-92 days were subjected to PCR assay after 45 and 120 days of incubation respectively. A similar tend was also observed in case of El Tor biotype. With non-culturable cell only 5 of 18 microcosms (of both Classical and El Tor biotypes) at pH 4.5 showed no viability. Since the PCR assay demonstrated positive results after 90 (in case of El Tor biotype) and 120 days (in case of Classical biotype), the total survival time thus ranges over 3 and 4 months. Thus PCR has an important advantage over the techniques those are used in routine laboratories for the identification of pathogenic organisms. Keywords: Physicochemical stresses, Survivality, Vibrio cholerae, Artificial microcosms, Polymerase chain reaction (PCR), Non-culturable state, Viable but non-culturable (VBNC)DOI: http://dx.doi.org/10.3329/bjm.v24i2.1258 Bangladesh J Microbiol, Volume 24, Number 2, December 2007, pp 133-136