Cross-reactivity of outer membrane proteins of Campylobacter species with cholera toxin.

Campylobacter jejuni is a foodborne pathogen and a leading cause of diarrhoea worldwide. It is believed that a cholera toxin-like toxin (CTLT) produced by C. jejuni may mediate watery diarrhoea. However, the production of a CTLT by C. jejuni is controversial. A cholera toxin gene (ctx) homologue has not been identified in Campylobacter species. We investigated the identity of the CT cross-reactive antigen from Campylobacter species previously and the results are reviewed here. Filtrates of C. jejuni grown in four different liquid media, reported to promote CTLT production, were tested by Chinese hamster ovary (CHO) cell elongation assay for functional toxin and for reactivity with CT antibody using GM1 ganglioside ELISA (GM1 ELISA) and immunoblotting. Protein sequence of the CT antibody-reactive band was determined by matrix-assisted laser desorption ionization-time of flight (MALDI TOF-TOF). Non-jejuni species (C. coli, C. lari, C. foetus, C. hyointestinalis and C. upsaliensis) were investigated by CHO cell assay and immunoblotting. Filtrates from seven C. jejuni reference strains reported to produce CTLT and from 80 clinical strains were negative in the CHO cell assay. However, filtrates from three reference strains and 16 clinical strains were positive by GM1 ELISA. All strains irrespective of GM1 ELISA reactivity, possessed a 53-kDa protein which reacted with CT antibody by immunoblotting. This band was identified as the major outer membrane protein (PorA) of C. jejuni. CT antibody reacted with a C. jejuni recombinant PorA on immunoblotting. All non-C. jejuni strains were negative by CHO cell assay, but the common 53-kDa proteins reacted with CT antibody on immunoblots. The cross-reactivity of PorAs of Campylobacter species with CT may lead to the erroneous conclusion that Campylobacter species produce a functional CTLT.

Effect of stress on production of heat labile enterotoxin by Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen responsible for secretory diarrhoea. The production of heat labile enterotoxin (LT), by ETEC, is largely responsible for the pathogenesis of diarrhoea. In the present study we investigated the effect of stress factors such as temperature, pH, osmotic stress and nutritional limitation on the production of LT by ETEC using in-house GMI-ELISA. Four strains of E. coli consisting, one standard strain MTCC 723 and three clinical isolates were used in the study. Maximum amount of LT (OD 3.285) was produced at 37 0 C followed by 40 0 C (OD 3.305). Growth of E. coli in medium with pH 8.6 resulted in maximum amount of LT production (OD 3.489). LT was not detectable when bacteria were grown in medium with pH ≤7.2 and ≥ 9.2. Sodium chloride concentration of 0.2 M stimulated maximum amount of LT production. Maximum amount of LT was produced when the bacteria were grown in medium containing 2.5g/l of glucose. All the stress factors had a significant effect on the LT production by E. coli , though quantitative differences in the various strains were observed.

Cloning and Expression of Vibrio cholerae CTB Gene and the Recombinant CTB Protein Activation Assay / 中国生物工程杂志

CTB protein possessed mucosal adjuvant immunoactivity. The CTB gene was amplified by PCR method from a strain V. cholerae. The nucleotide sequence of CTB gene was 375 bp and shared 96.0%~99.2% homology with other 6 CTB genes. The recombinant plasmid pTWIN1-CTB transformed E. coli strain BL21(DE3) expressed with 0.8 mmol/L IPTG. The molecular weight of expression products was identical with expectative weight by SDS-PAGE electrophoresis. The CTB fusion proteins mainly assembled inclusion bodies and the outputs of proteins were approximately 20% of the total bacterial proteins. The CTB proteins possessed mucosal immunoactivity by GM1-ELISA assay.