A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

Abstract

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.

A branch in the ToxR regulatory cascade of Vibrio cholerae revealed by characterization of toxT mutant strains

“…The pathway for CT secretion in V. cholerae includes disulphide bond formation catalysed by the TcpG(DsbA) protein (Yu et al, 1992;Peek and Taylor, 1992), originally identified as a ToxR-regulated gene product (Peterson and Mekalanos, 1988;Peek and Taylor, 1992). A strain deficient for TcpG(DsbA) activity cannot secrete the pentameric B subunit, which is the component of CT that is detected in the GM 1 -ELISA assay (Yu et al, 1992). We hypothesized that if tcpG transcription were under ToxT control, then the mutants might be deficient for CT in the culture supernatants, which is what we use for the GM 1 -ELISA.…”

“…To test whether toxin secretion was affected in the mutants, we analysed their ability to secrete the B subunit of heat-labile toxin (LT ) of E. coli, which is identical in structure to CT and which also requires TcpG(DsbA) to be secreted from V. cholerae (Yu et al, 1992). We introduced pMMB68, which expresses the gene encoding the Immunoblot of TcpA production in wild-type and toxT mutant V. cholerae.…”

A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K‐12

“…This gene (and, by association, the AE phenotype) is necessary for full virulence in humans, as an eae mutant shows a significantly diminished diarrhoeal attack rate in volunteers relative to the wild-type parent strain (Donnenberg et al, 1993a). Genes homologous to eae have been found in all AE bacteria with the exception of H. pylori, implying a common mechanism of AE lesion formation among these pathogens (Yu et al, 1992;Schauer and Falkow, 1993;Frankel et al, 1994).…”

Structural Basis for the Differential Toxicity of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

“…2 To facilitate controlled, high level expression of CtxAB, the EcoRV-SpeI fragment of pRC1 was subcloned into pTTQ18 (35) at the SmaI and XbaI sites to yield pRC9. Plasmid pTRH29 was described previously and is a pBluescript derivative containing the etxAB operon of E. coli enterotoxin flanked by EcoRV and SpeI restriction sites (36). A pTTQ18 derivative, harboring the EcoRV-SpeI fragment from pTRH29 and designated pAM29, has been described (37).…”

“…Wild-type and hybrid toxins. Plasmid pRC1 contains the entire ctxAB operon encoding wild-type CtxAB; plasmid pTRH29 (36) contains the entire etxAB operon encoding wild-type EtxAB; plasmid pATA14 encoding CtxA (RDEL) CtxB was generated by PCR amplification of upstream and downstream segments of the ctxAB operon followed by their ligation at an engineered EcoRI site to introduce a Lys-237 to Arg substitution; plasmid pRC18 encoding EtxA CtxA (225-240) CtxB was generated by ligating an upstream segment of etxA to a downstream segment of the ctxAB operon at an engineered KpnI site; pRC19 encoding CtxA EtxA (226 -240) EtxB was generated by ligating an upstream segment of ctxA to a downstream segment of the etxAB operon at an engineered KpnI site; and pCDR1 encoding CtxA (RDEL) EtxB was constructed by substituting the EcoRV-EcoRI fragment in pTRH29 for the corresponding fragment from pATA14. In all cases, the vector pBluescript II KS is not depicted.…”