Identification of a protein secretory pathway for the secretion of heat-labile enterotoxin by an enterotoxigenic strain of Escherichia coli - PubMed (original) (raw)

Identification of a protein secretory pathway for the secretion of heat-labile enterotoxin by an enterotoxigenic strain of Escherichia coli

Marija Tauschek et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen that causes cholera-like diarrhea in humans and animals. ETEC secretes a heat-labile enterotoxin (LT), which resembles cholera toxin, but the actual mechanism of LT secretion is presently unknown. We have identified a previously unrecognized type II protein secretion pathway in the prototypic human ETEC strain, H10407 (serotype O78:H11). The genes for this pathway are absent from E. coli K-12, although examination of the K-12 genome suggests that it probably once possessed them. The secretory pathway bears significant homology at the amino acid level to the type II protein secretory pathway required by Vibrio cholerae for the secretion of cholera toxin. With this in mind, we determined whether the homologous pathway of E. coli H10407 played a role in the secretion of LT. To this end, we inactivated the pathway by inserting a kanamycin-resistance gene into one of the genes (gspD) of the type II secretion pathway by homologous recombination. LT secretion by E. coli H10407 and the gspD mutant was assayed by enzyme immunoassay, and its biological activity was assessed by using Y-1 adrenal cells. This investigation showed that the protein secretory pathway is functional and necessary for the secretion of LT by ETEC. Our findings have revealed the mechanism for the secretion of LT by ETEC, which previously was unknown, and provide further evidence of close biological similarities of the LT and cholera toxin.

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Figures

Figure 1

Figure 1

(A) Schematic diagram of the genetic organization of the chromosomal region containing the genes (solid arrows) for the putative type II secretion pathway in ETEC H10407. *, Insertion site of the Kformula image gene in E. coli MT13. (B) Chromosomal region common to ETEC strain, H10407 and E. coli K-12 strain, MG1655, with the percentage identity/similarity of homologous proteins shown in parentheses. †, Percentage identity/similarity over 286 amino acid residues; ‡, percentage identity/similarity over 276 amino acid residues. (C) Arrangement of the genes encoding the type II pathway for the secretion of CT by V. cholerae [accession nos. AF109904 (30), AF055371 (31), and L33796 (10)], and, in parentheses, the percentage identity/similarity of homologous proteins of ETEC H10407 and V. cholerae, TRH7000. §, In V. cholerae TRH7000, the prepilin peptidase gene, pilD/vcpD, is not linked to the type II secretion gene cluster. ¶, There is no homologue of epsN in the ETEC H10407 type II secretion gene cluster. (D) Arrangement of the genes encoding the type II secretion pathway in E. coli K-12, strain MG1655 [accession no. AE000409 (15)], and, in parentheses, the percentage identity/similarity of homologous proteins of ETEC H10407 and E. coli K-12, strain MG1655. ∥, NSH, no significant homology.

Figure 2

Figure 2

(A) Growth curves of E. coli H10407 (interrupted line) and MT13 (solid line) in CAYE broth. (B) Concentration of LT (detected by *, EIA, and †, Y1 adrenal cell assay) in supernatants of CAYE broth, 4, 8, and 24 h after inoculation with E. coli H10407 or MT13. (C) Concentration of LT (detected by *, EIA, and †, Y1 adrenal cell assay) in centrifuged bacteria pellets obtained from the broth cultures depicted in B. Error bars indicate SD.

Figure 3

Figure 3

Phase-contrast photomicrographs of cultured Y1 adrenal cells 18 h after addition of a 1 in 40 dilution of cell free CAYE culture medium from 4-h cultures of (A) E. coli H 10407 or (B) E. coli MT13. C shows the effect of a 1 in 40 dilution of a periplasmic extract from a 4-h culture of E. coli MT13.

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