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. 2001 Jan;69(1):315-24.
doi: 10.1128/IAI.69.1.315-324.2001.

espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin

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espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin

J L Mellies et al. Infect Immun. 2001 Jan.

Abstract

At least five proteins are secreted extracellularly by enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea in developing countries. However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa. Here we determine that the espC gene is located within a second EPEC pathogenicity island at 60 min on the chromosome of E. coli. We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers. In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative E. coli eliminated EspC enterotoxin activity. Like the EAF plasmid, the espC pathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.

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Figures

FIG. 1
FIG. 1
Genetic map of espC pathogenicity island. The 15,195 bp of unique DNA at approximately 60 min of the EPEC chromosome, including the espC gene and identified ORFs, is presented. Sequences corresponding to probes A through G used in the hybridization studies are noted. ORFs found in E. coli K-12 strain MG1655 are in black. Restriction enzyme cleavage sites: S, SalI; K, KpnI; B, BglII; E, EcoRI; H, HindIII; P, PvuII.
FIG. 2
FIG. 2
Left and right junction sequences. (A) Comparison of DNA sequences immediately upstream of the left junction of the espC pathogenicity island (see arrows in Fig. 1) with section 241 of 400 of the complete E. coli K-12 strain MG1655 (accession no. AE000351) revealed 94% identity (6). (B) Comparison of DNA sequences downstream of the right junction of the espC pathogenicity island with section 237 of 400 of the complete E. coli K-12 strain MG1655 (accession no. AE000347) revealed 96% identity with the complete genome sequence of E. coli K-12 strain MG1655 (6). Position 1 of the espC pathogenicity island begins at the left arrow.
FIG. 3
FIG. 3
PCR analysis of the left and right junctions. (A) PCR analysis identified the left junction at 60.03 min adjacent to orf360 of the E. coli chromosome in strain E2348/69 and EPEC serotype O142:H6, both members of the EPEC1 category (48). PCR with primer pair 1 (K988 and K1072) resulted in an amplicon of 584 bp using K-12 strain MG1655 DNA as the template, whereas primer pair 2 (K988 and K1071) resulted in an amplicon of 716 bp using E2349/69 DNA as the template. (B) Similarly, the right junction was at 59.35 min within the ssrA gene of the the E. coli chromosome in strain E2348/69 and EPEC serotype O142:H6. PCR with primer pair 3 (J8 and J10) amplified a K-12-specific amplicon of 458 bp, whereas primer pair 4 (J8 and K1062) amplified an E2348/69-specific amplicon of 1,416 bp.
FIG. 4
FIG. 4
Enterotoxic activity of concentrated supernatant containing EspC protein. Changes in PD (A) and Isc (B) were measured in the presence of concentrated supernatants from HB101 (control), HB101(p2R), HB101(pHC79), HB101(pJLM174) grown with arabinose (ara) or glycerol and glucose (glu), and HB101(pJLM174) preincubated with antibodies against Pet protein. Concentrated supernatant protein (100 μg) was added to the mucosal hemichambers of rat jejunum preparations. The average values of experiments performed with preparations from different animals (n = 4) are presented.
FIG. 5
FIG. 5
Immunoblot analysis for detection of EspC protein. Concentrated supernatants from HB101 (B), E2348/69 (C), HB101(p2R) (D), and HB101(pJLM174) grown in the presence of arabinose (E) or glycerol and glucose (F) were subjected to electrophoresis in SDS–10% polyacrylamide gels and transferred to nitrocellulose membranes. Detection of the EspC protein was performed by incubation of the membranes with rabbit serum against Pet protein followed by immunostaining with alkaline phosphatase-labeled polyclonal antibodies to rabbit immunoglobulins (1:2,000 dilution). Positions of the molecular size markers in lane A are indicated at the left (in kilodaltons). The arrow at the right side indicates EspC protein.

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