Detection of intimins alpha, beta, gamma, and delta, four intimin derivatives expressed by attaching and effacing microbial pathogens

Abstract

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated alpha, beta, delta, and gamma, respectively.

FIG. 1

Immunoblotting analysis of polyclonal antisera against various EPEC strains. Each sample (optical density, 0.05) was loaded onto an SDS–7.5% PAGE gel, and the bacterial cell extracts were reacted with anti-Int280-H6 (A) or anti-Int280-H2 (B) serum. Molecular size markers (in kilodaltons) are shown in lane 1. Strain E2348/69 of serotype O127:H6 (lane 2) and strains of serotypes O142:H34 (lane 7), O55:H6 (lane 9), and O142:H6 (lane 13) reacted strongly with the anti-Int280-H6 serum, while strains of serotypes O111:H⁻ (lane 4), O114:H2 (lane 5), O119:H6 (lane 6), O111:H2 (lane 8), O119:H2 (lane 11), and O128:H2 (lane 15) reacted strongly with the anti-In280-H2 serum. Weak or no reactivity was observed with both antisera with strains CVD206 (lane 3) and strains of serotypes O55:H⁻ (lane 10), O86:H34 (lane 12), and O127:H40 (lane 14).

FIG. 2

Immunogold labelling of logarithmic-phase, DMEM-grown cultures of EPEC (a to c) and EHEC (d) and immunofluorescence labelling of HEp-2 cell-adherent EPEC (e to g) and EHEC (h to l) strains showing a serotype O127:H6 EPEC strain expressing intimin α (a and e), a serotype O114:H2 EPEC strain expressing intimin β (b and f), a serotype O86:H34 EPEC strain that expresses neither intimin α nor β (c and g), a serotype O157:H7 EHEC strain expressing neither intimin α nor β (d and h), a serotype O26:H⁻ EPEC strain expressing intimin β (i), and a serotype O26:H11 EHEC strain expressing intimin β (j) but not intimin α (k). Although not stained with anti-intimin α serum, the phase-contrast micrograph of field k shows cell-adherent bacteria (l). Original magnifications: a to d, ×30,000; e to l, ×5,500.

FIG. 3

Detection of intimins α and β by PCR. Representative strains are shown. PCR products obtained with primer Int-α were obtained from E2348/69 (A, lane 2) and from all of the serotype O55:H6 strains tested (A, lanes 4-9) but with none of the serotype O111:H2 strains (B, lanes 2 to 9) or strain CVD206 (A, lane 3). All of the serotype O119:H2 (C, lanes 2 and 4 to 7) and O119:H6 (D, lanes 2 to 7) strains tested, but not E2348/69 (C, lane 3), produced a positive PCR product with the Int-β primer. Molecular size markers (1-kb ladder; Bethesda Research Laboratories) were loaded in lanes 1. The complete DNA analysis is presented in Table 2.