Polymorphisms within EspA filaments of enteropathogenic and enterohemorrhagic Escherichia coli

Abstract

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) possess a filamentous type III secretion system (TTSS) employed to deliver effector proteins into host cells. EspA is a type III secreted protein which forms the filamentous extension to the TTSS and which interacts with host cells during early stages of attaching and effacing (A/E) lesion formation. By immunofluorescence, a polyclonal antibody previously raised to EspA from EPEC strain E2348/69 (O127:H6) stained approximately 12-nm-diameter EspA filaments produced by this strain but did not stain similar filaments produced by EHEC serotype O157:H7. Similarly, an antibody that we subsequently raised to EHEC strain 85-170 (O157:H7) EspA stained approximately 12-nm-diameter EspA filaments produced by strain 85-170 but did not stain E2348/69 EspA filaments. Given such heterogeneity between EPEC and EHEC EspA filaments, we examined polymorphisms of functional EspA filaments among different EPEC and EHEC serotypes. With use of the EPEC EspA antiserum, EspA filaments were observed only with EPEC serotypes O127:H6 and O55:H6, serotypes which encode an identical EspA protein. When stained with the EHEC EspA antiserum, EspA filaments were detected only on EHEC strains belonging to serotype O157:H7; the EHEC antiserum did, however, stain EspA filaments produced by the closely related EPEC serotype O55:H7 but not filaments of any other EPEC serotype tested. Such polymorphisms among functional EspA filaments of EPEC and EHEC would be expected to have important implications for the development of broad-range EspA-based vaccines.

FIG. 1.

Immunofluorescence staining of EspA filaments with EPEC EspA_E2348/69 (column 1) and EHEC EspA_85-170 antisera (column 2); HEp-2 cells were infected with EPEC and EHEC strains for 3 h, and bacteria were visualized by phase contrast. The EPEC antibody stained EspA filaments produced by EPEC E2348/69 (O127:H6) (row 1) and EPEC O55:H6 (row 3) while the EHEC antibody stained EspA filaments produced by EHEC 85-170 (O157:H7) (row 2) and EPEC O55:H7 (row 4). A positive FAS assay (column 3) indicated that all four strains produced A/E lesions on HEp-2 cells.

FIG. 2.

Scanning electron micrographs showing attachment of EPEC strains to RBC monolayers. Filamentous structures which did not react with the EPEC or EHEC EspA antisera but which were morphologically identical to EPEC E2348/69 EspA filaments identified by immunogold labeling (a) were seen to promote attachment of EHEC strain O26:H11 (b) and EPEC strain O119:H6 (c) to RBC membranes. Bar, 0.1 μm; inset bar, 0.01 μm.