Human response to Escherichia coli O157:H7 infection: antibodies to secreted virulence factors

Abstract

Vaccination has been proposed for the prevention of disease due to enterohemorrhagic Escherichia coli (EHEC), but the immune response following human infection, including the choice of potential antigens, has not been well characterized. To study this, sera were obtained from five pediatric patients with acute diarrhea caused by E. coli O157:H7 0, 8, and 60 days after hospitalization. These sera were used to examine the immune response to four different EHEC virulence factors: Tir (translocated intimin receptor, which is inserted into the host cell membrane), intimin (bacterial outer membrane protein which binds to Tir), EspA (secreted protein which forms filamentous structures on EHEC surface), and EspB (inserted into the host membrane and cytoplasm). The response to O157:H7 lipopolysaccharide was also examined. Sera were assayed against purified recombinant proteins using immunoblot analysis and by enzyme-linked immunosorbent assay to determine the sera's titers to each of the antigens in all patients. We found that there was little reaction to EspA, EspB, and intimin in the acute-phase sera, although there was some reactivity to Tir. By day 8, titers of antibody to all four virulence factors were present in all patients, with a very strong response against Tir (up to a titer of 1:256,000), especially in hemolytic-uremic syndrome patients, and lesser strong responses to the other three antigens. The titer to the antigens 60 days after hospitalization was decreased but was still highest for Tir. These results suggest that there is a strong immune response to Tir, and to a lesser extent to the other three virulence factors, following EHEC disease, indicating that these bacterial molecules are potential vaccine candidates for preventing EHEC disease. They also suggest that bacterial virulence factors that are inserted into host cells during infection by type III secretion systems (Tir or EspB) are still recognized by the host immune response.

FIG. 1

Immunoblot analysis of patient serum reactivity against EHEC secreted proteins. (A) Secreted proteins from wild-type EHEC O157:H7 and the type III secretion mutant, SepB. (B) Immunoreactivity of sera from patient 95-8001 against secreted proteins from O157:H7 or SepB at days 0, 8, and 60 posthospitalization. (C) Immunoblots showing reactivities of patients' sera at day 8 posthospitalization. The patient identification number is indicated beneath each blot. Culture supernatants were concentrated by trichloroacetic acid precipitation, resolved by SDS–12% PAGE, and either stained with Coomassie blue (A) or transferred to nitrocellulose and probed with patients' sera (B and C). Molecular mass markers (in kilodaltons) are shown to the left of each panel.

FIG. 2

Immunoblot analysis of patient serum reactivity against recombinant EHEC virulence proteins. (A) Purified GST fusions to EHEC virulence proteins separated by PAGE and stained with Coomassie blue. (B) Immunoreactivity of serum from patient 95-8001 against GST-EHEC fusion proteins at days 0, 8, and 60 posthospitalization. (C) Immunoreactivity of patients' sera at 8 days posthospitalization. GST and GST-EHEC fusion proteins were purified as described in Materials and Methods and resolved by SDS–12% PAGE, followed by either staining with Coomassie blue (A) or immunoblotting with patients' sera (B and C). Molecular mass markers (in kilodaltons) are shown to the left of each panel.

FIG. 3

Individual patient antibody response to EHEC antigens (y axis) at days 0, 8, and 60 posthospitalization (x axis). ELISAs were performed to detect the recombinant GST fusion proteins, and titers of the following were determined: Tir (squares), intimin (diamonds), EspA (circles), and EspB (triangles). The figure shows data from one representative experiment, and experiments were repeated at least three times.