MULTIPLE DETERMINANTS OF VEROTOXIN-PRODUCING ESCHERICHIA-COLI O157-H7ATTACHMENT-EFFACEMENT

Verotoxin-producing Escherichia coli strains of the serotype 0157:H7 b elong to a class of gastrointestinal pathogens that adhere to epitheli al cells in a characteristic pattern known as attaching and effacing. Recent insight into the nature of E. coli 0157:H7 adhesion was provide d by the cloning and sequencing of the chromosomal eaeA (for E. coli a ttaching and effacing) gene homolog (G. Beebakhee, M. Louie, J. De Aza vedo, and J. Brunton, FEMS Microbiol. Lett. 91:63-68, 1992, and J. Yu and J. B. Kaper, Mol. Microbiol. 6:411-417, 1992) and isolation of a 6 0-MDa plasmid referred to as pO157 (I. Toth, M. L. Cohen, H. S. Rumsch lag, L. W. Riley, E. H. White, J. H. Carr, W. W. Bond, and 1. K. Wachs muth, Infect. Immun. 58:1223-1231, 1990, and S. Tzipori, H. Karch, K. 1. Wachsmuth, R. M. Robins-Browne, A. D. O'Brien, H. Lior, M. L. Cohen , J. Smithers, and M. M. Levine, Infect. Immun. 55:3117-3125, 1987) an d an approximately 94-kDa outer membrane protein (94-kDa OMP; P. Sherm an, F. Cockerill III, R. Soni, and J. Brunton, Infect. Immun. 59:890-8 99, 1991). In this study, we examined the gene products of both eaeA a nd pO157 in relation to the 94-kDa OMP and as candidate effectors for 0157:H7 attachment-effacement. Peptide sequencing and immunoassay demo nstrated that the E. coli 0157:H7 eaeA gene product is distinct from t he 94-kDa OMP. Using ultrastructural analyses, we found that both pare nt and pO157 plasmid-cured 0157:H7 strains demonstrated attaching and effacing adhesion to host epithelial cells and reacted equally well to rabbit antiserum raised against the 94-kDa OMP. By, both transmission electron microscopy and light microscopy, E. coli HB101 transformed s eparately with the cloned eaeA gene and the pO157 plasmid did not form attaching and effacing lesions on cultured epithelial cells in vitro and rabbit intestinal tissues in vivo. Since additional determinants m ay mediate the attaching and effacing phenotype, we examined transposo n TnphoA mutants constructed from E. coli 0157:H7 strain CL8. Two Tnph oA mutants were found deficient in bacterial factors that are necessar y for 0157:H7 attachment-effacement and likely distinct from the eaeA gene product.