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Infection and Immunity, September 2003, p. 4985-4995, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.4985-4995.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Multiple Elements Controlling Adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells

Alfredo G. Torres and James B. Kaper^*

Center for Vaccine Development and Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received 21 January 2003/ Returned for modification 27 March 2003/ Accepted 6 June 2003

Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

Editor: A. D. O'Brien

Present address: Department of Microbiology and Immunology and Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555.

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