Heat-resistant agglutinin 1 (Hra1) is an accessory colonization factor of enteroaggregative Escherichia coli (EAEC) strain 042. Tia, a close homolog of Hra1, is an invasin and adhesin that has been described in enterotoxigenic E. coli. We devised a PCR-restriction fragment length polymorphism screen for the associated genes and found that they occur among 55 (36.7%) of the enteroaggregative E. coli isolates screened, as well as lower proportions of enterotoxigenic, enteropathogenic, enterohemorrhagic, and commensal E. coli isolates. Overall, 25%, 8%, and 3% of 150 EAEC strains harbored hra1 alone, tia alone, or both genes, respectively. One EAEC isolate, 60A, produced an amplicon with a unique restriction profile, distinct from those of hra1 and tia. We cloned and sequenced the full-length agglutinin gene from strain 60A and have designated it hra2. The hra2 gene was not detected in any of 257 diarrheagenic E. coli isolates in our collection but is present in the genome of Salmonella enterica serovar Heidelberg strain SL476. The cloned hra2 gene from strain 60A, which encodes a predicted amino acid sequence that is 64% identical to that of Hra1 and 68% identical to that of Tia, was sufficient to confer adherence on E. coli K-12. We constructed an hra2 deletion mutant of EAEC strain 60A. The mutant was deficient in adherence but not autoaggregation or invasion, pointing to a functional distinction from the autoagglutinin Hra1 and the Tia invasin. Hra1, Tia, and the novel accessory adhesin Hra2 are members of a family of integral outer membrane proteins that confer different colonization-associated phenotypes.Enteroaggregative Escherichia coli (EAEC) strains are increasingly implicated in human diarrhea, especially among children living in developing countries (18,33). EAEC strains are exceptional colonizers and are defined by a characteristic stacked-brick adherence pattern (43). This aggregative pattern of adherence is a convergent phenotype produced in different lineages by a variety of adhesins, only some of which have been described (35). To date, most studies of EAEC adherence have focused on structural adhesins known as aggregative adherence fimbriae. However, recent research has shown that EAEC strains also harbor an expanding repertoire of nonstructural outer membrane proteins that contribute to colonization (1,4,14,28).One such outer membrane protein is heat-resistant agglutinin 1 (Hra1), an accessory adhesin that we recently characterized in EAEC strain 042 (1). The hra1 gene (along with its 90% identical allelic variant hek, reported from uropathogenic E. coli and neonatal meningitic E. coli [11,39]) is predicted to encode a 29-kDa precursor, which is processed to a 25-kDa outer membrane protein. The 792-bp hra1 gene is sufficient to confer agglutination of human erythrocytes, bacterial autoaggregation, enhanced biofilm formation, and aggregative adherence to cultured HEp-2 cells (1, 25). Hra1 shares 67% identity with the previously characterized outer membrane invasin and adhesin Tia (12, 13). The tia...
