Identification and Characterization of lpfABCC ′ DE , a Fimbrial Operon of Enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is recognized as a significant enteric pathogen that has been implicated in numerous outbreaks worldwide (reviewed in reference 24). This organism colonizes the intestine and can cause bloody or nonbloody diarrhea and hemolytic uremic syndrome. A potent cytotoxin, Shiga toxin (Stx), is the best-characterized virulence factor, but many aspects of the pathogenesis of the disease associated with E. coli O157:H7 are poorly characterized. In particular, the mechanisms underlying the adherence of EHEC to intestinal epithelial cells are not well understood (24, 27). Colonization of the gastrointestinal tract, which is presumably mediated by specific adherence factors, is a key aspect of enteric infection caused by EHEC. Several potential virulence determinants of EHEC have been described, but the only adherence factor that has been demonstrated to play a role in intestinal colonization in vivo in an animal model is the outer membrane protein intimin (8, 22, 43). Most EHEC and all enteropathogenic E. coli (EPEC) strains produce this adhesin (17). Intimin, encoded by the eae gene, is located within the locus for enterocyte effacement (LEE) pathogenicity island, which is required for the classic attaching and effacing intestinal lesion produced by these organisms (14, 15, 21). The presence of a second adherence factor has been described in EPEC but not in EHEC strains. The type IV bundle-forming pilus (BFP) encoded by the plasmid of EPEC strains is involved in bacterium-to-bacterium adherence in the localized adherence pattern (4, 12) and potentially in direct interaction with the host epithelial cells (41, 42). Neither BFP nor analogous adhesins have been described in EHEC, but the existence of intestinal adherence factors distinct from intimin is suggested by the isolation of human EHEC strains of serotypes other than O157:H7 that lack the eae gene but are still associated with bloody diarrhea or hemolytic uremic syndrome (10). Several research groups have explored this hypothesis and proposed potential novel adhesin factors. Tarr et al. (37) identified Iha (Vibrio cholerae IrgA-homologue adhesin) in E. coli O157:H7. This protein was associated with adherence to HeLa cells while expressed in a nonfimbriated E. coli strain, but no difference in adherence was observed with an iha mutant of O157:H7. Nicholls et al. (25) characterized a chromosomal genetic locus termed efa1 (EHEC factor for adherence) in an EHEC strain serotype O111:H− and observed that this locus is associated with high levels of adherence to cultured Chinese hamster ovary (CHO) cells. They demonstrated that the efa1 locus was necessary for the in vitro adhesion to CHO cells and that the efa1 isogenic mutant strain lost its ability to adhere and also was defective in its hemagglutination and autoaggregation phenotypes. Tatsuno et al. (39) performed a transposon mutagenesis in the EHEC O157:H7 strain (O157Sakai), and the insertion mutants were screened for their ability to adhere to Caco-2 cells. Almost half of the insertion mutants were found within the LEE pathogenicity island, while the other half were mapped to open reading frames (ORFs) with unknown functions or with functions not directly associated with adherence. Their results suggested that EHEC might contain additional adherence-associated loci which are not contained within the LEE pathogenicity island. Recently, Brunder et al. (6) characterized a gene cluster in the large plasmid of a sorbitol-fermenting EHEC O157:H− strain which is required for the expression of fimbriae with homology to the P fimbriae of uropathogenic E. coli. The Sfp (for sorbitol-fermenting EHEC O157 fimbriae, plasmid encoded) fimbriae mediate mannose-resistant hemagglutination, but this sfp gene cluster is restricted to sorbitol-fermenting EHEC O157:H− strains and is absent in other EHEC serotypes, including O157:H7. In this work, we characterized a chromosomal fimbrial operon of E. coli O157:H7. Sequence analysis indicated that this operon showed high similarity to the long polar (LP) fimbria (lpf) operon of Salmonella enterica serovar Typhimurium and to a lesser degree to other well-characterized fimbrial operons. Introduction of the EHEC lpf operon into a nonfimbriated E. coli K-12 strain resulted in the expression of fimbriae and increased adhesion to tissue culture cells. We also provide evidence suggesting that LP fimbriae participate in the adherence of E. coli O157:H7 to eukaryotic cells and play a role in microcolony formation.