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

• DOI: 10.1128/iai.70.10.5416-5427.2002
• Publication Date: 2002-09-12T20:28:25Z
• Authors: Alfredo G. Torres; Jorge A. Giron; Nicole T. Perna; Valerie Burland; Fred R. Blattner; Fabiola Avelino-F...; James B. Kaper
• Keywords: DNA, Bacterial; 0301 basic medicine; Base Sequence; Gene Expression; Cross Reactions; Escherichia coli O157; Antibodies, Bacterial; Bacterial Adhesion; Enteritis; Cell Line; 3. Good health; Microscopy, Electron; 03 medical and health sciences; Genes, Bacterial; Fimbriae, Bacterial; Operon; Animals; Humans; Cloning, Molecular; Escherichia coli Infections; HeLa Cells

ABSTRACT

The mechanisms underlying the adherence of Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) strains to intestinal epithelial cells are poorly understood. We have identified a chromosomal region (designated lpfABCC ′ DE ) in EHEC O157:H7 containing six putative open reading frames that was found to be closely related to the long polar (LP) fimbria operon ( lpf ) of Salmonella enterica serovar Typhimurium, both in gene order and in conservation of the deduced amino acid sequences. We show that lpfABCC ′ DE is organized as an operon and that its expression is induced during the exponential growth phase. The lpf genes from EHEC strain EDL933 were introduced into a nonfimbriated (Fim − ) E. coli K-12 strain, and the transformed strain produced fimbriae as visualized by electron microscopy and adhered to tissue culture cells. Anti-LpfA antiserum recognized a ca. 16-kDa LpfA protein when expressed under regulation of the T7 promoter system. The antiserum also cross-reacted with the LP fimbriae in immunogold electron microscopy and Western blot experiments. Isogenic E. coli O157:H7 lpf mutants derived from strains 86-24 and AGT300 showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared with their wild-type parent strains. The adherence and microcolony formation phenotypes were restored when the lpf operon was introduced on a plasmid. We propose that LP fimbriae participate in the interaction of E. coli O157:H7 with eukaryotic cells by assisting in microcolony formation.