A new fimbrial putative colonization factor, PCFO20, in human enterotoxigenic Escherichia coli

Abstract

The ability to colonize the small intestine is essential for enterotoxigenic Escherichia coli (ETEC) to cause diarrhea. Several colonization factor antigens (CFAs) and putative colonization factors (PCFs) have been described for ETEC. However, there are still many ETEC strains isolated from patients with diarrhea which do not possess any of these antigens. To identify CFAs in ETEC lacking the above-mentioned antigens, we exploited the ability of ETEC to adhere to tissue-cultured cells from an enterocyte-like cell line, Caco-2. An ETEC strain producing heat-labile toxin and heat-stable toxin of serotype O20:K27:H- (ARG-2) that was isolated from a child with diarrhea in Argentina and bound to Caco-2 cells was studied in further detail. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of this strain revealed a band of 25 kDa when bacteria were grown at 37 degrees C that was missing when the same strain was cultured at 20 degrees C. Furthermore, electron microscopy examination revealed the presence of fimbriae on the surfaces of cells of this strain when cells were grown at 37 degrees C but not at 20 degrees C. Rabbit antiserum raised against purified fimbriae reacted with the 25-kDa protein in immunoblotting and bound specifically to the fimbriae, as shown by immunoelectron microscopy. The presence of fimbriae, adhesion to Caco-2 cells, and the 25-kDa band seen in the SDS-PAGE were all simultaneously lost by single-insertion mutations. The N-terminal amino acid sequence of the protein subunit of the fimbriae showed no relation with those of the known colonization factors of ETEC. Furthermore, the fimbriae of the ARG-2 strain did not cross-react immunologically with any of the previously described adhesive factors in human ETEC when specific antisera against colonization factor antigens and putative colonization factors were used. Moreover, a specific antiserum raised against the fimbriae in ARG-2 did not react with ETEC carrying known colonization factors. We propose to name these new fimbriae PCFO20.