Identification of IngA, the structural gene of longus type IV pilus of enterotoxigenic Escherichia coli

Human enterotoxigenic Escherichia coli (ETEC) produces a type IV pilus termed longus which is encoded on large plasmids in association with colonization factor antigens (CFAs) and enterotoxins. A plasmid-derived 7 kbp BamHI DNA fragment hybridizing with an oligonucleotide probe designed from the aminoterminal amino acid sequence of the denatured 22 kDa structural longus pilin subunit was subcloned and sequenced. DNA sequencing analysis revealed an open reading frame, designated IngA, whose predicted amino acid sequence matched perfectly the N-terminal sequence of LngA obtained by Edman degradation. IngA is the first gene described of the longus gene cluster. Cloned IngA encoded and expressed a prepilin protein of 236 residues with a calculated mass of 25.17 kDa. The prepilin is apparently processed into a mature pilin of 206 residues with a calculated mass of 21.5 kDa. The predicted peptide sequence of IngA showed 78.8 and 37% identity to CFA/III pilin (CofA) of ETEC and the toxin-coregulated pilus (TcpA) of Vibrio cholerae. Peptide sequence homology between IngA and cofA was more prominent towards the amino terminus than within the carboxy region. Like other type IV pilins, LngA contains two cysteine residues towards the carboxy-terminal region. Transmission electron microscopy and immunoblot analysis of ETEC strains expressing either longus or CFA/III detected antigenic differences between native and denatured epitopes of these pili. In addition, differential regulation of pilus expression was identified when ETEC strains were grown in different media. Our data indicate that longus and CPA/III are two distinct but yet highly related type IV pili of ETEC.