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 term ed longus which is encoded on large plasmids in association with colonizati on factor antigens (CFAs) and enterotoxins. A plasmid-derived 7 kbp BamHI D NA fragment hybridizing with an oligonucleotide probe designed from the ami noterminal amino acid sequence of the denatured 22 kDa structural longus pi lin subunit was subcloned and sequenced. DNA sequencing analysis revealed a n open reading frame, designated IngA, whose predicted amino acid sequence matched perfectly the N-terminal sequence of LngA obtained by Edman degrada tion. IngA is the first gene described of the longus gene cluster. Cloned I ngA encoded and expressed a prepilin protein of 236 residues with a calcula ted mass of 25.17 kDa. The prepilin is apparently processed into a mature p ilin of 206 residues with a calculated mass of 21.5 kDa. The predicted pept ide sequence of IngA showed 78.8 and 37% identity to CFA/III pilin (CofA) o f ETEC and the toxin-coregulated pilus (TcpA) of Vibrio cholerae. Peptide s equence homology between IngA and cofA was more prominent towards the amino terminus than within the carboxy region. Like other type IV pilins, LngA c ontains two cysteine residues towards the carboxy-terminal region. Transmis sion electron microscopy and immunoblot analysis of ETEC strains expressing either longus or CFA/III detected antigenic differences between native and denatured epitopes of these pill. In addition, differential regulation of pilus expression was identified when ETEC strains were grown in different m edia. Our data indicate that longus and CFA/III are two distinct but yet hi ghly related type IV pill of ETEC.