Identification of lngA, 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 amino-terminal 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 lngA, whose predicted amino acid sequence matched perfectly the N-terminal sequence of LngA obtained by Edman degradation. lngA is the first gene described of the longus gene cluster. Cloned lngA 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 lngA 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 lngA 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 CFA/III are two distinct but yet highly related type IV pili of ETEC.
Gómez-Duarte
Oscar G
OG
Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore St, Baltimore, MD 21201, USA.
Ruiz-Tagle
Alejandro
A
Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla, Apartado Postal 1622, Puebla, Pue., Mexico.
Gómez
Diana C
DC
Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore St, Baltimore, MD 21201, USA.
Viboud
Gloria I
GI
Instituto Nacional de Microbiología Dr. Carlos G. Malbrán, Ave Vélez Sarsfields 563 CAP.C.P. 1,281, Buenos Aires, Argentina.
Jarvis
Karen G
KG
Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore St, Baltimore, MD 21201, USA.
Kaper
James B
JB
Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore St, Baltimore, MD 21201, USA.
Girón
Jorge A
JA
Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla, Apartado Postal 1622, Puebla, Pue., Mexico.
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000378:metabolism; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D015151:Immunoblotting; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C121921:LngA protein, E coli; C085807:TcpA protein, Vibrio cholerae; C022924:colonization factor antigens; D036781:Fimbriae Proteins