Identification of a two-partner secretion locus of enterotoxigenic Escherichia coli.
Enterotoxigenic Escherichia coli (ETEC) remains a formidable cause of diarrheal illness worldwide. At present, there is no vaccine that provides broad-based protection against ETEC. A 'phoA-based self-cloning mutagenesis system, TnphoA.ts, employed to identify novel ETEC surface antigens, led to identification of an ETEC two-partner secretion locus (etpBAC) on the pCS1 virulence plasmid of prototype strain H10407. Cloning and expression of etpBAC in recombinant E. coli LMG194(pJY019) resulted in secretion of a high-molecular-weight (HMW) glycosylated exoprotein. This glycoprotein, EtpA, exhibits linear peptide sequence and predicted structural homologies with known HMW adhesins produced by other two-partner secretion loci. Antibodies directed against recombinant EtpA (anti-rEtpA.6H) recognized an HMW protein in culture supernatants of ETEC strains H10407 and LMG194(pJY019) but not in culture supernatant of strain H10407-P, which lacks the 92-kb pCS1 plasmid, or an isogenic etpA mutant. etpA mutants were deficient in adherence to intestinal epithelial cells in vitro, and anti-rEtpA.6H antibodies inhibited association of H10407 with target epithelial cells. Cloning and expression of etpB in recombinant E. coli were sufficient to confer adherence. Screening of multiple ETEC isolates for the etpBAC locus by colony hybridization and by EtpA immunoblotting suggested that EtpA is one of the most common antigens secreted by these pathogens. Together, these results indicate that the newly identified ETEC two-partner secretion locus directs the secretion of a high-molecular-weight glycosylated protein, EtpA, that in concert with the putative EtpB transporter participates in adherence of H10407 to epithelial cells, thereby expanding the repertoire of potential ETEC virulence proteins and vaccine candidates.
Medicine Service (151), Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104, USA. jflecke1@tennessee.edu
James M
JM
Fleckenstein
Koushik
K
Roy
Julia F
JF
Fischer
Michael
M
Burkitt
D018830:Adhesins, Escherichia coli / Q000096:biosynthesis / Q000235:genetics; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics*; D001422:Bacterial Adhesion / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D018938:Caco-2 Cells; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D005819:Genetic Markers; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism / Q000382:microbiology; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D037521:Virulence Factors / Q000096:biosynthesis / Q000235:genetics
D018830:Adhesins, Escherichia coli; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C510011:EtpA protein, E coli; C510012:EtpB protein, E coli; C510013:EtpC protein, E coli; D005819:Genetic Markers; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D037521:Virulence Factors