title: The gaf fimbrial gene cluster of Escherichia coli expresses a full-size and a truncated soluble adhesin protein.
abstract: The GafD lectin of the G (F17) fimbriae of diarrhea-associated Escherichia coli was overexpressed and purified from the periplasm of E. coli by affinity chromatography on GlcNAc-agarose. The predicted mature GafD peptide comprises 321 amino acids, but the predominant form of GafD recovered from the periplasm was 19,092 Da in size and corresponded to the 178 N-terminal amino acid residues, as judged by mass spectrometry and amino acid sequencing, and was named DeltaGafD. Expression of gafD from the cloned gaf gene cluster in DegP-, Lon-, and OmpT-deficient recombinant strains did not significantly decrease the formation of DeltaGafD. The peptide was also detected in the periplasm of the wild-type E. coli strain from which the gaf gene cluster originally was cloned. We expressed gafD fragments encoding C-terminally truncated peptides. Peptides GafD1-252, GafD1-224, GafD1-189, and the GafD1-178, isolated from the periplasm by affinity chromatography, had apparent sizes closely similar to that of DeltaGafD. Only trace amounts of truncated forms with expected molecular sizes were detected in spheroplasts. In contrast, the shorter GafD1-157 peptide was detected in spheroplasts but not in the periplasm, indicating that it was poorly translocated or was degraded by periplasmic proteases. Pulse-chase assays using gafD indicated that DeltaGafD was processed from GafD and is not a primary translation product. The DeltaGafD peptide was soluble by biochemical criteria and exhibited specific binding to GlcNAc-agarose. Inhibition assays with mono- and oligosaccharides gave a similar inhibition pattern in the hemagglutination by the G-fimbria-expressing recombinant E. coli strain and in the binding of [(14)C]DeltaGafD to GlcNAc-agarose. DeltaGafD bound specifically to laminin, a previously described tissue target for the G fimbria. Our results show that a soluble, protease-resistant subdomain of GafD exhibits receptor-binding specificity similar to that for intact G fimbriae and that it is formed when gafD is expressed alone or from the gaf gene cluster.
authors.affiliation: Division of General Microbiology, Department of Biosciences, FIN-00014 University of Helsinki, Finland.
authors.forename: J
authors.initials: J
authors.lastname: Tanskanen
authors.forename: S
authors.initials: S
authors.lastname: Saarela
authors.forename: S
authors.initials: S
authors.lastname: Tankka
authors.forename: N
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: M
authors.initials: M
authors.lastname: Rhen
authors.forename: T K
authors.initials: TK
authors.lastname: Korhonen
authors.forename: B
authors.initials: B
authors.lastname: Westerlund-Wikström
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D018829:Adhesins, Bacterial / Q000235:genetics* / Q000276:immunology; D000907:Antibodies, Bacterial; D000918:Antibody Specificity; D002451:Cell Compartmentation; D004926:Escherichia coli / Q000235:genetics* / Q000472:pathogenicity; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial; D037102:Lectins / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D005810:Multigene Family; D010446:Peptide Fragments; D019897:Periplasm; D011993:Recombinant Fusion Proteins; D012995:Solubility; D013104:Spheroplasts; D014774:Virulence / Q000235:genetics
chemical_list: D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D029968:Escherichia coli Proteins; C092493:GafD protein, E coli; D037102:Lectins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D036781:Fimbriae Proteins; D000117:Acetylglucosamine