The gaf fimbrial gene cluster of Escherichia coli expresses a full-size and a truncated soluble adhesin protein

The GafD lectin of the G (F17) fimbriae of diarrhea-associated Escherichia coli was overexpressed and purified-from the periplasm of E. coli by affini ty chromatography on GlcNAc-agarose. The predicted mature GafD peptide comp rises 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 amin o acid residues, as judged by mass spectrometry and: amino acid sequencing, and was named Delta GafD. Expression of gafD from the cloned gaf gene clus ter in DegP-, Lon-, and OmpT-deficient recombinant strains did not signific antly decrease the formation of Delta GafD. The peptide was also detected i n the periplasm of the wild-type E. coil strain from which the gaf gene clu ster originally was cloned. We expressed gafD fragments encoding C-terminal ly truncated peptides, Peptides GafD1-252, GafD1-224, GafD1-189, and the Ga fD1-178, isolated from the periplasm by affinity chromatography, had appare nt sizes closely similar to that of Delta GafD. Only trace amounts of trunc ated forms with expected molecular sizes were detected in spheroplasts. In contrast, the shorter GafD1-157 peptide was detected in spheroplasts but no t in the periplasm, indicating that it was poorly translocated or was degra ded by periplasmic proteases, Pulse-chase assays using gafD indicated that Delta GafD was processed from GafD and is not a primary translation product . The Delta GafD peptide was soluble by biochemical criteria and exhibited specific binding to GlcNAc-agarose, Inhibition assays with mono- and oligos accharides gave a similar inhibition pattern in the hemagglutination by the G-fimbria-expressing recombinant E. coli strain and in the binding of [C-1 4]Delta GafB to GlcNAc-agarose, Delta GafD) bound specifically to laminin, a previously described tissue target for the G fimbria. Our results show th at a soluble, protease-resistant subdomain of GafD exhibits receptor-bindin g specificity similar to that for intact G fimbriae and that it is formed w hen gafD is expressed alone or from the gaf gene cluster.