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.

FIG. 1

Purification of GafD from E. coli BL21 λDE3(pKJ1) periplasm by affinity chromatography and analysis of the reactivities of fimbrial preparations with anti-G-fimbria and anti-GafD antibodies. (A) Binding of ¹⁴C-labeled GafD to GlcNAc-agarose. Lane 1, pulse-labeled periplasmic peptides from rifampin-treated E. coli BL21 λDE3(pKJ1); lane 2, peptides not bound to GlcNAc-agarose; lane 3, peptides bound to GlcNAc-agarose. (B) Coomassie blue-stained SDS-PAGE gel of GafD peptides eluted with 5% (wt/vol) GlcNAc from the GlcNAc-agarose column. The peptides were prepared from periplasm of nonlabeled E. coli BL21 λDE3(pKJ1) (lane 1) and E. coli BL21 λDE3 expressing the truncated GafD1-178 (lane 2). (C) Western blot of fimbrial preparations. Lanes 1 to 3, reactivities of fimbrial peptides with anti-G-fimbria antibodies; lanes 4 to 6, reactivities with anti-GafD antibodies. The fimbrial preparations were the G fimbria isolated from E. coli HB101(pRR-5) (lanes 1 and 4), the mutated G fimbria without lectin activity isolated from E. coli HB101(pHUB110) (lanes 2 and 5), and the type 1 fimbria from E. coli EH826 (lanes 3 and 6). The migration distances of molecular weight markers are indicated on the left; those of the GafD and the GafA peptides are indicated on the right.

FIG. 2

Expression of gafD peptides in rifampin-treated pulse-labeled E. coli cells and in the periplasm of G fimbria-expressing E. coli strains. (A) Autoradiographic analysis of ¹⁴C-labeled peptides of E. coli BL21 λDE3(pKJ1) immediately after addition of ¹⁴C-amino acids (lane 1) and 1 h 30 min later (lane 2). (B) Western blotting with anti-GafD antibodies of periplasmic peptides prepared from recombinant E. coli strains BL21 λDE3(pRR-5), KS474(pRR-5), IHE11088(pRR-5), BL21 λDE3(pKJ1) (lanes 1 to 4, respectively) and from wild-type E. coli strain IHE11165 (lane 5). Plasmid pRR-5 contains the entire gaf gene cluster, and pKJ1 encodes GafD1-321. The migration distance of the ΔGafD peptide is indicated on the right.

FIG. 3

Expression in E. coli BL21 λDE3 of gafD constructs. (A) Schematic presentation of GafD and the truncated GafD peptides and their recovery from the periplasm by affinity chromatography. Black arrow, position of the cleavage site in ΔGafD; ΔGT, position of the in-frame deletion abolishing binding activity. (B) Autoradiographic analysis of pulse-labeled peptides in the whole cells (C; lanes 1, 4, 7, and 10), spheroplasts (S; lanes 2, 5, 8, and 11), and the periplasm (P; lanes 3, 6, 9, and 12) of E. coli BL21 λDE3 expressing gafD constructs. The DNA fragments encoded full-length GafD1-321 (lanes 1 through 3) and the truncated forms GafD1-157 (lanes 4 through 6), GafD1-252 (lanes 7 through 9), and GafD1-178 (lanes 10 through 12). The migration distances of molecular weight markers are indicated on the left; those of the GafD and the ΔGafD peptides are indicated on the right. Open arrow, migration distance of β-lactamase.

FIG. 4

Binding of ΔGafD to proteins immobilized on microtiter plates. (A) Binding to immobilized laminin (●) and BSA (▴). (B) Binding to immobilized laminin in the absence of carbohydrates (bar 1), in the presence of 50 mM GalNAc (bar 2), and in the presence of 50 mM GlcNAc (bar 3).