Abstract
The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome encodes 12 intestinal colonization factors of the chaperone/usher fimbrial assembly class; however, the binding specificity is known for only one of these adhesins, known as type 1 fimbriae. Here we explored the utility of glycomics to determine the carbohydrate binding specificity of plasmid-encoded fimbriae from S. Typhimurium. A cosmid carrying the pef operon was introduced into Escherichia coli and expression of fimbrial filaments composed of PefA confirmed by flow cytometry and immune-electron microscopy. Plasmid-encoded fimbriae were purified from the surface of E. coli, and the resulting preparation was shown to contain PefA as the sole major protein component. The binding of purified plasmid-encoded fimbriae to a glycanarray suggested that this adhesin specifically binds the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc, also known as the Lewis X (Le(x)) blood group antigen. Results from the glycanarray were validated by enzyme-linked immunosorbent assay (ELISA) in which plasmid-encoded fimbriae bound Le(x)-coated wells in a concentration-dependent manner. The binding of plasmid-encoded fimbriae to Le(x)-coated wells could be inhibited by co-incubation with soluble Le(x) antigen. Our results establish glycomic analysis as a promising new approach for determining the carbohydrate binding specificity of bacterial adhesins.
Highlights
Expression of plasmid-encoded fimbriae is regulated by phase variation [14], which provides a plausible explanation as to why expression of PefA was only detected in a fraction of cells in the population
The thin flexible fibrillae expressed by ORN172(pFB11) could be labeled with rabbit anti-PefA serum and goat anti-rabbit 10-nm gold conjugate (Fig. 1D), but no labeling was observed with the ORN172-negative control (Fig. 1E)
These data suggested binding to the Gal1– 4(Fuc␣1–3)Gal 3Ј-su-LacNAc 6-O-su-LacNAc T␣␣ TF 3Ј-su-Lec (GlcNAc) moiety, known as the Lewis X (Lex) blood group antigen, because various carbohydrates present on the glycanarray were conjugated to biotin via the same linker as Lex (-O(CH2)2NH(O)(CH2)5NHC(O)(CH2)5NHC(O)-)
Summary
Bacterial Strains and Growth Conditions—E. coli strains BL21(DE3) and TOP10 were obtained from Stratagene and Invitrogen, respectively. The E. coli strain ORN172 carries a ⌬fimBEACDFGH::Km allele and is phenotypically non-fimbriate [16]. Bacteria were routinely grown at 37 °C in Luria Bertani (LB) broth (with shaking) or on LB agar plates. Bacteria were grown statically in LB broth at 37 °C overnight. Generation of Anti-PefD Serum—A DNA region encoding an internal portion of PefD was amplified with primers: 5Ј-CGGGATCCGGCACGCGTTTTATCTATGAGGAAGG-3Ј and 5ЈGGAATTCTCACTTCAGCGTGTAGTCCTGGGTG-3Ј and cloned into the plasmid vector pGEX4T-2 [17](Amersham Biosciences) to generate pCWD41, which encodes a PefD-GST fusion protein. The PefD-GST fusion protein was purified from E. coli strain BL21(DE3) using affinity chromatography as described previously [11].
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