Discovery of Bacterial Fimbria-Glycan Interactions Using Whole-Cell Recombinant Escherichia coli Expression

Abstract

Chaperone-usher (CU) fimbriae are the most abundant Gram-negative bacterial fimbriae, with 38 distinct CU fimbria types described in Escherichia coli alone. Some E. coli CU fimbriae have been well characterized and bind to specific glycan targets to confer tissue tropism. For example, type 1 fimbriae bind to α-d-mannosylated glycoproteins such as uroplakins in the bladder via their tip-located FimH adhesin, leading to colonization and invasion of the bladder epithelium. Despite this, the receptor-binding affinity of many other E. coli CU fimbria types remains poorly characterized. Here, we used a recombinant E. coli strain expressing different CU fimbriae, in conjunction with glycan array analysis comprising >300 glycans, to dissect CU fimbria receptor specificity. We initially validated the approach by demonstrating the purified FimH lectin-binding domain and recombinant E. coli expressing type 1 fimbriae bound to a similar set of glycans. This technique was then used to map the glycan binding affinity of six additional CU fimbriae, namely, P, F1C, Yqi, Mat/Ecp, K88, and K99 fimbriae. The binding affinity was determined using whole-bacterial-cell surface plasmon resonance. This work describes new information in fimbrial specificity and a rapid and scalable system to define novel adhesin-glycan interactions that underpin bacterial colonization and disease.IMPORTANCE Understanding the tropism of pathogens for host and tissue requires a complete understanding of the host receptors targeted by fimbrial adhesins. Furthermore, blocking adhesion is a promising strategy to counter increasing antibiotic resistance and is enabled by the identification of host receptors. Here, we use a defined E. coli heterologous expression system to identify glycan receptors for six chaperone-usher fimbriae and identify novel receptors that are consistent with their known function. The same system was used to measure the kinetics of binding to the identified glycan, wherein bacterial cells were immobilized onto a biosensor chip and the interactions with glycans were quantified by surface plasmon resonance. This novel, dual-level analysis, where screening for the repertoire of glycan binding and the hierarchy of affinity of the identified ligands is determined directly from a natively expressed fimbrial structure on the bacterial cell surface, is superior in both throughput and biological relevance.

Keywords: Escherichia coli; FimH; fimbriae; glycomics; glycoproteins.

FIG 1

Glycan array analysis of a range of fimbrial proteins from E. coli. This figure provides a graphical representation of the pattern of binding of fimbrial proteins to the 375 glycans present on the Institute for Glycomics glycan microarray to identify similarities and differences in binding patterns between these proteins. Full data showing the identity of each glycan bound is shown in Data Set S1 and is discussed in detail in the text. Red indicates binding above background; white indicates no binding. Mono, monosaccharides; Gal, terminal galactose; GalNAc, terminal N-acetylgalactosamine; Fuc, fucose-containing glycans; Sia, sialylated glycans; Man, mannose-containing glycans; GlcNAc: terminal N-acetylglucosamine; Glc, repeating glucose; LMW GAGs, low-molecular-weight glycosaminoglycans; HMW GAGs, high-molecular-weight GAGs.

FIG 2

Workflow of the comparison between purified FimH^LD and type 1 fimbria-expressing E. coli. From left to right, glycan array analysis compares the purified protein detected with fluorescent antibodies (red) to fluorescent dye-labeled bacteria (red). This provides fluorescent signals on the array that can be detected and presented as a yes/no binding across 400 glycans. Surface plasmon resonance analysis takes the positive binding and allows for the determination of the affinity (K_D) using either the purified protein (CM5 chip, high-density dextran layer) or the whole bacteria (C1 chip, no dextran layer, binding very close to the gold surface). Each analysis requires a blank flow cell (FC1) with an ethanolamine-blocked dextran layer used for protein and immobilized MS428 (fim negative) for the type 1 fimbria-expressing strain.