Detection of Bacterial α-l-Fucosidases with an Ortho-Quinone Methide-Based Probe and Mapping of the Probe-Protein Adducts.

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria.

Development of a 1,2-difluorofucoside activity-based probe for profiling GH29 fucosidases.

GH29 α-l-fucosidases catalyze hydrolysis of terminal α-l-fucosyl linkages with varying specificity and are expressed by prominent members of the human gut microbiota. Both homeostasis and dysbiosis at the human intestinal microbiota interface have been correlated with altered fucosidase activity. Herein we describe the development of a 2-deoxy-2-fluoro fucosyl fluoride derivative with an azide mini-tag as an activity-based probe (ABP) for selective in vitro labelling of GH29 α-l-fucosidases. Only catalytically active fucosidases are inactivated by this ABP, allowing their functionalization with a biotin reporter group via the CuAAC reaction and subsequent in-gel detection at nanogram levels. The ABP we present here is shown to be active against a GH29 α-l-fucosidase from Bacteroides fragilis and capable of labeling two other GH29 α-l-fucosidases with different linkage specificity, illustrating its broader utility. This novel ABP is a valuable addition to the toolbox of fucosidase probes by allowing identification and functional studies of the wide variety of GH29 fucosidases, including those in the gut microbiota.

Bacteroides fragilis fucosidases facilitate growth and invasion of Campylobacter jejuni in the presence of mucins.

The enteropathogenic bacterium, Campylobacter jejuni, was considered to be non-saccharolytic, but recently it emerged that l-fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l-fucose utilisation. In the intestinal tract, l-fucose is abundantly available in mucin O-linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l-fucose. We set out to determine how C. jejuni can gain access to these intestinal l-fucosides. Growth of the fuc + C. jejuni strains, 129,108 and NCTC 11168, increased in the presence of l-fucose while fucose permease knockout strains did not benefit from additional l-fucose. With fucosidase assays and an activity-based probe, we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. Campylobacter jejuni invaded Caco-2 intestinal cells that express complex O-linked glycan structures that contain l-fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc + strains are dependent on exogenous fucosidases for increased growth and invasion.

MUC1 is a receptor for the Salmonella SiiE adhesin that enables apical invasion into enterocytes.

The cellular invasion machinery of the enteric pathogen Salmonella consists of a type III secretion system (T3SS) with injectable virulence factors that induce uptake by macropinocytosis. Salmonella invasion at the apical surface of intestinal epithelial cells is inefficient, presumably because of a glycosylated barrier formed by transmembrane mucins that prevents T3SS contact with host cells. We observed that Salmonella is capable of apical invasion of intestinal epithelial cells that express the transmembrane mucin MUC1. Knockout of MUC1 in HT29-MTX cells or removal of MUC1 sialic acids by neuraminidase treatment reduced Salmonella apical invasion but did not affect lateral invasion that is not hampered by a defensive barrier. A Salmonella deletion strain lacking the SiiE giant adhesin was unable to invade intestinal epithelial cells through MUC1. SiiE-positive Salmonella closely associated with the MUC1 layer at the apical surface, but invaded Salmonella were negative for the adhesin. Our findings uncover that the transmembrane mucin MUC1 is required for Salmonella SiiE-mediated entry of enterocytes via the apical route.