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 utilization. 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. C. 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. This article is protected by copyright. All rights reserved

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

Exploring the dynamic role of L-fucose and α-L-fucosidases in the cross talk between gut bacteria and host

As for all cells on earth, both human gut epithelial cells and our gut microbiota are covered by a dense coat of glycans, called the glycocalyx. The unique glycans in this glycocalyx are not encoded in the genome and the biosynthesis of their complex structures is not template-driven. The regular molecular biology tools to study and manipulate the biomolecules DNA and proteins at the molecular level can therefore not be easily applied to elucidate the functional role of glycans and their interacting proteins at this human-gut microbiota interface. The application of techniques from the field of chemical biology however has resulted in a successful strategy for this through the development of smart tailor-made probes – carbohydrate-based bioactive small molecular tools – that target a specific carbohydrate or interacting protein class in a cell or whole organism. In the past decades such glycan-based probes have increasingly been used to unravel glycan-related biological processes. The focus of the research reported in this thesis was on studying fucosylated glycans and their interacting enzymes at the human-gut microbiota interface. Fucosylated glycoproteins are abundant at the gut-microbiota interface and have been implicated in critical biological processes such as immune response, signal transduction, and adhesion of pathogens. Quantification and visualization of fucosidases, the enzymes involved in altering this fucosylation pattern, will help us unravel their biological importance. The research described in this thesis includes the development of two different fucosidase-targeting activity-based probes (ABPs). These new tools can in the future potentially be used in high throughput methods to study the relationship between fucosylation and the maintenance of homeostasis at the human-gut microbiota interface

Exploring the dynamic role of L-fucose and α-L-fucosidases in the cross talk between gut bacteria and host

As for all cells on earth, both human gut epithelial cells and our gut microbiota are covered by a dense coat of glycans, called the glycocalyx. The unique glycans in this glycocalyx are not encoded in the genome and the biosynthesis of their complex structures is not template-driven. The regular molecular biology tools to study and manipulate the biomolecules DNA and proteins at the molecular level can therefore not be easily applied to elucidate the functional role of glycans and their interacting proteins at this human-gut microbiota interface. The application of techniques from the field of chemical biology however has resulted in a successful strategy for this through the development of smart tailor-made probes – carbohydrate-based bioactive small molecular tools – that target a specific carbohydrate or interacting protein class in a cell or whole organism. In the past decades such glycan-based probes have increasingly been used to unravel glycan-related biological processes. The focus of the research reported in this thesis was on studying fucosylated glycans and their interacting enzymes at the human-gut microbiota interface. Fucosylated glycoproteins are abundant at the gut-microbiota interface and have been implicated in critical biological processes such as immune response, signal transduction, and adhesion of pathogens. Quantification and visualization of fucosidases, the enzymes involved in altering this fucosylation pattern, will help us unravel their biological importance. The research described in this thesis includes the development of two different fucosidase-targeting activity-based probes (ABPs). These new tools can in the future potentially be used in high throughput methods to study the relationship between fucosylation and the maintenance of homeostasis at the human-gut microbiota interface

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

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

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