Abstract
C-type lectin receptors (CLRs) are carbohydrate-binding receptors that recognize their ligands often in a Ca2+-dependent manner. Upon ligand binding, myeloid CLRs in innate immunity trigger or inhibit a variety of signaling pathways, thus initiating or modulating effector functions such as cytokine production, phagocytosis, and antigen presentation. CLRs bind to various pathogens, including viruses, fungi, parasites, and bacteria. The bacterium Campylobacter jejuni (C. jejuni) is a very frequent Gram-negative zoonotic pathogen of humans, causing severe intestinal symptoms. Interestingly, C. jejuni expresses several glycosylated surface structures, for example, the capsular polysaccharide (CPS), lipooligosaccharide (LOS), and envelope proteins. This “Methods” paper describes applications of CLR–Fc fusion proteins to screen for yet unknown CLR/bacteria interactions using C. jejuni as an example. ELISA-based detection of CLR/bacteria interactions allows a first prescreening that is further confirmed by flow cytometry-based binding analysis and visualized using confocal microscopy. By applying these methods, we identified Dectin-1 as a novel CLR recognizing two selected C. jejuni isolates with different LOS and CPS genotypes. In conclusion, the here-described applications of CLR–Fc fusion proteins represent useful methods to screen for and identify novel CLR/bacteria interactions.
Highlights
C-type lectin receptors (CLRs) are pattern recognition receptors and are known to sense pathogenassociated molecular patterns as well as danger-associated molecules
Generation and Detection of CLR–hFc Fusion Proteins Used in This Study
The first step was the cloning of the cDNA fragment encoding for the extracellular part of each CLR and its fusion to the Fc fragment of human IgG1 in the pFuse-hIgG1-Fc expression vector [1]
Summary
C-type lectin receptors (CLRs) are pattern recognition receptors and are known to sense pathogenassociated molecular patterns as well as danger-associated molecules. CLRs trigger a variety of functions, including the production of inflammatory mediators, the phagocytosis of pathogens, or intracellular signaling [1, 2]. Screening for CLR/Bacteria Interactions the binding of CLRs to their specific ligands. One well-described example for a CLR–ligand pair is the CLR Mincle and its ligand trehalose-6,6'-dimycolate (TDM), a unique glycolipid present in the cell wall of mycobacteria [3, 4]. Crystal structure analyses of the bovine [5] and human [6] Mincle CRD revealed that the two glucose moieties and one acyl chain of TDM and its synthetic analog trehalose-6,6'-dibehenate interact with Mincle. For the majority of CLRs, their glycan ligands and binding mode to their respective ligands are still incompletely understood
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