Significance: The interplay between glycan binding proteins and their carbohydrate ligands is fundamental to all aspects of life. Printed glycan arrays have helped to elucidate the role of glycans in many biological processes with monosaccharide resolution, particularly those of mammalian origin, over the past 15 y. Current microbial glycan arrays contain specialized small synthetic collections or isolated carbohydrates but better coverage is needed as microbes are enveloped in complex layers of carbohydrates. The chemically diverse microbial glycans play crucial roles in immunity and infectious diseases. We present an extensive synthetic glycan collection focused on microbial glycans as a tool to investigate microbial glycobiology.

Recognition of viral glycans by pattern recognition receptors (PRRs) in innate immunity contributes to antiviral immune responses. C-type lectin receptors (CLRs) are PRRs capable of sensing glycans present in viral pathogens to activate antiviral immune responses such as phagocytosis, antigen processing and presentation, and subsequent T cell activation. The ability of CLRs to elicit and shape adaptive immunity plays a critical role in the inhibition of viral spread within the host. However, certain viruses exploit CLRs for viral entry into host cells to avoid immune recognition. To block CLR interactions with viral glycoproteins, antiviral strategies may involve the use of multivalent glycan carrier systems. In this review, we describe the role of CLRs in antiviral immunity and we highlight their dual function in viral clearance and exploitation by viral pathogens.

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.

La Crosse virus (LACV) is a mosquito-transmitted arbovirus and the main cause of virus-mediated neurological diseases in children. To date, little is known about the role of C-type lectin receptors (CLRs)—an important class of pattern recognition receptors—in LACV recognition. DC-SIGN remains the only well-described CLR that recognizes LACV. In this study, we investigated the role of additional CLR/LACV interactions. To this end, we applied a flow-through chromatography method for the purification of LACV to perform an unbiased high-throughput screening of LACV with a CLR-hFc fusion protein library. Interestingly, the CARD9-associated CLRs Mincle, Dectin-1, and Dectin-2 were identified to strongly interact with LACV. Since CARD9 is a common adaptor protein for signaling via Mincle, Dectin-1, and Dectin-2, we performed LACV infection of Mincle^−/− and CARD9^−/− DCs. Mincle^−/− and CARD9^−/− DCs produced less amounts of proinflammatory cytokines, namely IL-6 and TNF-α, albeit no reduction of the LACV titer was observed. Together, novel CLR/LACV interactions were identified; however, the Mincle/CARD9 axis plays a limited role in early antiviral responses against LACV.

The macrophage galactose-type lectin (MGL) recognizes glycan moieties exposed by pathogens and malignant cells. Particularly, mucin-1 (MUC1) glycoprotein presents an altered glycosylation in several cancers. To estimate the ability of distinct MGL orthologs to recognize aberrant glycan cores in mucins, we applied evanescent-field detection to a versatile MUC1-like glycopeptide microarray platform. Here, as binding was sequence-dependent, we demonstrated that not only sugars but also peptide region impact the recognition of murine MGL1 (mMGL1). In addition, we observed for all three MGL orthologs that divalent glycan presentation increased the binding. To assess the utility of the glycopeptide binders of the MGL orthologs for MGL targeting, we performed uptake assays with fluorescein-MUC1 using murine dendritic cells. A diglycosylated MUC1 peptide was preferentially internalized in an MGL-dependent fashion, thus showing the utility for divalent MGL targeting. These findings may be relevant to a rational design of antitumor vaccines targeting dendritic cells via MGL.

C-type lectin receptor (CLR)/carbohydrate recognition occurs through low affinity interactions. Nature compensates that weakness by multivalent display of the lectin carbohydrate recognition domain (CRD) at the cell surface. Mimicking these low affinity interactions in vitro is essential to better understand CLR/glycan interactions. Here, we present a strategy to create a generic construct with a tetrameric presentation of the CRD for any CLR, termed TETRALEC. We applied our strategy to a naturally occurring tetrameric CRD, DC-SIGNR, and compared the TETRALEC ligand binding capacity by synthetic N- and O-glycans microarray using three different DC-SIGNR constructs i) its natural tetrameric counterpart, ii) the monomeric CRD and iii) a dimeric Fc-CRD fusion. DC-SIGNR TETRALEC construct showed a similar binding profile to that of its natural tetrameric counterpart. However, differences observed in recognition of low affinity ligands underlined the importance of the CRD spatial arrangement. Moreover, we further extended the applications of DC-SIGNR TETRALEC to evaluate CLR/pathogens interactions. This construct was able to recognize heat-killed Candida albicans by flow cytometry and confocal microscopy, a so far unreported specificity of DC-SIGNR. In summary, the newly developed DC-SIGNR TETRALEC tool proved to be useful to unravel novel CLR/glycan interactions, an approach which could be applied to other CLRs.