Discoidins are cytosolic lectins that shape intracellular host defence by sensing virulence-associated mycobacterial glycolipids and glycopeptidolipids
Mycobacteria are surrounded by a dynamic, glycolipid-rich envelope that controls both virulence and immune recognition, yet how cytosolic lectins detect and interpret the organization of surface glycans during intracellular infection remains unclear. Here, we show that Dictyostelium discoideum discoidins act as cytosolic sensors of mycobacterial envelope organization. During Mycobacterium marinum infection, Discoidin A and Discoidin E assemble into foci on intracellular bacilli and bind surface-associated glycan patches. Using transposon mutagenesis, synthetic mycobacterial glycan arrays, biochemical fractionation and targeted envelope mutants, we find that discoidins recognize a restricted set of lipid-linked glycans enriched in methylated L-rhamnose motifs, including structures associated with LOS, PGL and GPL. Binding depends on the H-type lectin {beta}-galactoside-binding pocket and is inhibited by point mutations and TDG, a soluble disaccharide competitor, demonstrating glycan-dependent recognition. Specific assays led to exclusion of major structural carbohydrates, including AG-PG, AM/LAM, -glucan and TDM, while protease treatment of capsular material and fractionation of polar lipids, identified both GPL and LOS-like glycolipids as dominant discoidin ligands. Disruption of LOS biosynthesis or PDIM/PGL-dependent envelope organization reduced discoidin binding to intact bacteria even though some ligands remained detectable by dot blot in various envelope extracts. Thus, discoidins do not simply detect ligand abundance, but binding depends on envelope perturbations and unmasking of glycolipids and glycopeptidolipids. These findings reveal pathogen envelope glycans as direct targets of cytosolic lectin surveillance and establish discoidins as probes of mycobacterial envelope remodelling during intracellular infection. Author SummaryMany disease-causing bacteria are surrounded by a protective outer layer that helps them survive inside host cells and avoid being eliminated. This surface is not static, it can be remodelled during infection, changing which molecules are exposed to the host. How host cells detect these changes is still not fully understood. We studied this question using Dictyostelium discoideum, a single-celled amoeba that shares many cellular defence mechanisms with human immune phagocytes. We focused on discoidins, a family of proteins that bind sugars. We found that discoidins accumulate on intracellular Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, the bacterium that causes tuberculosis, and recognize specific sugar-containing molecules exposed at the bacterial surface. Importantly, discoidins do not simply detect whether these molecules are present. Instead, they respond to how they are displayed and exposed on the bacterial surface. Changes in the organization of the bacterial outer layer strongly affected discoidin binding, masking or revealing specific target molecules. Our findings show that discoidins act as sensors of bacterial surface remodelling during infection. More broadly, they reveal an ancient mechanism by which host cells can monitor pathogens by detecting changes in the sugars exposed on their surface.