Discoidins are cytosolic lectins that recognize methylated L-rhamnose epitopes on mycobacterial glycolipids and glycopeptidolipids upon vacuole damage

Mycobacteria are surrounded by a dynamic, glycolipid-rich envelope that governs virulence and immune recognition, yet how cytosolic lectins sense its organization during intracellular infection remains unclear. Here, we show that Dictyostelium discoideum discoidins act as cytosolic sensors of mycobacterial envelope exposure. During infection, Mycobacterium marinum damages the limiting vacuolar membrane, triggering recruitment of all canonical isoforms (DscA, DscC, DscD, and DscE) to distinct foci around bacilli. Discoidins are not recruited after sterile endolysosomal damage, indicating that, unlike galectins, they recognize non-self surface-associated bacterial glycans rather than exposed host glycans. DscA and DscE display distinct glycan specificities. Combining transposon mutagenesis, synthetic glycan arrays, biochemical fractionation, and envelope mutants, we identify a restricted set of lipid-associated determinants enriched in methylated L-rhamnose motifs within LOS-, PGL-, and GPL-related structures. Recognition requires the conserved beta-galactoside-binding pocket of the H-type lectin domain, and isoform-specific synthetic disaccharides inhibit binding to intact bacteria. Analyses exclude AG-PG, AM/LAM, alpha-glucan, and TDM as major ligands and identify LOS and GPL-like glycolipids as dominant targets. PDIM/PGL or LOS disruption reduces discoidin binding to intact bacteria, although some ligands remain detectable in envelope extracts, underscoring the importance of accessibility. LOS deficiency also reduces DscA recruitment to intracellular bacilli. Therefore, binding of discoidins to glycan epitopes depends on ligand presence and accessibility, as well as on envelope perturbations and unmasking of specific glycolipids and glycopeptidolipids. These findings reveal pathogen envelope glycans as direct targets of cytosolic lectin surveillance and establish discoidins as probes of mycobacterial envelope remodeling during infection.