The Bactericidal Activity of the C-type Lectin RegIIIβ against Gram-negative Bacteria involves Binding to Lipid A

RegIII beta is a member of the C-type lectin family called RegIII. It is known to bind peptidoglycan, and its bactericidal activity shapes the interactions with commensal and pathogenic gut bacteria. However, little is known about its carbohydrate recognition specificity and the bactericidal mechanism, particularly against Gram-negative bacteria. Here, we show that RegIII beta can bind directly to LPS by recognizing the carbohydrate moiety of lipid A via a novel motif that is indispensable for its bactericidal activity. This bactericidal activity of RegIII beta could be inhibited by preincubation with LPS, lipid A, or gentiobiose. The latter is a disaccharide composed of two units of beta-(1 -> 6)-linked D-glucose and resembles the carbohydrate moiety of lipid A. Therefore, this structural element may form a key target site recognized by RegIII beta. Using point-mutated RegIII beta proteins, we found that amino acid residues in two structural motifs termed loop 1 and loop 2, are important for peptidoglycan and lipid A binding (Arg-135, Asp-142) and for the bactericidal activity (Glu-134, Asn-136, Asp-142). Thus, the ERN motif and residue Asp-142 in the loop 2 are of critical importance for RegIII beta function. This provides novel insights into the carbohydrate recognition specificity of RegIII beta and explains its bactericidal activity against Gram-negative bacteria.