Metabolic Labeling of Legionaminic Acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase

Campylobacter jejuni is the major human food‐borne pathogen. Its bipolar flagella are heavily O‐glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide‐modified Leg precursors. The hereby azido‐Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC–MS detection of labeled CMP‐Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness. Probes used to metabolically label legionaminic acid on bacterial flagella substantiated the identification of Maf4 as a putative legionaminyl transferase in Campylobacter jejuni (see picture). The labeled glycan could be detected intracellularly as its activated CMP analogue, on flagella by Western blot analysis, and on intact bacteria by confocal microscopy.