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

Abstract

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.

Chemical Reporters for Bacterial Glycans: Development and Applications

“…Indeed, a 2021 study by the Wennekes and Wosten groups showed that azide-modified legionaminic acid precursors label flagellin glycoproteins in C. jejuni and enabled the identification of a glycosyltransferase likely to be responsible for legionaminic acid installation into flagellin (see Section 4.1.2). 193 3.2.3. Pseudaminic Acid Precursor Derivatives.…”

“…202 The designed compounds thus had azidoacetyl groups at either the 2-or 4-position, or both, and were prepared in either nonacetylated or peracetylated form (compounds 79−84, Figure 28). 193 All six compounds were prepared in >10 steps from D-fucose via chemical synthesis, which was highlighted by sequential S N 2 reactions to establish the diamino core structure with proper stereochemistry (not shown). 193 Although lengthy compared to the chemoenzymatic synthesis of the related compound AltNAc4NAz (75, Figure 27A), the chemical synthesis route did exhibit the versatility required to access all six target compounds 79− 84.…”

“…193 All six compounds were prepared in >10 steps from D-fucose via chemical synthesis, which was highlighted by sequential S N 2 reactions to establish the diamino core structure with proper stereochemistry (not shown). 193 Although lengthy compared to the chemoenzymatic synthesis of the related compound AltNAc4NAz (75, Figure 27A), the chemical synthesis route did exhibit the versatility required to access all six target compounds 79− 84. 193 Evaluation of compounds 79−84 for incorporation into flagellin glycoprotein was performed mainly in C. jejuni strain 11168 using experimental approaches similar to those of Tanner and Logan described above.…”

“…193 Although lengthy compared to the chemoenzymatic synthesis of the related compound AltNAc4NAz (75, Figure 27A), the chemical synthesis route did exhibit the versatility required to access all six target compounds 79− 84. 193 Evaluation of compounds 79−84 for incorporation into flagellin glycoprotein was performed mainly in C. jejuni strain 11168 using experimental approaches similar to those of Tanner and Logan described above. C. jejuni was cultured in the compounds and subjected to SPAAC reaction with a cyclooctyne-PEG-biotin reagent, and whole-cell lysates were obtained and analyzed by Western blot using streptavidin-HRP and antiflagellin antibody.…”

Sweet impersonators: Molecular mimicry of host glycans by bacteria

“…2011 ; Tra and Dube 2014 ), for example, bacillosamine in N -linked glycans of C. jejuni , Legionaminic acid (Leg) in Legionella Pneumophilia ( Pons et al. 2014 ) and C. jejuni ( Meng et al 2021 ) and Pseudaminic acid (Pse) in C. jejuni , Pseudomonas aeruginosa , Acinetobacter baumannii and Vibrio vulnificus ( Liu et al. 2009 ; Andolina et al.…”