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.

Stereoisomer‐specific reprogramming of a bacterial flagellin sialyltransferase

“…Since flagellin glycosylation was far more efficient when cells only produced Leg, we infer that a functional Pse biosynthesis pathway could interfere with Leg production, perhaps at the junction that consumes UDP-GlcNAc. Alternatively, CMP-Pse and CMP-Leg could both competitively bind to FlmG Cc-Bs It will be interesting to conduct similar dissection experiments with the other class of fGTs that are wide-spread in bacteria, the Mafs (van Alphen et al, 2008;Parker et al, 2014;Sulzenbacher et al, 2018;Khairnar et al, 2020;Meng et al, 2021), to test if a similar re-programming in donor specificity is possible once the corresponding determinants have been identified. An X-ray structure determined for the Maf from M. magneticum (Sulzenbacher et al, 2018) revealed a tripartite domain architecture with central GT-A domain bearing clear resemblance to the GT29 and GT42 family of sialyltransferases.…”

Genetic glyco-profiling and rewiring of insulated flagellin glycosylation pathways

“…Similar dissection experiments should be conducted with the other class of fGTs that are wide-spread in bacteria, the Mafs [10, 26, 28, 47, 48], to reveal if analogous mechanisms and determinants underpin flagellin glycosylation in these systems. It stands to reason that donor and acceptor specificities exist in Mafs as well, however, the flagellin recognition determinants remain unknown.…”

“…Pse or Leg are constituents of capsular polysaccharides (CPS or K-antigen) [4] or the Oantigen of lipopolysaccharide (LPS) [5], but they often also occur conjugated to proteinaceous surface appendages, for example on the subunits of S-layer arrays [6], pilus adhesins [7] or flagellar filaments (the H-antigen) [8,9]. Pse and Leg derivatives synthesized in vitro can be added exogenously in metabolic labeling experiments to be incorporated into bacterial surface structures [10,11]. Moreover, Pse and Leg are attractive vaccine targets as shown by the recent report that mice immunized with Pse chemically conjugated to a carrier protein were protected against the Pse-containing pathogenic Acinetobacter baumannii strain Ab-00.191 [12].…”

“…The structure-function relationship and specificities of Maf and FlmG fGTs is poorly understood. Some Maf enzymes have been linked to flagella glycosylated with Pse, while other Maf affect modification of flagella with Leg [10,[26][27][28]. The determinants conferring donor or acceptor specificities in these enzymes have not been elucidated.…”

“…Expression of this chimeric FlmG Cc-Bs variant in C. crescentus ΔflmG cells that had been engineered to synthesize Leg resulted in the modification of the C. crescentus flagellin, whereas the WT version of FlmG Bs had poor activity (Figure 5C). Conversely, FlmG Cc-Bs was unable to support glycosylation of C. crescentus flagellins with Pse, likely because it no longer possesses the Pse-specific GT-B domain of FlmG Cc .Similar dissection experiments should be conducted with the other class of fGTs that are wide-spread in bacteria, the Mafs[10,26,28,47,48], to reveal if analogous mechanisms and determinants underpin flagellin glycosylation in these systems. It stands to reason that donor and acceptor specificities exist in Mafs as well, however, the flagellin recognition determinants remain unknown.…”