Elucidation of the glycan structure of the b-type flagellin of Pseudomonas aeruginosa PAO1

Hensbergen, P. J.; van Huijkelom, L.; van Angeren, J.; de Ru, A. H.; Claushuis, B.; van Veelen, P. A.; Smits, W. K.; Corver, J.

doi:10.1101/2024.09.23.614423

bioRxiv · 10.1101/2024.09.23.614423

Elucidation of the glycan structure of the b-type flagellin of Pseudomonas aeruginosa PAO1

Hensbergen, P. J.·van Huijkelom, L.·van Angeren, J.·de Ru, A. H.·Claushuis, B.·van Veelen, P. A.·Smits, W. K.·Corver, J.

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Abstract

Flagella are essential for motility and pathogenicity in many bacteria. The main component of the flagellar filament, flagellin, often undergoes post-translational modifications, with glycosylation being a common occurrence. In Pseudomonas aeruginosa PAO1, the b-type flagellin is O-glycosylated with a structure that includes a rhamnose, a phospho-group and a previous unknown moiety. This structure resembles the well-characterized glycan (Type A) in Clostridioides difficile strain 630, which features an N-acetylglucosamine linked to an N-methylthreonine via a phosphodiester bond. This study aimed to characterize the b-type glycan structure in Pseudomonas aeruginosa PAO1 using a set of mass spectrometry experiments. For this purpose, we used wildtype P. aeruginosa PAO1 and several gene mutants from the b-type glycan biosynthetic cluster. Moreover, we compared the mass spectrometry characteristics of the b-type glycan with those of in vitro modified Type A-peptides from C. difficile strain 630{Delta}erm. Our results demonstrate that the thus far unknown moiety of the b-type glycan in P. aeruginosa consists of an N,N-dimethylthreonine. These data allowed us to refine our model of the flagellin glycan biosynthetic pathway in both P. aeruginosa PAO1 and C. difficile strain 630.

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Hensbergen, P. J., van Huijkelom, L., van Angeren, J., de Ru, A. H., Claushuis, B., van Veelen, P. A., Smits, W. K., Corver, J.. 2024-09-23. Elucidation of the glycan structure of the b-type flagellin of Pseudomonas aeruginosa PAO1. https://doi.org/10.1101/2024.09.23.614423

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