Polar flagellin glycan heterogeneity of Aeromonas hydrophila strain ATCC 7966^T

Motile pathogens often rely upon flagellar motility as an essential virulence factor and in many species the structural flagellin protein is glycosylated. Flagellin glycosylation has been shown to be important for proper function of the flagellar filament in a number of bacterial species. Aeromonas hydrophila is a ubiquitous aquatic pathogen with a constitutively expressed polar flagellum. Using a suite of mass spectrometry techniques, the flagellin FlaA and FlaB structural proteins of A. hydrophila strain ATCC 7966^T were shown to be glycosylated with significant microheterogeneity, macroheterogeneity, and metaheterogeneity. The primary linking sugar in this strain was a novel and previously unreported pseudaminic acid derivative with a mass of 422 Da. The pseudaminic acid derivative was followed in sequence by two hexoses, an N-acetylglucosamine (with additional variable secondary modification), and a deoxy N-acetylglucosamine derivative. These pentasaccharide glycans were observed modifying all eight modification sites. Hexasaccharides, which included an additional N-acetylhexosamine residue as the capping sugar, were observed exclusively modifying a pair of isobaric peptides from FlaA and FlaB. Interestingly, these isobaric peptides are immediately adjacent to a toll-like receptor 5 binding site in both protein sequences. Glycosylation status was also linked to motility, a critical bacterial virulence factor.