Hensbergen Paul J, van Huijkelom Loes, van Angeren Jordy, de Ru Arnoud H, Claushuis Bart, van Veelen Peter A, Smits Wiep Klaas, Corver Jeroen
Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden 2333 ZA, The Netherlands.
Leiden University Center for Infectious Diseases, Leiden University Medical Center, Leiden 2333 ZA, The Netherlands.
ACS Infect Dis. 2025 Feb 14;11(2):518-528. doi: 10.1021/acsinfecdis.4c00896. Epub 2025 Jan 24.
Flagella are essential for motility and pathogenicity in many bacteria. The main component of the flagellar filament, flagellin (FliC), often undergoes post-translational modifications, with glycosylation being a common occurrence. In PAO1, the b-type flagellin is -glycosylated with a structure that includes a deoxyhexose, a phospho-group, and a previous unknown moiety. This structure resembles the well-characterized glycan (Type A) in strain 630, which features an -acetylglucosamine linked to an -methylthreonine via a phosphodiester bond. This study aimed to characterize the b-type glycan structure in PAO1 using a set of mass spectrometry experiments. For this purpose, we used wild-type 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 modified Type A-peptides from strain 630Δ. Our results demonstrate that the thus far unknown moiety of the b-type glycan in consists of an -dimethylthreonine. These data allowed us to refine our model of the flagellin glycan biosynthetic pathway in both PAO1 and strain 630.
鞭毛对于许多细菌的运动性和致病性至关重要。鞭毛丝的主要成分鞭毛蛋白（FliC）通常会经历翻译后修饰，糖基化是常见的修饰方式。在铜绿假单胞菌PAO1中，b型鞭毛蛋白被糖基化，其结构包括一个脱氧己糖、一个磷酸基团和一个此前未知的部分。这种结构类似于630菌株中特征明确的聚糖（A型），该聚糖的特点是通过磷酸二酯键将N-乙酰葡糖胺与O-甲基苏氨酸相连。本研究旨在通过一系列质谱实验来表征PAO1中b型聚糖的结构。为此，我们使用了野生型PAO1和来自b型聚糖生物合成簇的几个基因突变体。此外，我们将b型聚糖的质谱特征与630Δ菌株中修饰后的A型肽段的质谱特征进行了比较。我们的结果表明，PAO1中b型聚糖迄今未知的部分由O-二甲基苏氨酸组成。这些数据使我们能够完善PAO1和630菌株中鞭毛蛋白聚糖生物合成途径的模型。
