van Alphen Lieke B, Wuhrer Manfred, Bleumink-Pluym Nancy M C, Hensbergen Paul J, Deelder André M, van Putten Jos P M
Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Albinusdreef 2, 2300 RC Leiden, The Netherlands.
Microbiology (Reading). 2008 Nov;154(Pt 11):3385-3397. doi: 10.1099/mic.0.2008/019919-0.
Flagellin of Campylobacter jejuni is extensively modified with (derivatives of) pseudaminic acid. The flagellar glycosylation locus contains several genes with homopolymeric G-tracts prone to slipped-strand mispairing, some of which belong to the maf gene family. We investigated the function of the putative phase-variable maf4 gene of C. jejuni strain 108. A constructed maf4 mutant displayed unaltered flagella assembly and bacterial motility. 2D-PAGE analysis revealed that the flagellin of strain 108 migrated at a more acidic pI than the protein of the Maf4 mutant. MS-MS in combination with high-resolution matrix-assisted laser desorption/ionization Fourier transform ion cyclotron MS (MALDI-FT-ICR-MS) on flagellin-derived glycopeptides showed that the flagellins of the mutant lacked two previously unidentified modifications of pseudaminic acid. These glycoforms carried additional CO(2) and C(2)H(2)O(2) groups, consistent with the more acidic pI of the wild-type flagellin. Phenotypically, the maf4 mutant displayed strongly delayed bacterial autoagglutination. Collectively, our results suggest that the presence of a functional Maf4 expands the flagellin glycan repertoire with novel glycoforms of pseudaminic acid and, in the event of phase variation, alters the population behaviour of C. jejuni.
空肠弯曲菌的鞭毛蛋白被假氨基糖酸（及其衍生物）广泛修饰。鞭毛糖基化位点包含几个带有易于发生滑链错配的同聚物G序列的基因，其中一些属于maf基因家族。我们研究了空肠弯曲菌108株假定的相变异maf4基因的功能。构建的maf4突变体显示出未改变的鞭毛组装和细菌运动性。二维聚丙烯酰胺凝胶电泳分析表明，108株的鞭毛蛋白在比Maf4突变体的蛋白更酸性的等电点处迁移。对鞭毛蛋白衍生的糖肽进行串联质谱分析并结合高分辨率基质辅助激光解吸/电离傅里叶变换离子回旋共振质谱（MALDI-FT-ICR-MS）表明，突变体的鞭毛蛋白缺乏两种先前未鉴定的假氨基糖酸修饰。这些糖型带有额外的CO₂和C₂H₂O₂基团，这与野生型鞭毛蛋白更酸性的等电点一致。从表型上看，maf4突变体表现出强烈延迟的细菌自凝作用。总体而言，我们的结果表明，功能性Maf4的存在通过假氨基糖酸的新型糖型扩展了鞭毛蛋白聚糖库，并且在发生相变异时，改变了空肠弯曲菌的群体行为。
