Glycosylation of Campylobacter flagella

描述(申请人提供)：空肠弯曲杆菌是北美食源性疾病的主要原因，也是全球细菌性腹泻的主要原因之一。空肠弯曲菌肠道定植和侵袭肠道上皮细胞需要鞭毛和运动，鞭毛蛋白是一种免疫优势抗原，可能是一种保护性抗原。空肠弯曲菌81-176菌株和弯曲杆菌VC167菌株的鞭毛蛋白分别在19个和16个丝氨酸或苏氨酸残基上糖基化，其中9个碳糖称为伪氨基酸及其衍生物。这些修饰约占这些糖蛋白重量的10%，表面暴露在鞭毛细丝上，可能参与鞭毛蛋白与真核宿主的相互作用。遗传分析表明，假氨基酸的生物合成途径在81-176和VC167中都是保守的。两个菌株的Flagellin都含有轻微的修饰，是伪氨基酸(质量为315 Da)的乙酰氨基形式。然而，由81-176和VC167合成的315Da基团在结构和免疫上是不同的，在每个生物体中都是通过独立的途径合成的。这些数据表明，弯曲杆菌鞭毛蛋白需要糖基化才能出口和/或组装成细丝。81-176的一个突变株不能合成乙酰氨基形式的伪氨基酸，其毒力似乎已减弱。这项研究的目的是进一步阐明合成不同形式的伪氨基酸的途径，并研究这些糖基化基因调节的独特方面。将对鞭毛蛋白进行定点突变，以顺序消除修饰位点，以确定对鞭毛功能和位点占用规则至关重要的位点。鞭毛糖基化的生物学作用将通过在体外和体内毒力测试中检测一系列突变体来研究。

DESCRIPTION (provided by applicant): Campylobacter jejuni is the leading cause of foodborne illness in North America and is among the major causes of bacterial diarrhea worldwide. Flagella and motility are required for intestinal colonization and invasion of intestinal epithelial cells by C. jejuni, and flagellin is an immunodominant and possibly a protective antigen. Flagellin from C. jejuni strain 81-176 and Campylobacter coli strain VC167 are glycosylated at 19 and 16 serine or threonine residues, respectively, with a 9 carbon sugar called pseudaminic acid and derivatives of pseudaminic acid. The modifications, which account for approximately 10% of the weight of these glycoproteins, are surface exposed on the flagella filament and are likely involved in interaction of flagellin with the eukaryotic host. Genetic analyses indicate that the pathway for biosynthesis of pseudaminic acid is conserved in both 81-176 and VC167. Flagellins from both strains contain minor modifications that are acetamidino forms of pseudaminic acid (mass 315 Da). However, the 315 Da group synthesized by 81-176 and VC 167 are structurally and immunologically distinct and are synthesized by independent pathways in each organism. The data suggest that campylobacter flagellin needs to be glycosylated in order to be exported and/or assembled into a filament. A mutant in 81-176 that is unable to synthesize the acetamidino form of pseudaminic acid appears to be attenuated in virulence. The aim of this study is to further elucidate the pathways by which the different forms of pseudaminic acid are synthesized and to study unique aspects of the regulation of these glycosylation genes. Site-specific mutagenesis will be done on flagellin to eliminate modification sites sequentially in order to determine sites that are critical for flagella function and the rules of site occupancy. The biological role of flagella glycosylation will be studied by examining a series of mutants in in vitro and in vivo assays of virulence.