Glycosylation of Campylobacter flagella

描述（由申请人提供）：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。空肠梭菌定殖和侵袭肠上皮细胞需要鞭毛和运动，鞭毛蛋白是免疫优势抗原，可能是一种保护性抗原。空肠C. 81-176菌株和大肠弯曲杆菌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被称为伪胺酸的9碳糖及其衍生物糖基化。这些修饰约占这些糖蛋白重量的10%，它们表面暴露在鞭毛丝上，可能与鞭毛蛋白与真核宿主的相互作用有关。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。两种菌株的鞭毛蛋白都含有少量的修饰，这些修饰是假氨基酸的乙酰氨基形式（质量315 Da）。然而，由81-176和VC 167合成的315 Da基团在结构和免疫学上是不同的，它们在每个生物体中通过独立的途径合成。数据表明弯曲杆菌的鞭毛蛋白需要糖基化才能输出和/或组装成细丝。81-176突变体不能合成假氨基酸的乙酰氨基形式，其毒力似乎减弱了。本研究的目的是进一步阐明不同形式假胺酸合成的途径，并研究这些糖基化基因调控的独特方面。将对鞭毛蛋白进行位点特异性诱变，依次消除修饰位点，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检测一系列突变体在体外和体内的毒力测定来研究。

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.