MECHANISMS OF GROUP B STREPTOCOCCUS CAPSULE EXPRESSION

针对荚膜多糖的抗体可有效保护新生儿免受B群链球菌（GBS）感染，这是新生儿中危及生命的细菌感染的主要原因。然而，至少有五种抗原不同的GBS荚膜类型是感染的重要原因，免疫是类型特异性的。对III型GBS胶囊合成的遗传学和生物化学研究表明，在染色体的16kb区域内可能存在几个基因。然而，只有两个胶囊合成基因具有特定的功能。这项提议的目的是确定参与GBS胶囊表达的许多其他基因的功能。研究人员建议对Ia型和Ib型GBS染色体的荚膜基因区域进行表征，这两种血清型的多糖结构与III型GBS荚膜含有相同的糖，但具有独特的糖苷键。克隆Ia型和Ib型GBS的荚膜基因区域将允许对荚膜位点的结构和单个基因的序列进行详细的比较，以便鉴定编码所有血清型共同蛋白质的保守基因以及编码血清型特异性糖基转移酶的独特基因。每个基因的功能将通过构建等基因突变体和确定所得荚膜多糖的结构来研究。糖基转移酶将通过在大肠杆菌中表达单个重组酶，然后在体外测定系统中鉴定其糖脂产物来研究。糖基转移酶特异性的结构基础将通过位点特异性诱变和突变来确定负责糖苷键特异性的蛋白质结构域。体外胶囊生物合成试验将用于表征糖脂中间体和参与多糖聚合和出口的基因产物的功能。这些研究将促进对GBS新血清型出现的分子基础的理解，并可能为药物治疗提供新的靶点。

Antibodies to the capsular polysaccharide effectively protect newborn infants against infection with group B Streptococcus (GBS), the leading cause of life-threatening bacterial infection in neonates. However, at least five antigenically distinct capsular types of GBS are important causes of infection and immunity is type-specific. Studies on the genetics and biochemistry of capsule synthesis in type III GBS have implicated several putative genes spanning a 16 kb region of the chromosome. However, specific functions have been assigned to only two capsule synthesis genes. The goal of this proposal is to determine the function of the many other genes involved in GBS capsule expression. It is proposed to characterize the capsule gene region of the GBS chromosome for types Ia and Ib GBS, serotypes whose polysaccharide structures contain identical sugars as the type III GBS capsule but unique glycosidic linkages. Cloning the capsule gene regions of types Ia and Ib GBS will permit detailed comparison of the architecture of the capsule loci and the sequences of individual genes in order to identify conserved genes encoding proteins common to all serotypes as well as unique genes encoding serotype-specific glycosyltransferases. The function of each gene will be studied by constructing isogenic mutants and determining the structure of the resultant capsular polysaccharides. Glycosyltransferases will be investigated by expressing individual recombinant enzymes in E. coli then identifying their glycolipid products in an in vitro assay system. The structural basis for the specificity of glycosyltransferases will be determined using site-specific mutagen&sis to identify domains of the protein responsible for glycosidic bond specificity. An in vitro capsule biosynthesis assay will be used to characterize glycolipid intermediates and the function of gene products involved in polysaccharide polymerization and export. These studies will advance understanding of the molecular basis for emergence of new serotypes of GBS and may suggest new targets for pharmacologic therapy.