BASIS OF BACTERIAL KILLING BY COMPLEMENT

描述(改编自申请人的摘要)： 补体介导的对革兰氏阴性细菌的杀灭仍然难以捉摸。 这个项目的长期目标是揭示分子基础。 补体攻膜复合体杀菌活性的研究 (C5b-9)以及细菌产生抗药性的变化。最直接的 目标是确定C9中需要毒性的区域，以了解 C9跨周质转运的分子要求 空间及其插入内(质膜)膜，以及 描述使细菌对其影响产生抗药性的细菌突变。 提出了三个具体目标：在具体目标1中，最小结构 将确定细菌细胞毒性所需的C9中的元素。 各种形式的重组C9将被表达和分泌到 缺乏形成二硫键的酶的大肠杆菌菌株的周质。 添加氧化还原试剂将影响二硫键配对和折叠 导致C9介导的细胞死亡。在特定目标2中，细菌外膜 或C9转运所需的周质成分 细胞壁将被识别出来。原生C9将被引入 完整细胞的周质。放射性标记C9与非标记C9的结合 分子(或该序列的其他排列)将被执行以 了解为什么细菌溶解需要聚C9。在具体目标3中， 与成人分离的补体抗性大肠杆菌相似的菌株 会被产生，因此导致血清耐药性的突变基因可以 被刻画出来。双臂筛选和遴选机制将是 用来鉴定对沙门氏菌的细胞毒作用具有抵抗力的突变体 重组C9输出到周质和存在于血清中的C9。

DESCRIPTION (Adapted from applicant's abstract): The mechanism of complement-mediated killing of Gram-negative bacteria has remained elusive. The long-term objectives of this project are to uncover the molecular basis of the bactericidal activity of the membrane attack complex of complement (C5b-9) and the changes made by bacteria to become resistant. The immediate goals are to identify the regions in C9 required for toxicity, to understand the molecular requirements for translocation of C9 across the periplasmic space and its insertion into the inner (plasma) membrane, and to characterize bacterial mutations that render them resistant to its effects. Three specific aims are proposed: In Specific Aim 1, the minimal structural elements in C9 required for bacterial cytotoxicity will be determined. Various forms of recombinant C9 will be expressed and secreted into the periplasm of E. coli strains that lack enzymes which form disulfide bonds. The addition of redox reagents will effect disulfide pairing and folding and lead to C9-mediated cell death. In Specific Aim 2, bacterial outer membrane or periplasmic constituents necessary for translocation of C9 across the cell wall will be identified. Native C9 will be introduced into the periplasm of intact cells. Binding of radiolabeled C9 followed by unlabeled molecules (or other permutations of this sequence) will be performed to understand why poly C9 is required for bacteriolysis. In Specific Aim 3, complement-resistant E. coli strains resembling those isolated from adults will be generated so that mutated genes contributing to serum resistance can be characterized. A two-arm screening and selection mechanism will be employed to identify mutants that are resistant to the cytotoxic action of recombinant C9 exported into the periplasm and to C9 present in serum.