BASIS OF BACTERIAL KILLING BY COMPLEMENT

描述（改编自申请人摘要）： 补体介导的对革兰氏阴性菌的杀伤仍然是难以捉摸的。 这个项目的长期目标是揭示 补体膜攻击复合物的杀菌活性 （C5 b-9）和细菌产生耐药性的变化。 立即 目标是确定C9中毒性所需的区域， C9跨周质易位的分子要求 空间及其插入内（血浆）膜，并 表征细菌突变，使它们对它的影响产生抵抗力。 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将被表达并分泌到细胞中。 E.缺乏形成二硫键的酶的大肠杆菌菌株。 氧化还原试剂的添加将影响二硫键配对和折叠， 导致C9介导细胞死亡。 在特定目标2中，细菌外膜 或C9跨膜转运所必需的周质成分。 细胞壁将被识别。 原生C9将被引入 完整细胞的周质。 放射性标记的C9与未标记的C9的结合 分子（或该序列的其他排列）将被执行以 了解为什么聚C9需要用于细菌溶解。 在具体目标3中， 补体抗性E.大肠杆菌菌株类似于从成人分离的菌株 这样有助于抵抗血清的突变基因就可以 被定性。 一个两方面的筛选和选择机制将 用于鉴定对以下细胞毒性作用具有抗性的突变体： 将重组C9输出到周质中并输出到存在于血清中的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.