OXIDATIVE NEUTROPHIL MICROBICIDAL MECHANISMS

该项目的主要目的是表征氧化杀微生物剂 人类中性粒细胞（PMN）的机制，特别是当它们与 嗜天青颗粒酶髓过氧化物酶（MPO）。在这份更新提案中， 重点已经从研究被破坏的微生物 能量代谢的重点是抑制微生物DNA复制。 改变的基础是一系列使用MPO的实验，以及 其他无细胞氧化（黄嘌呤氧化酶加乙醛）和 非氧化性（庆大霉素）杀微生物系统，证明，仅用于 MPO系统，生存能力丧失， DNA合成和抑制微生物DNA-膜相互作用。的 最后一个效应的显著性与以下发现有关： 在微生物膜和染色体复制起点之间， oriC是启动染色体DNA复制所必需的。的 该提案的中心假设是，MPO衍生的氧化剂以某种方式 干扰染色体复制起始，可能是通过 膜效应，并使细菌不能存活。 初步 实验表明，完整的中性粒细胞抑制微生物的DNA合成， 类似于无细胞MPO系统的方式。 PMN效应需要 功能性MPO系统。 具体目标是： PMN介导的DNA合成抑制，并确定是否 具有缺陷MPO系统的PMN的代谢重建（慢性 肉芽肿病，MPO缺乏）允许这些PMN抑制 微生物DNA合成更正常。（2）确定是否 抑制微生物DNA合成确实与染色体 复制起始。 E.具有致死温度的大肠杆菌突变体 敏感的，复制起始缺陷（dnaA[ts]）将与 MPO处理的正常细胞在染色体复制方面， 噬菌体和质粒DNA对oriC具有不同的依赖性， 复制起始。 目的3）确定哪些复制元件 引发被MPO衍生的氧化剂改变。 主要候选人是 膜磷脂和dnaA起始蛋白，dnaA的产物 基因 候选结构将从MPO处理的生物体中提取 并在oriC的无细胞测定中测试功能完整性， 依赖DNA合成。目的4）将获得的知识与 无细胞髓过氧化物酶系统的完整的中性粒细胞的MPO功能的评估。 的 该项目的预期好处是提高对PMN的理解- 介导宿主对细菌感染的防御。

The broad aim of the project is to characterize oxidative microbicidal mechanisms of human neutrophils (PMN), especially as they relate to the azurophil granule enzyme myeloperoxidase (MPO). In this renewal proposal, the emphasis has been shifted from investigations of disrupted microbial energy metabolism to a focus on inhibition of microbial DNA replication. The basis for the change is a series of experiments using MPO, as well as other cell free oxidative (xanthine oxidase plus acetaldehyde) and nonoxidative (gentamicin) microbicidal systems, that demonstrate, only for the MPO system, a close correlation among loss of viability, cessation of DNA synthesis, and inhibition of microbial DNA-membrane interactions. The significance of the last effect relates to findings that an interaction between the microbial membrane and the chromosomal origin of replication, oriC, is essential for initiation of chromosomal DNA replication. The proposal's central hypothesis is that MPO-derived oxidants somehow interfere with chromosomal replication initiation, presumably through a membrane effect, and render the bacterium non-viable. Preliminary experiments suggest that intact PMNs inhibit microbial DNA synthesis in a fashion similar to the cell-free MPO system. The PMN effect requires a functional MPO system. Specific aims are: Aim l) To complete studies of PMN-mediated inhibition of DNA synthesis and to determine whether metabolic reconstitution of PMNs that have defective MPO systems (chronic granulomatous disease, MPO deficiency) permits these PMN to inhibit microbial DNA synthesis more normally. Aim 2) To determine whether the inhibition of microbial DNA synthesis is indeed related to chromosomal replication initiation. E. coli mutants with a lethal, temperature sensitive, defect in replication initiation (dnaA[ts]) will be compared to MPO-treated normal cells with respect to replication of chromosomal, phage, and plasmid DNAs which have different dependencies on oriC for replication initiation. Aim 3) To determine which elements of replication initiation are altered by MPO-derived oxidants. Principal candidates are membrane phospholipids and the dnaA initiator protein, product of the dnaA gene. Candidate structures will be extracted from MPO-treated organisms and tested for functional integrity in a cell-free assay for oriC- dependent DNA synthesis. Aim 4) To relate knowledge obtained with the cell-free MPO system to an assessment of MPO function in intact PMNs. The anticipated benefit of the project is an improved understanding of PMN- mediated host defense against bacterial infection.