OXIDATIVE NEUTROPHIL MICROBICIDAL MECHANISMS

该项目的主要目标是表征氧化型杀微生物剂。 人中性粒细胞(PMN)的机制，特别是当它们与 天青颗粒酶髓过氧化物酶(MPO)。在这份续签提案中， 重点已经从调查被破坏的微生物转移到 能量代谢的重点是抑制微生物的DNA复制。 这一变化的基础是使用MPO的一系列实验，以及 其他细胞自由氧化(黄嘌呤氧化酶加乙醛)和 非氧化性(庆大霉素)杀菌系统，仅适用于 MPO系统，与生存能力丧失、停止 DNA合成，以及抑制微生物DNA-膜相互作用。这个 最后一个效应的意义与发现相互作用有关 在微生物膜和复制的染色体起源之间， ORIC是启动染色体DNA复制所必需的。这个 Proposal的中心假设是MPO衍生的氧化剂 干扰染色体复制启动，推测是通过一种 膜效应，使细菌无法存活。初步 实验表明，完整的中性粒细胞抑制微生物DNA的合成 时尚类似于无手机MPO系统。PMN效应需要一个 功能强大的MPO系统。具体目标是：目标：完成对L的研究 中性粒细胞介导的DNA合成抑制并确定 MPO系统有缺陷的PMN的代谢重建(慢性 肉芽肿性疾病，MPO缺乏)允许这些PMN抑制 微生物DNA合成更正常。目标2)确定是否 微生物DNA合成的抑制确实与染色体有关 复制启动。具有致死温度的大肠杆菌突变体 复制启动中的敏感缺陷(Dna A[ts])将与 MPO处理的正常细胞在染色体复制方面， 噬菌体和对ORIC有不同依赖关系的质粒DNA 复制启动。目标3)确定复制的哪些要素 引发被MPO衍生的氧化剂改变。主要候选人是 膜磷脂和DNAA的产物DNAA启动子蛋白 吉恩。候选结构将从MPO处理的生物体中提取 并在ORIC-的无细胞检测中测试功能完整性 依赖DNA合成。目标4)将获得的知识与 无细胞MPO系统用于评估完整PMN的MPO功能。这个 该项目的预期收益是对PMN的更好理解- 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.