MECHANISMS OF NITRIC OXIDE CYTOTOXICITY IN SALMONELLOSIS

描述（改编自申请人摘要）：一氧化氮（NO） 是一种具有显著广谱抗菌活性生物介质 活性NO可以以不同的氧化还原形式存在，每种形式都具有不同的 稳定性、反应性和生物学作用。 虽然没有 在许多感染的动物模型中研究， NO在微生物上的特定氧化还原形式还没有被广泛地 在分子水平上进行研究。 该提案分析了 NO抗菌活性的机制和生物学意义 鼠沙门氏菌病模型。 初步数据显示， S-亚硝基硫醇是重要的内源性抗微生物氧化还原形式 NO的作用，并且微生物高半胱氨酸拮抗细胞生长抑制作用 S-亚硝基硫醇的影响。 这项建议的具体目标是： 以下内容： A）确定沙门氏菌易感性的沙门氏菌基因的鉴定 体外不同氧化还原形式的一氧化氮; B）鉴定一氧化氮调节的沙门氏菌基因; C）沙门氏菌对体外一氧化氮的易感性的相关性 对细胞来源的一氧化氮和毒性敏感的供体。 S-亚硝基谷胱甘肽、SIN-1和DETA/NO将用作 亚硝鎓（NO+）、过氧亚硝酸根（OONO-）和NO自由基（NO-）， 分别 影响NO易感性的细菌基因或 其调节受NO影响，将被突变、定位、克隆， 测序和补充。 在前期工作中， 对特定氧化还原形式敏感性改变的同基因突变体 已经被鉴定和表征。 的 突变体对细胞来源的NO的敏感性和这些突变体的毒力 将检查小鼠中的菌株以确定生物学意义 NO的特定氧化还原形式。 同型半胱氨酸水平和同型半胱氨酸调节的基因融合的应用 将被用来检验微生物 同型半胱氨酸合成拮抗吞噬体内抗菌剂 虽然这个项目利用了先进的知识， 沙门氏菌遗传学在利用这个特定的模型系统， 这项工作的意义绝不限于沙门氏菌病。 的 初步研究结果已经确定了新的途径， 内源性抗菌介质，以及微生物 对这些中介的抵制。 拟议的研究将提供 进一步深入了解NO细胞毒性的分子基础， 这种活性在广泛的感染中的潜在重要性， 特别是由细胞内病原体引起的那些。

DESCRIPTION (Adapted from the applicant's abstract): Nitric oxide (NO) is a biological mediator with remarkably broad-spectrum antimicrobial activity NO can exist in different redox forms, each with distinctive stability, reactivity, and biological actions. Although NO has been investigated in numerous animal models of infection, the effects of specific redox forms of NO on microbes have not been extensively investigated at a molecular level. This proposal analyzes the molecular mechanism and biological relevance of NO antimicrobial activity in a murine salmonellosis model. Preliminary data are presented suggesting that S-nitrosothiols are important endogenous antimicrobial redox forms of NO, and that microbial homocysteine antagonizes the cytostatic effects of S-nitrosothiols. The specific aims of this proposal are the following: A) Identification of Salmonella genes which determine susceptibility to different redox forms of nitric oxide in vitro; B) Identification of nitric oxide-regulated Salmonella genes; C) Correlation of Salmonella susceptibility to in vitro nitric oxide donors with susceptibility to cell-derived nitric oxide and virulence. S-nitrosoglutathione, SIN-1, and DETA/NO will be used as donors of nitrosonium (NO+), peroxynitrite (OONO-), and NO radical (NO-), respectively. Bacterial genes which influence NO susceptibility or whose regulation is influenced by NO will be mutated, mapped, cloned, sequenced, and complemented. In preliminary work, a considerable number of isogenic mutants with altered susceptibility to specific redox forms of NO have already been identified and characterized. The susceptibility of mutants to cell-derived NO and virulence of these strains in mice will be examined to define the biological significance of specific redox forms of NO. Genetic manipulation of intracellular homocysteine levels and the use of homocysteine-regulated gene fusions will be employed to test the provocative thesis that microbial homocysteine synthesis antagonizes the intraphagosomal antimicrobial activity of NO. Although this project exploits the advanced knowledge of Salmonella genetics in utilizing this specific model system, the implications of this work are by no means limited to salmonellosis. The preliminary findings have already identified novel pathways used by endogenous antimicrobial mediators, and novel mechanisms of microbial resistance to these mediators. The proposed studies will provide further insights into the molecular basis of NO cytotoxicity and the potential importance of this activity in a wide range of infections, particularly those caused by intracellular pathogens.