The role of manganese homeostasis in the nitric oxide stress response of Salmonella enterica serovar Typhimurium

总结/摘要 一氧化氮（NO·）是哺乳动物先天免疫细胞产生的一种自由基分子 系统作为防御病原体。当肠道病原体如 革兰氏阴性菌沙门氏菌被NO·抑制， 抑菌作用只是部分了解。沙门氏菌编码一种黄素血红蛋白，Hmp， 作为对NO的防御，但其他途径和细胞过程也参与其中。 一氧化氮应激反应。本提案的目标是确定 过渡金属离子锰在促进对亚硝化的抗性和从亚硝化的恢复中起作用， 沙门氏菌的压力目的1旨在研究潜在的可能机制， 在锰限制性沙门氏菌中观察到长时间的抑菌作用。基于电极 将使用探针和分子测定来监测呼吸活动和细胞ATP水平。 该目标还将调查获得锰的能力是否能保护DNA 通过确定复制阻断病变的频率和突变率来确定损伤。了作用 锰需要的肽酶在促进受损蛋白质的周转将进行研究 使用基因突变体。目标2&3将使用生长测定和酶活性测定， 基因背景目的2实验将确定金属酶的活性， 是NO·抑制的假定靶点，并且这些酶在NO· 在锰充足和锰限制条件下的暴露。Aim 3将进行调查 后期锰的外排和铁、镁的输入要求 从亚硝化应激中恢复过来目的4：利用RNA提取和qPCR技术， 亚硝化过程中交替锰依赖酶的表达和活性 应激反应表征NO·的分子靶点将有助于了解NO·如何 这种先天免疫防御分子发挥其抑菌作用， 可以避免这些行为。这项工作的长期目标是确定新的 未来抗微生物疗法抑制的候选者。

Summary/Abstract Nitric oxide (NO·) is a radical molecule produced by cells of the mammalian innate immune system as a defense against pathogens. While replication of enteric pathogens such as the Gram-negative bacterium Salmonella is inhibited by NO·, the mechanisms by which NO· exerts bacteriostatic effects are only partly understood. Salmonella encodes a flavohemoglobin, Hmp, as a defense against NO· but other pathways and cellular processes are also involved in the nitric oxide stress response. The goal of this proposal is to characterize the role that the transition metal ion manganese plays in promoting resistance to, and recovery from, nitrosative stress in Salmonella. Aim 1 seeks to investigate possible mechanisms underlying the prolonged period of bacteriostasis observed in manganese-limited Salmonella. Electrode-based probes and molecular assays will be used to monitor respiratory activity and cellular ATP levels. This aim will also investigate whether the ability to acquire manganese protects against DNA damage by determining the frequency of replication blocking-lesions and mutation rates. A role for manganese-requiring peptidases in promoting turnover of damaged proteins will be studied using genetic mutants. Aims 2&3 will use growth assays and enzyme activity assays in various genetic backgrounds. Aim 2 experiments will determine the activities of metalloenzymes that are putative targets of NO· inhibition and the repair rates of these enzymes following NO· exposure under manganese-replete and manganese-limited conditions. Aim 3 will investigate the requirements for efflux of manganese and import of iron and magnesium during late stages of recovery from nitrosative stress. Aim 4 will use RNA isolation and qPCR to investigate the expression and activity of alternative manganese-dependent enzymes during the nitrosative stress response. Characterizing the molecular targets of NO· will improve understanding of how this innate immune defense molecule exerts its bacteriostatic effects as well as how Salmonella can circumvent these actions. The long-term objective of this work is to identify novel candidates for inhibition by future antimicrobial therapies.