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

锰稳态在鼠伤寒沙门氏菌一氧化氮应激反应中的作用

• 批准号: 10514182
• 负责人: Elaine R. Frawley
• 金额: $ 30.69万
• 依托单位: RHODES COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10514182
• 关键词: Adult; Affect; Africa South of the Sahara; Amino Acids; Antibiotics; Bacterial Infections; Bacterial Physiology; Biological Assay; Biotin; Carbon; Cell Death; Cell physiology; Cells; Child; Citrates; DNA; DNA Damage; DNA Sequence Alteration; DNA biosynthesis; Defect; Disease Outbreaks; Domestic Fowls; Drug Metabolic Detoxication; Electrodes; Enzymes; Frequencies; Future; Genetic; Goals; Gram-Negative Bacteria; Growth; HIV; Homeostasis; Immune; Immune system; Infection; Infection Control; Innate Immune System; Ions; Iron; Lesion; Magnesium; Mammalian Cell; Manganese; Measures; Mediating; Metabolism; Metalloproteins; Metals; Molecular; Molecular Target; Monitor; Mononuclear; Mutation; Nitric Oxide; Nucleotides; Nutritional Requirements; Pathway interactions; Pattern; Peptide Hydrolases; Phagocytes; Phenotype; Play; Protein Import; Proteins; Public Health; RNA; Recovery; Regulation; Research Personnel; Resistance; Respiration; Role; Salmonella; Salmonella infections; Salmonella typhimurium; Source; Spectrum Analysis; Stress; Supplementation; System; Techniques; Time; Toxic effect; Transition Elements; United States; Work; Zinc; antimicrobial; base; biological adaptation to stress; diarrheal disease; enteric pathogen; enzyme activity; experimental study; foodborne; improved; metalloenzyme; mutant; nitrosative stress; novel; pathogen; pathogenic bacteria; prevent; protein degradation; repaired; respiratory; response; undergraduate student

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.

摘要/摘要 一氧化氮 (NO·) 是一种由哺乳动物先天免疫细胞产生的自由基分子 系统作为抵御病原体的防御系统。虽然肠道病原体的复制，例如 NO· 抑制革兰氏阴性菌沙门氏菌，NO· 发挥作用的机制 抑菌作用仅被部分了解。沙门氏菌编码黄素血红蛋白 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。该提案的目标是描述以下角色的特征： 过渡金属离子锰可促进亚硝化的抵抗和恢复 沙门氏菌应激。目标 1 旨在研究潜在的机制 在限锰沙门氏菌中观察到长时间的抑菌作用。基于电极 探针和分子检测将用于监测呼吸活动和细胞 ATP 水平。 该目标还将调查获取锰的能力是否可以预防 DNA 通过确定复制阻断损伤的频率和突变率来损伤。一个角色 将研究需要锰的肽酶促进受损蛋白质的周转 使用基因突变体。目标 2 和 3 将在各种领域使用生长测定和酶活性测定 遗传背景。目标 2 实验将确定金属酶的活性 是 NO· 抑制的假定目标以及这些酶在 NO· 后的修复率 在充满锰和限制锰的条件下暴露。目标 3 将进行调查 后期锰的流出和铁、镁的进口需求 从亚硝化应激中恢复。目标 4 将使用 RNA 分离和 qPCR 来研究 亚硝化过程中替代锰依赖性酶的表达和活性 应激反应。表征 NO· 的分子靶点将加深对 NO· 分子靶标的理解 这种先天免疫防御分子发挥其抑菌作用以及沙门氏菌如何发挥作用 可以规避这些行为。这项工作的长期目标是确定新颖的 未来抗菌疗法抑制的候选者。