Chemical Biology of Nitroxyl (HNO) in Bacillus Subtilis

枯草芽孢杆菌中硝酰基 (HNO) 的化学生物学

• 批准号: 10730746
• 负责人: S BRUCE King
• 金额: $ 40.7万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10730746
• 关键词: Address; Anabolism; Analytical Biochemistry; Analytical Chemistry; Anthrax disease; Antibiotics; Bacillus anthracis; Bacillus subtilis; Back; Bacteria; Biochemical; Biochemistry; Biological; Biology; Chemicals; Chemistry; Chromatography; Cysteine; Development; Environment; Enzymes; Generations; Goals; Gram-Positive Bacteria; Growth; Health; Human; Hydrogen Sulfide; Impairment; In Vitro; Incubated; Institution; Investigation; Iron; Measures; Mentors; Metabolism; Methods; Microbial Physiology; Modification; Molecular; Molecular Weight; Mycobacterium tuberculosis; Natural Products; Nitric Oxide; Nitrogen; Organic Chemistry; Organic Synthesis; Outcome; Oxidation-Reduction; Pathogenicity; Pathway interactions; Phenotype; Physiological; Physiology; Principal Investigator; Production; Protein Biosynthesis; Proteins; Publishing; Reaction; Reactive Nitrogen Species; Reactive Oxygen Species; Records; Recycling; Research; Role; Sodium Chloride; Source; Stress; Sulfate; Sulfhydryl Compounds; Sulfur; Sulfur Metabolism Pathway; System; Training; Tuberculosis; Universities; Vitamins; Work; cofactor; college; design; experience; experimental study; forest; in vivo; inhibitor; methicillin resistant Staphylococcus aureus; microbial; model organism; nitroxyl; persulfides; programs; response; undergraduate student

PROJECT SUMMARY Many pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), Bacillus anthracis and Mycobacterium tuberculosis, mobilize sulfur through redox conversion of sulfate (SO4-) to hydrogen sulfide (H2S) that supports cysteine biosynthesis for protein and low molecular weight (LMW) thiol production. These redox reactions are required for normal metabolic processes. The versatile chemistry of nitrogen and sulfur allows their participation in many biochemical redox pathways; as intermediary reactive nitrogen and sulfur species (RNS and RSS), they often act together with reactive oxygen species (ROS). Various lines of evidence indicate nitroxyl (HNO) sits at the intersection of RNS and RSS crosstalk and this intersection inspires the proposed work. The interplay of bacterial sulfur mobilization and human health remains poorly explored but presents an opportunity for understanding chemical redox biology and the exploitation of new antibiotic strategies. The goal of the proposed research consists of defining HNO’s role in RSS and RNS crosstalk and its significance in microbial physiology. The proposed mechanistic study combines several approaches to define the reactivity of the key chemical components—HNO, H2S, and bacillithiol, (BSH)—in vitro and in Bacillus subtilis, a model organism capable of endogenous nitric oxide (NO) and H2S generation that produces BSH as its predominant LMW thiol. In conjunction with these chemical investigations, biochemical studies will delineate HNO’s effect on phenotypic outcomes. This research goal is based on the hypothesis that “Bacteria lacking BSH demonstrate redox sensitivity to HNO”. This hypothesis will be probed using synthetic organic and analytical chemistry to define the reactivity of BSH and HNO both in vitro and in vivo by identifying the products of this reaction including the sulfinamide and the persulfide modified versions of BSH (Specific Aim 1). Microbiological growth curve experiments will show the effect of HNO on B. subtilis phenotype and analytical biochemistry will reveal the amount of oxidized thiol content. Specific Aim 2 builds on Specific Aim 1 to design and synthesize compounds that inhibit BSH biosynthesis as potential antibiotics activated in the presence of an HNO donor. This aim will be approached by organic synthesis, analytical and mechanistic biochemistry, and standard microbiological methods. Our team is well poised to address these questions and Wake Forest University provides an ideal setting for undergraduate training at the interface of a strong undergraduate college and small graduate program. Achieving a complete understanding of this fundamental chemistry will inform a wide range of physiological responses modulated by HNO-thiol reactions and potentially lead to the development of HNO-based antibiotics that target microbial sulfur metabolism/biochemistry not present in humans.

项目摘要 许多致病性革兰氏阳性细菌，包括甲氧西林金黄色葡萄球菌（MRSA）， 炭疽芽孢杆菌和结核分枝杆菌通过硫酸盐的氧化还原转化（SO4-）动员硫 硫化氢（H2S）支持半胱氨酸生物合成的蛋白质和低分子量（LMW）硫醇 生产。这些氧化还原反应是正常代谢过程所必需的。多功能化学 氮和硫可以参与许多生化氧化还原途径。作为中间反应性 它们经常与活性氧（ROS）一起起作用。 表明硝氧基（HNO）的各种证据位于RN和RSS串扰的交集，这 交叉点激发了拟议的工作。细菌硫和人类健康的相互作用 探索仍然很差，但为了解化学氧化还原生物学和 开发新的抗生素策略。拟议研究的目标包括定义HNO在 RSS和RNS串扰及其在微生物生理中的重要性。提出的机械研究 结合了几种定义关键化学成分的反应性的方法 - HHO，H2S和 芽孢杆菌醇（BSH） - 在枯草芽孢杆菌中，一种能够内源性一氧化氮的模型生物（NO） H2S的生成产生BSH为主要的LMW硫醇。与这些化学物质结合 研究，生化研究将描绘HNO对表型结果的影响。这个研究目标是 基于“缺乏BSH的细菌表现出对HNO的氧化还原敏感性”的假设。这个假设 将使用合成有机和分析化学探测，以定义BSH和HNO的反应性 通过识别包括磺胺酰胺和硫硫化物在内的该反应的产物，体外和体内 BSH的修改版本（特定目标1）。微生物生长曲线实验将显示 枯草芽孢杆菌表型和分析生物化学的HNO将揭示氧化硫醇含量的量。 特定的目标2建立在特定目标1上，以设计和合成抑制BSH生物合成为的化合物 在HNO供体存在下激活了潜在的抗生素。这个目标将由有机 合成，分析和机械生物化学以及标准微生物方法。我们的团队很好 被毒解决这些问题，维克森林大学为本科提供了理想的环境 在强大的本科学院和小型研究生课程的界面上进行培训。实现完整 对这种基本化学的理解将为由 HNO-硫醇反应，并有可能导致基于HNO的抗生素的发展，这些抗生素靶向微生物 硫代谢/生物化学不存在于人类中。