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.

项目概要 许多致病性革兰氏阳性菌，包括耐甲氧西林金黄色葡萄球菌 (MRSA)、 炭疽杆菌和结核分枝杆菌通过硫酸盐 (SO4-) 的氧化还原转化来动员硫 生成硫化氢 (H2S)，支持蛋白质和低分子量 (LMW) 硫醇的半胱氨酸生物合成 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 氮和硫允许它们参与许多生化氧化还原途径；作为中介反应 氮和硫物质（RNS 和 RSS），它们通常与活性氧物质 (ROS) 一起作用。 各种证据表明硝酰基 (HNO) 位于 RNS 和 RSS 串扰的交叉点，这 交叉点激发了拟议的工作。细菌硫动员与人类健康的相互作用 仍然缺乏探索，但提供了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 在以下方面的作用： RSS 和 RNS 串扰及其在微生物生理学中的意义。拟议的机制研究 结合了多种方法来定义关键化学成分的反应性 - HNO、H2S 和 杆菌硫醇 (BSH)——体外和枯草芽孢杆菌中，枯草芽孢杆菌是一种能够产生内源性一氧化氮 (NO) 的模型生物体 生成 BSH 作为其主要 LMW 硫醇的 H2S 生成。与这些化学品一起 调查、生化研究将描述 HNO 对表型结果的影响。本研究目标是 基于“缺乏 BSH 的细菌表现出对 HNO 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进行探测，以确定 BSH 和 HNO 的反应性 通过鉴定该反应的产物（包括亚磺酰胺和过硫化物）来进行体外和体内实验 BSH 的修改版本（具体目标 1）。微生物生长曲线实验将显示效果 HNO 对枯草芽孢杆菌表型和分析生物化学的影响将揭示氧化硫醇含量的多少。 Specific Aim 2 以 Specific Aim 1 为基础，设计并合成抑制 BSH 生物合成的化合物： 潜在的抗生素在 HNO 供体存在下被激活。这一目标将通过有机的方式实现 合成、分析和机械生物化学以及标准微生物学方法。我们的团队很好 维克森林大学致力于解决这些问题，为本科生提供了理想的环境 在强大的本科学院和小型研究生课程的结合处进行培训。实现完整的 对这一基本化学的理解将指导一系列由以下因素调节的生理反应： HNO-硫醇反应并可能导致针对微生物的 HNO 抗生素的开发 人类中不存在硫代谢/生物化学。