Chemical Biology of Nitroxyl (HNO) in Bacillus Subtilis

项目摘要 许多致病性革兰氏阳性细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（SO 4-）的氧化还原转化来移动硫 到硫化氢（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。多功能的化学物质 氮和硫允许它们参与许多生物化学氧化还原途径;作为中间反应物， 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，并且这 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 仍然很少探索，但提供了一个机会，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确定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 结合了几种方法来定义关键化学成分的反应性-HNO，H2S， bacillithiol，（BSH）-在体外和枯草芽孢杆菌中，一种能够内源性一氧化氮（NO） 和H2S生成，其产生BSH作为其主要的LMW硫醇。与这些化学物质一起 通过研究，生化研究将描述HNO对表型结果的影响。本研究的目标是 基于“缺乏BSH的细菌表现出对HNO的氧化还原敏感性”的假设。这一假设 将使用合成有机化学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， BSH的修改版本（具体目标1）。微生物生长曲线实验将显示 HNO在B。枯草杆菌表型和分析生物化学将揭示氧化硫醇含量的量。 具体目标2建立在具体目标1的基础上，设计和合成抑制BSH生物合成的化合物， 在HNO供体存在下活化的潜在抗生素。这一目标将通过有机 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 准备解决这些问题，维克森林大学为本科生提供了理想的环境 在一个强大的本科学院和小型研究生课程的接口培训。实现完全 对这种基本化学的理解将告知广泛的生理反应， HNO-硫醇反应，并可能导致开发基于HNO的抗生素， 硫代谢/生物化学不存在于人类。

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.