Chemical Biology of Nitroxyl (HNO) in Bacillus Subtilis

项目总结 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)， 炭疽芽孢杆菌和结核分枝杆菌通过硫酸盐(SO4-)的氧化还原转化来活化硫 到支持半胱氨酸生物合成蛋白质和低分子量硫醇的硫化氢(H_2S) 制作。这些氧化还原反应是正常代谢过程所必需的。多才多艺的化学 氮和硫允许它们参与许多生化氧化还原途径；作为中间反应 氮和硫物种(RNS和RSS)，它们经常与活性氧物种(ROS)一起作用。 各种证据表明，氮氧基(HNO)位于RNS和RSS串扰的交叉点上，这 交叉点的灵感来自于提议的工作。细菌固硫与人体健康的相互作用 仍然没有得到很好的探索，但为理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 RSS和RNS串扰及其在微生物生理学中的意义。拟议的机械论研究 结合几种方法来定义关键化学成分的反应性-HNO、H2S和 杆菌硫醇(BSH)-在体外和在枯草芽孢杆菌中，一种能够内源性一氧化氮(NO)的模式生物 以及产生BSH作为其主要低分子硫醇的硫化氢生成。与这些化学品结合使用 调查，生化研究将描绘HNO对表型结果的影响。本研究的目标是 基于“缺乏BSH的细菌对HNO表现出氧化还原敏感性”的假设。这一假设 将使用合成有机化学和分析化学来确定BSH和HNO在 通过鉴定该反应的产物，包括亚磺酰胺和过硫化物，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其效果 HNO对枯草杆菌表型和分析生化的影响将揭示氧化硫含量的多少。 特定目标2建立在特定目标1的基础上，以设计和合成抑制BSH生物合成的化合物 在HNO供体在场的情况下激活潜在的抗生素。这一目标将由有机组织来实现 合成、分析和机械生物化学，以及标准微生物学方法。我们队打得很好 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 在强大的本科学院和小型研究生项目的交界处进行培训。实现一个完整的 对这一基础化学的理解将提供广泛的生理反应，由 HNO-硫醇反应，并可能导致以HNO为基础的针对微生物的抗生素的开发 硫磺代谢/生物化学在人类中不存在。

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.