New mechanisms of sulfur sensing and trafficking in Staphylococcus aureus.

描述(由申请人提供)：革兰氏阳性条件致病菌金黄色葡萄球菌是严重得多的医院和社区获得性疾病的轻微皮肤感染的病原体。因此，迫切需要针对新的代谢途径的新的抗生素疗法，而硫代谢就是这样一种经过验证的抗菌药物和疫苗策略。金黄色葡萄球菌以独特的硫醇代谢为特征，其获得无机硫以制造必需氨基酸半胱氨酸的能力受到强烈限制。我们已经在金黄色葡萄球菌Newman菌株中发现了铜敏感转录抑制因子CSOR(铜敏感操纵子抑制因子)的类似序列，我们将其命名为CSTR，即CSOR样硫基转移酶抑制因子。CSTR调控一个新的操纵子Cst的表达，该操纵子编码一个假定的亚硫酸盐/磺酸盐流出物(TauE)，以及两个含有典型的硫代硫酸盐硫代转移酶或罗丹氏结构域(Csta，CstB)的多结构域蛋白，其生物学功能尚不清楚。我们的具体目标是(1)阐明CSTR介导的抑制和去抑制CST操纵子转录的机制基础；(2)阐明多结构域硫代硫酸盐硫转移酶csta的结构和机制，我们假设它含有三个硫中继模块，共同介导半胱氨酸过硫化物从供体到受体的载体转移。(3)利用“自下而上”的蛋白质组学方法鉴定瞬时携带34S-过硫基的细胞蛋白质组；(4)开始阐明CSTR调节基因产物CstB和SQR的酶活性；(3)利用“自下而上”的蛋白质组学方法鉴定瞬时携带34S-过硫基的细胞蛋白质；(4)开始阐明CSTR调控基因产物CstB和SQR的酶活性。该项目将为人类重要病原体的硫代谢机制和对硫代谢物毒性的抗性提供新的见解。这些研究将极大地扩展其他细菌中基于半胱氨酸过硫化物的硫穿梭系统的新兴范例，使之成为一种潜在的新型抗菌干预策略。 公共卫生相关性：金黄色葡萄球菌是公认的人类病原体，是无数严重的医院和社区获得性感染的病原体。在这个项目中，我们建议研究一种新的硫代谢操纵子在金黄色葡萄球菌中的调节和功能，该操纵子可能成为抗菌治疗的新靶点。

DESCRIPTION (provided by applicant): The Gram-positive opportunistic pathogen Staphylococcus aureus is the causative agent of minor skin infections to far more severe hospital and community-acquired diseases. New antibiotic therapies that target novel metabolic pathways are therefore urgently needed and sulfur metabolism represents one such validated antimicrobial and vaccine strategy. Staphylococcus aureus is characterized by a unique thiol metabolism and is strongly restricted in its ability to obtain inorganic sulfur to make cysteine, an essential amino acid. We have discovered a paralog of a copper-sensing transcriptional repressor CsoR (copper-sensitive operon repressor) in S. aureus strain Newman that we denote CstR, for CsoR-like sulfurtransferase repressor. CstR regulates the expression of a novel operon, cst, which encodes a putative sulfite/sulfonate effluxer (TauE) as well as two multidomain proteins that harbor canonical thiosulfate sulfurtransferase or rhodanese domains (CstA, CstB) whose biological functions are unknown. Our specific objectives are to (1) Elucidate the mechanistic basis of CstR-mediated repression and derepression of cst operon transcription; (2) Elucidate the structure and mechanism of the multidomain thiosulfate sulfurtransferase CstA, which we hypothesize harbors three sulfur relay modules that collectively mediate vectorial transfer of cysteine persulfides from donor to acceptor. NMR structural studies and a novel 34S-32SO32- pulse-32S-32SO32- chase experiment are proposed; (3) Identify cellular proteins that transiently carry 34S-persulfide groups originating with extracellular sodium 34S- 32SO3 thiosulfate using a "bottom-up" proteomics approach; and 4) Begin to elucidate the enzymatic activities of CstR-regulated gene products CstB, a putative rhodanese-containing sulfur dioxygenase and SQR, a putative sulfide:quinone oxidoreductase. This project will provide new insights into the mechanisms of sulfur metabolism and resistance to sulfur metabolite toxicity in an important human pathogen. These studies will significantly extend the emerging paradigm of a cysteine persulfide-based sulfur shuttling system in other bacteria to a new metabolic process as a potential novel antimicrobial intervention strategy. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus is a recognized human pathogen and the causative agent of myriad severe hospital and community acquired infections. In this project, we propose studies to investigate the regulation and function of a novel sulfur metabolic operon in S. aureus that may serve as a new target for antimicrobial therapeutics.