Role and regulation of Resistance-Nodulation-Division family efflux pumps in physiology and resistance mechanisms of Staphylococcus aureus

RND外排泵包括一个古老的转运蛋白家族，在微生物生理学中起着关键作用。我们首次发表了对金黄色葡萄球菌RND泵功能的描述，包括TetR家族调节因子（TFR） FarR和发散转录的外排泵FarE，后者赋予对抗菌不饱和游离脂肪酸（uFFA）的抗性。这种分散的排列通常表明TFR具有抑制外排泵的功能，但我们发现FarR具有激活剂的功能。我们的项目重点关注FarR和FarE作为RND外排系统的模型，以获得对关键微生物过程的新见解，包括新的调控范式和对RND泵如何促进细胞稳态的更深入的基本理解。我们将：1；进行FarR的结构和生物物理表征，以了解DNA结合是如何被酰基磷酸配体调节的。2.确定FarE 3的生理功能范围。定义代谢信号如何调节FarE表达。目的1定义了FarR如何激活FarE表达。基于FarR与亚油酸复合物的模型结构，我们将评估特定氨基酸在结合酰基磷酸中的作用以及它如何修饰DNA结合。这将得到长期目标的支持，以解决FarR的晶体结构，以及FarR与酰基磷酸和DNA的复合物。目标2是基于RND泵可以容纳一系列底物的知识，以及a ？farER突变体在化学定义的最小培养基（CDM）中长时间孵育后表现出活力丧失。我们将使用脂质组学和代谢组学来鉴定由于farE失活或组成表达而改变的细胞和分泌代谢物。这些实验应该确定FarE如何有助于维持膜完整性和有毒代谢物的排出。Aim 3扩展了FarE功能与代谢活性的相关性。我们将确定farE在CDM中的最佳表达时间，并通过RNAseq来确定当farE失活时基因表达是如何改变的。在确定了具有不同营养限制或抗菌uFFA的CDM中farE的最佳表达条件后，我们将使用farE启动子探针从细胞裂解物中捕获DNA结合蛋白，并确定控制其表达的调节因子的光谱。影响和意义：该项目将定义一种通过TetR家族调节因子激活RND外排泵表达的新调控模式，同时定义FarE在细胞稳态中的新作用，并确定在环境应激和细菌营养状况下调节FarE表达的其他调节因子。这将有助于更好地理解RND外排泵如何促进葡萄球菌的基本生理过程，以及这是如何通过响应代谢信号的转录调节因子实现的。

RND efflux pumps comprise an ancient family of transporters with critical roles in microbial physiology. We published the first description of RND pump function in Staphylococcus aureus, comprised of a TetR family regulator (TFR) FarR, and divergently transcribed efflux pump FarE that confer resistance to antimicrobial unsaturated free fatty acids (uFFA). This divergent arrangement normally signifies that the TFR functions to repress the efflux pump, but we found that FarR functions as an activator. Our program focuses on FarR and FarE as a model RND efflux system to obtain new insight into critical microbial processes, including a novel regulatory paradigm and deeper fundamental understanding of how RND pumps contribute to cellular homeostasis. We will: 1.Conduct structural and biophysical characterization of FarR to understand how DNA binding is modulated by an acyl-phosphate ligand. 2.Determine the scope of physiologic functions of FarE 3.Define how metabolic signals modulate FarE expression. Aim 1 defines how FarR activates FarE expression. Based on a model structure of FarR in complex with linoleic acid, we will assess the role of specific amino acids in binding acyl-phosphate and how this modifies DNA binding. This will be supported by longer term goals to resolve crystal structures of FarR, and FarR in complex with acyl-phosphate and DNA. Aim 2 is based on knowledge that RND pumps can accommodate a range of substrates, and preliminary data that a ?farER mutant exhibits loss of viability on extended incubation in a chemically defined minimal medium (CDM). We will use lipidomics and metabolomics to identify cellular and secreted metabolites that are altered as a consequence of farE inactivation, or constitutive expression. These experiments should define how FarE contributes to maintenance of membrane integrity and efflux of toxic metabolites. Aim 3 expands the correlation of FarE function with metabolic activity. We will determine when farE is optimally expressed in CDM, and conduct RNAseq to determine how gene expression is altered when farE is inactivated. After determining conditions for optimal expression of farE in CDM with different nutrient limitations or antimicrobial uFFA, we will use a farE promoter probe to capture DNA binding proteins from cell lysates, and identify the spectrum of regulators that control its expression. Impact and Significance: This program will define a novel regulatory paradigm for activation of RND efflux pump expression through a TetR family regulator, while defining new roles for FarE in cellular homeostasis, and identifying other regulators that modulate FarE expression in response to environmental stress and the nutritional status of the bacteria. This will contribute to a greater understanding of how RND efflux pumps facilitate fundamental physiologic processes in Staphylococci, and how this is achieved through transcriptional regulators that respond to metabolic signals.