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

RND外排泵是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。我们发表了金黄色葡萄球菌RND泵功能的第一个描述，包括一个TetR家族调节因子(TfR)FARR和不同转录的外排泵功能，使其对抗菌素不饱和游离脂肪酸(UFFA)产生耐药性。这种不同的排列通常意味着TFR起到抑制外排泵的作用，但我们发现FARR起着激活剂的作用。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，包括新的调节范式和对RND泵如何有助于细胞内稳态的更深层次的基础理解。我们将：1.进行FARR的结构和生物物理表征，以了解酰基磷酸配体是如何调节DNA结合的。2.确定FIGH的生理功能范围3.明确代谢信号如何调节FIGH的表达。目标1定义了FARR如何激活票价表达。基于FARR与亚油酸络合物的模型结构，我们将评估特定氨基酸在与酰基磷酸结合中的作用以及这如何改变DNA结合。这将得到长期目标的支持，即解析FARR的晶体结构，以及FARR与酰基磷酸和DNA的络合物。AIM 2是基于RND泵可以适应一系列底物的知识，以及初步数据表明，较少的突变株在化学定义的最低限度培养基(CDM)中延长孵育时间后表现出活力丧失。我们将使用脂质组学和代谢组学来鉴定细胞和分泌的代谢物，这些代谢物是FARE失活或结构性表达的结果。这些实验应该确定FARE如何有助于维持膜的完整性和有毒代谢物的外流。目的3扩展FARE功能与代谢活动的关系。我们将确定何时在CDM中最佳表达FRAGE，并进行RNAseq以确定当FIGH被灭活时基因表达如何改变。在确定了在不同营养限制或抗菌剂UFFA的CDM中最佳表达FET的条件后，我们将使用FARE启动子探针从细胞裂解产物中捕获DNA结合蛋白，并鉴定控制其表达的调控因子的谱。影响和意义：该计划将为通过TetR家族调节器激活RND外排泵表达定义一个新的调控范例，同时定义FET在细胞内稳态中的新角色，并确定其他调节FET表达的调节器，以响应环境压力和细菌的营养状态。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，以及如何通过对代谢信号做出反应的转录调节来实现这一点。

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.