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

耐药-结瘤-分裂家族外排泵在金黄色葡萄球菌生理和耐药机制中的作用和调节

• 批准号: RGPIN-2022-03934
• 负责人: McGavin, Martin
• 金额: $ 2.48万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761039
• 关键词: Role; regulation; Resistance; Nodulation; Division

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.

RND外排泵包括一个古老的转运蛋白家族，在微生物生理中具有关键作用。我们在金黄色葡萄球菌中发表了RND泵功能的第一个描述，该葡萄球菌由TERM家族调节器（TFR）FARR组成，并发出了发散的排出泵票价，该泵允许对抗菌不饱和的非饱和游离脂肪酸（UFFA）的耐药性。这种发散的布置通常表示TFR功能抑制外排泵，但我们发现Farr充当激活器。我们的计划将FARR和票价作为RND外排系统的重点，以获得对关键微生物过程的新见解，包括新型的调节范式以及对RND泵如何促进细胞稳态的更深入的基本了解。我们将：1。FARR的结构和生物物理表征的结构和生物物理表征，以了解DNA结合如何通过磷酸酰基配体调节。 2.确定票价生理功能的范围3.定义代谢信号如何调节票价表达。 AIM 1定义Farr如何激活票价表达。基于FARR与亚油酸复合物中的模型结构，我们将评估特定氨基酸在结合酰基磷酸盐中的作用，以及如何修饰DNA结合。这将由长期目标来支持FARR的晶体结构，以及与酰基磷酸酰基和DNA的复合物的支持。 AIM 2是基于知道RND泵可以容纳一系列底物的知识，并且初步数据表明，在化学定义的最小培养基（CDM）中，较大的突变体在扩展孵育时表现出生存力丧失。我们将使用脂肪组学和代谢组学来识别由于票价失活或构成表达而改变的细胞和分泌代谢产物。这些实验应定义票价如何有助于维持有毒代谢物的膜完整性和外排。 AIM 3扩大了票价功能与代谢活性的相关性。我们将确定何时在CDM中最佳表达票价，并进行RNASEQ以确定票价失活时的基因表达如何改变。在确定具有不同养分限制或抗菌UFFA的CDM中最佳表达的条件之后，我们将使用票价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剂。 影响和意义：该程序将定义一个新型的调节范式，以通过TERT家族调节器激活RND外排泵表达，同时定义了在细胞体内稳态中票价的新作用，并确定其他调节器，这些调节器可调节票价表达，以响应环境压力和对环境压力和响应细菌的营养状况。这将有助于更了解RND外排泵如何促进葡萄球菌中的基本生理过程，以及如何通过对代谢信号响应的转录调节器来实现这一目标。