Grant: Discovery Grants Program - Individual

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.