Evaluating the role of a lipoic acid transfer protein in Staphylococcus aureus metabolism and virulence

PROJECT SUMMARY Staphylococcus aureus asymptomatically colonizes 30% of the human population. However, the bacterium can breach innate host defenses to gain access to deeper tissues and vasculature allowing it to infect diverse tissue sites such as the heart, lungs, blood, bones, and skin. Despite distinct nutritional limitations at these sites, we know comparatively little about how S. aureus adapts its metabolism to survive and cause infection. To better combat infections caused by S. aureus, it is imperative to understand how the bacterium changes metabolic flux to adapt to nutritional restrictions imposed by host tissues. Several metabolic enzyme complexes involved in S. aureus central metabolism are activated via the covalent linkage of the cofactor lipoic acid to E2 subunits. The transfer of lipoic acid to E2 subunits is mediated by the amidotransferase, LipL. Notably, LipL transfers lipoic acid to a critical enzyme used in glycolysis, pyruvate dehydrogenase. Pyruvate dehydrogenase use pyruvate to generate acetyl-CoA, a molecule that is crucial for several downstream metabolic pathways. Our prior studies and preliminary data established that LipL is genetically and functionally coupled to the phosphotransacetylase, Pta. Pta catalyzes the generation of acetyl-phosphate from pyruvate dehydrogenase-derived acetyl-CoA. Acetyl- phosphate is subsequently delivered to acetate kinase, AckA, to generate ATP during overflow metabolism. We recently demonstrated a potential interaction between Pta and LipL and found that a ∆lipL mutant produces negligible acetate in culture. Furthermore, the loss of either the pta or lipL gene results in significant attenuation in the host. Thus, we hypothesize that a functional link is established between Pta and LipL that enhances glycolytic flux by coupling pyruvate dehydrogenase activity (lipoylation) to acetogenesis (Pta-AckA) to promote energy balance and survival during infection (Aim 1). Additionally, we found that acetate production still occurs in a ∆pta mutant, suggesting compensatory enzymes - such as the pyruvate oxidase CidC - could be promoting acetogenesis and might affect virulence. Furthermore, we found that expression of lipL under the control of high and low expressing promoters regulated the transition from acetogenesis to TCA cycle activity. These results led us to hypothesize that LipL levels/activity establish a metabolite signature that controls (i) acetogenesis via CidC and (ii) TCA cycle activity to promote energy balance and S. aureus survival during infection (Aim 2). In Aim 1, we will determine how LipL interfaces with Pta to control energy balance during overflow metabolism. In Aim 2, we will investigate how LipL governs metabolic flux through CidC and the TCA cycle. Together, these Aims will lead to a better understanding of how LipL promotes metabolism, energy balance, and virulence in Sa.