The fadXDEBA locus of Staphylococcus aureus is required for metabolism of exogenous palmitic acid and in vivo growth.

In Staphylococcus aureus, genes that should confer the capacity to metabolize fatty acids by ß-oxidation occur in the fadXDEBA locus, but their function has not been elucidated. Previously, incorporation into phospholipid through the fatty acid kinase FakA pathway was thought to be the only option available for S. aureus to metabolize exogenous saturated fatty acids. We now find that in S. aureus USA300, a fadX::lux reporter was repressed by glucose and induced by palmitic acid but not stearic acid, while in USA300ΔfakA basal expression was significantly elevated, and enhanced in response to both fatty acids. When cultures were supplemented with palmitic acid, palmitoyl-CoA representing the first metabolite in the ß-oxidation pathway was detected in USA300, but not in a fadXDEBA deletion mutant USA300Δfad, which relative to USA300 exhibited increased incorporation of palmitic acid into phospholipid accompanied by a rapid loss of viability. USA300Δfad also exhibited significantly reduced viability in a murine tissue abscess infection model. Our data are consistent with FakA-mediated incorporation of fatty acids into phospholipid as a preferred pathway for metabolism of exogenous fatty acids, while the fad locus is critical for metabolism of palmitic acid, which is the most abundant free fatty acid in human plasma.

Staphylococcus aureus Fatty Acid Kinase FakA Modulates Pathogenesis during Skin Infection via Proteases.

Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro, and that the ΔfakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla ΔfakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the ΔfakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the ΔfakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the ΔfakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in ΔfakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δaur and ΔsspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the ΔfakA mutant during skin infection.

Inactivation of the exogenous fatty acid utilization pathway leads to increased resistance to unsaturated fatty acids in Staphylococcus aureus.

The human pathogen Staphylococcus aureus produces saturated fatty acids, but can incorporate both exogenous saturated and unsaturated fatty acids into its lipid membrane. S. aureus encounters unsaturated fatty acids in the host skin where they serve as an innate immune defence due to their toxicity. Previously, we identified a fatty acid kinase in S. aureus that is necessary for the utilization of exogenous fatty acids. The goal of this study was to determine the effects of fatty acids on mutants deficient in the exogenous fatty acid utilization machinery. We have demonstrated that mutants lacking a functional fatty acid kinase (fakA) or both fatty acid carrier proteins (fakB1 fakB2) are more resistant to unsaturated fatty acids. Previous studies suggested a role for ammonia-producing enzymes in resistance to unsaturated fatty acids, but these enzymes do not contribute to the resistance of the fakA mutant, despite increased urease transcription and protein activity in the mutant. Additionally, while pigment is altered in mutants unable to use exogenous fatty acids, staphyloxanthin does not contribute to fatty acid resistance of an fakA mutant. Because exposure to unsaturated fatty acids probably initiates a stress response, we investigated the role of the alternative sigma factor σB and determined if it is necessary for the fatty acid resistance observed in the fakA mutant. Collectively, this study demonstrates that the inability to incorporate unsaturated fatty acids leads to increased resistance to those fatty acids, and that resistance requires a σB stress response.

Redirection of Metabolism in Response to Fatty Acid Kinase in Staphylococcus aureus.

Staphylococcus aureus is capable of phosphorylating exogenous fatty acids for incorporation into the bacterium's membrane via the fatty acid kinase, FakA. Additionally, FakA plays a significant role in virulence factor regulation and skin infections. We previously showed that a fakA mutant displays altered growth kinetics in vitro, observed during the late-exponential phase of growth. Here, we demonstrate that the absence of FakA leads to key metabolic changes. First, the fakA mutant has an altered acetate metabolism, with acetate being consumed at an increased rate than in the wild-type strain. Moreover, the growth benefit was diminished with inactivation of the acetate-generating enzyme AckA. Using a mass spectrometry-based approach, we identified altered concentrations of tricarboxylic acid (TCA) cycle intermediates and both intracellular and extracellular amino acids. Together, these data demonstrate a change in carbohydrate carbon utilization and altered amino acid metabolism in the fakA mutant. Energy status analysis revealed the mutant had a similar ADP/ATP ratio to that of the wild type, but a reduced adenylate energy charge. The inactivation of fakA changed the NAD+/NADH and NADP+/NADPH ratios, indicating a more oxidized cellular environment. Evidence points to the global metabolic regulatory proteins CcpA and CodY being important contributors to the altered growth in a fakA mutant. Indeed, it was found that directing amino acids from the urea cycle into the TCA cycle via glutamate dehydrogenase was an essential component of S. aureus growth after glucose depletion. Together, these data identify a previously unidentified role of FakA in the global physiology of S. aureus, linking external fatty acid utilization and central metabolism.IMPORTANCE The fatty acid kinase, FakA, of Staphylococcus aureus plays several important roles in the cell. FakA is important for the activation of the SaeRS two-component system and secreted virulence factors like α-hemolysin. However, the contribution of FakA to cellular metabolism has not been explored. Here, we highlight the metabolic consequence of removal of FakA from the cell. The absence of FakA leads to altered acetate metabolism and altered redox balance, as well as a change in intracellular amino acids. Additionally, the use of environmental amino acid sources is affected by FakA. Together, these results demonstrate for the first time that FakA provides a link between the pathways for exogenous fatty acid use, virulence factor regulation, and other metabolic processes.