VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System

Abstract

In previous studies, we identified the fatty acid kinase virulence factor regulator B (VfrB) as a potent regulator of ␣-hemolysin and other virulence factors in Staphylococcus aureus. In this study, we demonstrated that VfrB is a positive activator of the SaeRS two-component regulatory system. Analysis of vfrB, saeR, and saeS mutant strains revealed that VfrB functions in the same pathway as SaeRS. At the transcriptional level, the promoter activities of SaeRS class I (coa) and class II (hla) target genes were downregulated during the exponential growth phase in the vfrB mutant, compared to the wild-type strain. In addition, saePQRS expression was decreased in the vfrB mutant strain, demonstrating a need for this protein in the autoregulation of SaeRS. The requirement for VfrB-mediated activation was circumvented when SaeS was constitutively active due to an SaeS (L18P) substitution. Furthermore, activation of SaeS via human neutrophil peptide 1 (HNP-1) overcame the dependence on VfrB for transcription from class I Sae promoters. Consistent with the role of VfrB in fatty acid metabolism, hla expression was decreased in the vfrB mutant with the addition of exogenous myristic acid. Lastly, we determined that aspartic acid residues D38 and D40, which are predicted to be key to VfrB enzymatic activity, were required for VfrB-mediated ␣-hemolysin production. Collectively, this study implicates VfrB as a novel accessory protein needed for the activation of SaeRS in S. aureus.IMPORTANCE The SaeRS two-component system is a key regulator of virulence determinant production in Staphylococcus aureus. Although the regulon of this twocomponent system is well characterized, the activation mechanisms, including the specific signaling molecules, remain elusive. Elucidating the complex regulatory circuit of SaeRS regulation is important for understanding how the system contributes to disease causation by this pathogen. To this end, we have identified the fatty acid kinase VfrB as a positive regulatory modulator of SaeRS-mediated transcription of virulence factors in S. aureus. In addition to describing a new regulatory aspect of SaeRS, this study establishes a link between fatty acid kinase activity and virulence factor regulation.

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

“…We also provide evidence supporting the FakA-dependent activity of CcpA, as shown through the altered transcription of gltA observed in the fakA mutant. It should be noted that we have recently reported that FakA is an activator of the SaeRS two-component system (30), while another group demonstrated that CodY binds to the sae P1 promoter (39). Thus, it was reasonable to postulate that Sae would be important for the growth phenotype observed in the fakA mutant.…”

“…The resulting PCR fragment was digested with EcoRI and SalI and ligated into the same sites of pJB185 to produce pZD3. ␤-Galactosidase activity was determined as previously described (30). Briefly, 1 ml of bacterial culture was collected after 3 or 6 h of growth.…”

“…FakA has been shown to regulate virulence factor production, such as that of ␣-hemolysin and several proteases (27), as well as impacting the type VII secretion system (29). We have shown that FakA mediates at least some of these effects through the activation of the SaeRS two-component system (30). In addition, FakA influences biofilm formation (31) by an unknown mechanism.…”

DNA Binding and Sensor Specificity of FarR, a Novel TetR Family Regulator Required for Induction of the Fatty Acid Efflux Pump FarE in Staphylococcus aureus

“…Although our data demonstrate that FakA is needed to induce farE in response to linoleic acid, paradoxically, USA300 ΔfakA exhibited an elevated basal level of farE expression sufficient to promote increased resistance to linoleic acid. This may be due to the pleiotropic phenotype ascribed to USA300 ΔfakA, including an elevated pool of nonesterified fatty acids, leading to reduced transcription of the SaeRS two-component regulator and defective production of Hla toxin (32)(33)(34). Broader metabolic changes were also noted, including altered carbon and amino acid metabolism (35).…”

Role of Fatty Acid Kinase in Cellular Lipid Homeostasis and SaeRS-Dependent Virulence Factor Expression in Staphylococcus aureus

“…A genome-wide analysis showed that FA kinase-null strains were specifically deficient in the expression of all virulence factors controlled by the SaeRS system ( 29 ). The fact that acetyl-phosphates are known to phosphorylate response regulators suggests that FA kinase may participate in the regulatory phosphorylation cascade in the SaeRS system ( 29 , 37 ). Although it is clear that transcription of the SaeRS virulence regulon is supported by a functional FA kinase, the connection between FA kinase and SaeRS has not been established.…”