| Staphylococcus aureus is a major human pathogen that can cause a variety of infections due to the diverse array of virulence factors it produces. In recent years, the number of S. aureus infections has dramatically increased for a variety of reasons including more invasive surgeries. Because of this, new anti-staphylococcal therapies need to be developed. Expression of virulence factors is coordinated temporally with surface proteins being produced during early exponential growth and exoproteins being produced during late exponential and stationary growth. This switch in gene expression is primarily regulated by the loci sarA (staphylococcal accessory regulator) and agr (accessory gene regulator). SarA, a DNA-binding protein produced by the sarA locus, regulates expression of many virulence genes including hla, sspA, cna, and fnbA. SarA also interacts with the agr promoter region and enhances expression of the RNAIII transcript, a regulatory RNA molecule that upregulates exoprotein production. In this study, we identified two amino acids, Cys9 and Lys28, necessary for SarA function in vivo and in vitro. Cys9 is the only cysteine present in SarA and is located in the DNA-binding cleft, and Lys28 may be involved in SarA dimerization. To identify SarA/agr interactions necessary for P3 activity, we introduced mutations into SarA binding sites within the agr regulatory region. The A2 half-site is required for P3 activation, and mutagenesis of this site abolished promoter activity and reduced SarA binding. We examined SarA activity by EMSA during the in vitro growth cycle and concluded that SarA was capable of DNA-binding throughout growth. Finally, we refined a consensus sequence for SarA binding by SELEX (systematic evolution of ligands by exponential enrichment). The consensus site ATTTTAT was located within sarA-regulated promoter regions, and mutagenesis of this site within agr resulted in a decrease in affinity by SarA. These studies shed new light on SarA function and SarA gene targets and contribute to a better understanding of how SarA is involved in virulence gene regulation. |
