Development of a dual fluorescent reporter system to identify inhibitors of Staphylococcus aureus virulence factors.

Staphylococcus aureus is a leading cause of bacterial infections due to its resistance to most antibiotics and the production of diverse virulence factors. Various alternative approaches to treating staphylococcal infections have been proposed, including targeting the virulence factors or mechanisms that regulate their synthesis. The accessory gene regulator (agr) and the S. aureus exoprotein expression (sae) are two major virulence regulators of S. aureus. Here, we describe a dual reporter system capable of simultaneously monitoring the transcriptional activities of agr and sae in S. aureus. The reporter plasmid contained two fluorescent protein cassettes arranged back-to-back, with the mCherry cassette driven by the agrP3 promoter and the GFP cassette driven by the sbi promoter. We examined the fluorescent responses of wild-type, agr, and sae mutant strains using fluorescence microscopy and found that the activities of agr and sae systems could be detected without significant crosstalk in the dual reporter strain. The dual reporter system could be used to monitor the dynamic changes in agr and sae activities during batch growth in S. aureus. Additionally, we evaluated the responsiveness of the reporter strain to virulence inhibitors by treating it with several known inhibitors, including autoinducing peptides (AIPs) containing culture supernatants, indole, and flavone. The results suggested that the dual reporter system was sensitive to both agr and sae inhibitors. This reporter construct enables the characterization of the activity of a substance against two different targets in one screening round, significantly reducing screening time and expense.IMPORTANCEStaphylococcus aureus is a formidable pathogen responsible for a wide range of infections, and the emergence of antibiotic-resistant strains has posed significant challenges in treating these infections. In this study, we have established a novel dual reporter system capable of concurrently monitoring the activities of two critical virulence regulators in S. aureus. By incorporating both reporters into a single screening platform, we provide a time- and cost-efficient approach for assessing the activity of compounds against two distinct targets in a single screening round. This innovative dual reporter system presents a promising strategy for the identification of molecules capable of modulating virulence gene expression in S. aureus, potentially expediting the development of antivirulence therapies.