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.

Automated summary

Here, a dual reporter system capable of monitoring the activities of two major virulence regulators in Staphylococcus aureus, agr and sae, was described. The system showed no significant crosstalk and could be used to monitor the dynamic changes in agr and sae activities during batch growth. The reporter strain was also responsive to virulence inhibitors, making it a valuable tool for screening potential drugs.