Abstract 1197 NorA efflux pump inhibition in S. aureus using novel lead compounds in a DiOC3 real-time efflux assay

Quorum sensing is a mechanism utilized in bacterial strains to communicate between neighboring colonies and assess the strain's population density. Upon surpassing a population density threshold, this mechanism can trigger a population response, such as biofilm production, through the release of signal molecules. Biofilm is an extracellular collection of polysaccharides and proteins that has been observed to provide pathogenic bacteria protection from antibiotic treatment and the body's immune responses. Our lab synthesized and tested quorum sensing mimetic drug compounds for their potential to reduce or inhibit biofilm production in Staphylococcus aureus (S. aureus) through potential inhibition of receptor signaling. However, we predicted these tested drug compounds may also be reducing biofilm through inhibition of the NorA efflux pump, which has been observed to pump out biofilm forming components. Drug compounds Proline-61, Proline-68, and Tyrosine-16 were tested using a Crystal Violet Assay against three strains of S. aureus, a wildtype (SA-1199), an overexpressor of the NorA efflux pump (SA-1199B), and a knockout strain missing the NorA efflux pump (SA-K1758). Proline-61, Proline-68, and Tyrosine-16 displayed an inhibition level of 40.2%, 75.7%, and 46.3% respectively, compared to a DMSO control in the wildtype (SA-1199) strain. Inhibition in the overexpressor was observed at 55.1%, 46.4%, and 44.1% respectively. No significant inhibition in the knockout was observed. Further studies are needed to assess the specific activity of the NorA efflux pump. We have developed a fluorescence assay utilizing 3-dipropyloxacarbocyanine iodide, a known substrate of NorA, which will allow us to directly quantify NorA activity and its corresponding inhibition following exposure to Proline-61, Proline-68, and Tyrosine-16 in S. aureus.