Efflux pump modulation by Montelukast and its roles in restoring antibiotic susceptibility in multidrug-resistant Staphylococcus aureus

Staphylococcus aureus and its drug-resistant mutants are mentioned among the WHO's high-priority list of pathogens. Antibiotics like fluoroquinolones and cephalosporins are used to treat multidrug-resistant S. aureus infections. However, a higher expression of efflux pumps (NorA, NorB, and AbcA) induces multidrug resistance. The master regulator, MgrA, regulates the expression of most of these efflux pumps in S. aureus. The phosphorylation status of MgrA is determined by the cellular PknB/RsbU ratio, where PknB, a serine-threonine kinase, and RsbU, a serine-threonine phosphatase, are critical for MgrA functioning. An FDA-approved drug library was screened using an EtBr-accumulation assay to identify efflux pump inhibitors (EPIs). The synergy of EPIs with antibiotics was studied in vitro and in vivo in the murine skin infection model of female BALB/c mice. The effect of EPIs on mgrA, norB, pknB, and rsbU gene expression, interaction with MgrA, and effects on MgrA phosphorylation were studied. We identified Montelukast as an effective EPI, which showed synergy with moxifloxacin, a substrate of the NorB efflux pump, both in vitro and in the murine skin infection model. Further, Montelukast decreased norB expression and increased the pknB/rsbU expression ratio. Our in vitro results demonstrated that Montelukast strongly interacted with MgrA, facilitated MgrA phosphorylation, and enhanced its affinity for the norB promoter. Our study showed that Montelukast repressed MgrA expression and promoted MgrA phosphorylation to suppress norB expression and efflux pump activity, leading to the restoration of antibiotic susceptibility in multidrug-resistant S. aureus. The study was supported by SERB-DST, India (CRG/2021/005069), and the BRIC-ILS core.