Subinhibitory concentration of clarithromycin targets SaeR to reduce the haemolytic activity of Staphylococcus aureus

OBJECTIVES: This study aimed to investigate the anti-virulence effects of subinhibitory concentration of clarithromycin on Staphylococcus aureus and to explore the underlying molecular mechanism. METHODS: Haemolytic activity was measured using rabbit erythrocytes. α-haemolysin production was assessed by Western blot analysis. Transcriptional changes were analysed by RT-qPCR and promoter activity assays. To verify the target mechanism, a ΔsaeR mutant and a complemented strain were constructed. Molecular docking and DARTS assay were employed to evaluate the potential clarithromycin-SaeR interaction. Substitution of a single residue, Arg154, with alanine was performed to determine its functional role. In vivo efficacy of clarithromycin was assessed using the Galleria mellonella infection model. RESULTS: Clarithromycin at 1/8MIC reduced haemolytic activity in methicillin-resistant S. aureus strains MR200 and MR271 from 83.61% ± 0.86% to 9.05% ± 4.13% and from 85.12% ± 1.95% to 45.24% ± 1.06%, respectively, and significantly decreased α-haemolysin production. Clarithromycin selectively downregulated saeR, with a 1.87-fold reduction in MR200 and 1.54-fold reduction in MR271, while the agr pathway remained largely unaffected. Promoter activity assays confirmed suppression of the saeR-P1 promoter. Haemolytic activity and α-haemolysin production were significantly reduced in the ΔsaeR mutant, and clarithromycin caused no additional inhibition, and these effects were restored upon saeR complementation. Molecular docking and DARTS assay suggested a potential SaeR-clarithromycin binding. Arg154 substitution eliminated both SaeR regulatory activity and drug-mediated virulence suppression. In vivo, 1/8MIC clarithromycin improved larval survival to over 90%. CONCLUSIONS: This study demonstrated that clarithromycin exerted an anti-virulence effect against S. aureus by targeting the SaeR, highlighting a potential strategy for repurposing antibiotics to mitigate bacterial pathogenicity.