Subminimum inhibitory concentrations of rifampicin attenuate methicillin-resistant staphylococcus aureus virulence by suppressing SaeRS two-component system and arginine metabolism-related pathways

Objectives

Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant threat to global public health, prompting the exploration of alternative strategies to mitigate its virulence. This study investigates the impact of subminimum inhibitory concentrations (sub-MICs) of rifampicin on MRSA virulence, aiming to provide insights for optimizing antibiotic treatment strategies.

Methods

Enzyme-linked immunosorbent assay and western blot analysis were used to assess α-hemolysin expression. Transcriptomic sequencing and RT-qPCR analyzed gene expression changes in MRSA treated with sub-MICs of rifampicin. Mutant strains (ΔsaeR and ΔargGH) were constructed to validate the roles of the SaeRS system and arginine metabolism. Thermal shift assays evaluated the interaction between L-arginine and SaeR protein. In vivo murine models and Galleria mellonella infection models were used to assess the anti-virulence effects of rifampicin.

Results

Our findings reveal that sub-MICs of rifampicin significantly reduce the expression of MRSA α-hemolysin. Transcriptomic sequencing and RT-qPCR analysis suggest a dual-pathway mechanism, wherein rifampicin suppresses virulence by indirectly inhibiting the SaeRS two-component system and disrupting arginine metabolism-related pathways. The construction of a saeR knockout mutant (ΔsaeR) and an arginine biosynthesis deficient mutant (ΔargGH) further supports this mechanism. Notably, exogenous l-arginine supplementation reverses rifampicin's inhibitory effect on α-hemolysin expression, underscoring the pivotal role of l-arginine metabolism in MRSA virulence regulation. Thermal shift assays demonstrate a direct interaction between l-arginine and SaeR protein, elucidating the intricate interplay between metabolic pathways and virulence regulation. In vivo studies confirm that sub-MICs of rifampicin attenuate the severity of skin abscesses in a murine model, improve survival rates in bloodstream infection models, and mitigate inflammation in both skin and lung tissues.

Conclusion

This study highlights the potential of rifampicin as an anti-virulence agent and pave the way for the development of innovative therapeutic strategies targeting MRSA infections.