Quorum Sensing Inhibitor-Antibiotic Conjugate Suppresses Virulence and Eradicates Intracellular Staphylococcus aureus

Staphylococcus aureus ( S. aureus ) employs an array of virulence factors, predominantly governed by the accessory gene regulator (agr) quorum sensing (QS) system, to potentiate infection severity. Particularly challenging is the capacity of intracellular S. aureus to evade both antibiotic treatment and host immune clearance, often resulting in chronic or recurrent infections that complicate pathogen eradication in vivo . To overcome these limitations, we report the rational design of a QS inhibitor-antibiotic conjugate (QSI-A) for simultaneous attenuation of virulence and significant reduction of intracellular S. aureu s. This conjugate integrates synthetic autoinducing peptide (AIP) analogues-potent inhibitors of agr QS-with a rifalogue antibiotic via cathepsin-cleavable linkers, supporting intracellular antibiotic release within the proteolytic milieu of host cells. Using qRT-PCR, hemolysis assays, and LC-MS, we demonstrate that QSI-A markedly suppresses the expression of key agr components ( agrA and RNAIII ) and major virulence genes (e.g., psmα , hla , pvl ), while substantially reducing δ -toxin production and markedly suppressing hemolytic and cytolytic activities. Mechanistic studies reveal that QSI-A efficiently enters infected host cells-either through direct uptake or co-internalization with bacteria-and liberates the rifalogue intracellularly, leading to effective killing of intracellular S. aureus . In a murine bacteremia model, QSI-A treatment significantly reduced bacterial loads, underscoring its therapeutic potential. These results establish QSI-A as a dual-mechanism anti-infective strategy that concurrently silences virulence expression and targets intracellular reservoirs. Moreover, this QSI-antibiotic conjugate platform holds broad applicability for treating other pathogens dependent on QS-regulated virulence.