Staphylococcus aureus is an opportunistic and formidable human pathogen implicated in a wide array of infections, ranging from benign cutaneous lesions to severe systemic diseases such as bacteremia, which are frequently associated with secondary complications such as pneumonia and endocarditis [1,2]. Antibiotic therapies are designed to combat S. aureus infections, which frequently fail due to antibiotic resistance [3]. To date, 4.95 million deaths have been attributed to antibiotic-resistant bacteria, of which 1.27 million have been directly caused by antibiotic-resistant bacteria, making them the third leading cause of death after stroke and heart disease [4]. Among them, multidrug-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) can be transmitted between humans, animals, and the environment, sounding an alarm for public health [5,6]. The increasing difficulty of treating such infections necessitates the exploration of additional control strategies. In response, several treatments, including antimicrobial peptides, immunotherapy and antivirulence agents, have been developed [7].

In this study, we focused on antivirulence strategies that impair S. aureus virulence without affecting its growth and exert less evolutionary pressure on bacteria than traditional strategies do, thereby reducing the risk of developing resistance. The complex and versatile virulence of S. aureus contributes to host adhesion and invasion [8,9]. By suppressing virulence factors, the ability of bacteria to colonize the host is reduced, which may enable host natural immunity to eliminate the attenuated pathogen. As it is difficult to attenuate the pathogenicity of S. aureus by inhibiting any single virulence gene or regulatory factor [10], simultaneous inhibition of multiple virulence factors would guard against infections via nongermicidal gateways, representing a potential strategy for controlling S. aureus infections.

On the basis of the above findings, we expect to develop a compound that targets multiple virulence factors. Therefore, a screening platform was established and applied to systematically screen inhibitors of large numbers of targets. In the screening process of hundreds of natural small-molecule libraries, we were pleasantly surprised that esculetin simultaneously inhibited sortase A (SrtA), coagulase (Coa) and von Willebrand factor-binding protein (vWbp), which all facilitate evasion of host immune defense [11]. By processing the LPXTG motif at specific threonine and glycine residues, SrtA covalently tethers surface proteins to the peptidoglycan matrix, enabling bacterial adherence, invasion, biofilm establishment, and immunity [12,13].

In addition, S. aureus alters the coagulation cascade reaction by secreting Coa and vWbp to usurp a coagulation factor (prothrombin), causing exuberant, uncontrolled polymerization of fibrin, which leads to blood clotting [14,15]. Fibronectin coats S. aureus and protects it from host immune phagocytosis, allowing it to survive for long periods and causing persistent infection [16]. Therefore, simultaneous inhibition of SrtA, Coa and vWbp impedes immune escape and protects the host against infection.

Virulence in S. aureus is governed by interlocked, partially redundant circuits; consequently, blocking a single regulator is often buffered by compensatory pathways. In contrast, agents that concurrently suppress multiple regulators or effector modules can achieve broader and more durable attenuation, underscoring the rationale for multitarget antivirulence therapy. After three rounds of panning through natural small-molecule libraries, esculetin (6,7-dihydroxycoumarin) was identified as the principal active ingredient in the traditional Chinese medicine Cortex Fraxini. Esculetin has been previously reported to possess anti-inflammatory, anticancer [17], and antihepatotoxic properties [18], as well as the ability to disrupt microbial biofilm formation [19]. However, a systematic evaluation of the effects of esculetin on multiple virulence factors was not conducted until our study. By inhibiting these three virulence factors, SrtA, Coa and vWbp, esculetin effectively disrupts a pathogen's ability to adhere, evade immune defenses, and form protective barriers, thus diminishing its overall virulence and pathogenicity (Graphical Abstract). This finding is particularly noteworthy, as esculetin is currently the only known natural product-derived multitarget inhibitor of these virulence factors. This discovery provides a new impetus for anti-Staphylococcus aureus research and highlights the broad potential applications of esculetin in this field.