Active components of Patrinia scabiosifolia and mechanisms of action against methicillin-resistant Staphylococcus Epidermidis.

INTRODUCTION: Staphylococcus epidermidis is a significant cause of hospital bloodstream infections, often transmitted through medical devices. Methicillin-resistant Staphylococcus epidermidis (MRSE) exhibits multidrug resistance, challenging clinical treatment. Current options are limited, with vancomycin as the primary choice despite its negative effects. Traditional Chinese Medicine (TCM) is a valuable resource for novel antibiotics; Our research showed Patrinia scabiosifolia has potent anti-MRSE activity. TCM's efficacy stems from active components, but active components and mechanisms of Patrinia scabiosifolia are unclear. Arginine deiminase (AD) directly promotes bacterial growth by participating in protein synthesis, energy metabolism, and intermediate product formation, serving as a key substance for bacterial adaptation and survival. Our preliminary results demonstrated that Patrinia scabiosifolia can effectively inhibit the expression of AD in MRSE. Thus, we propose that AD serves as the antibacterial target of Patrinia scabiosifolia against MRSE. TCM's efficacy stems from active components, but active components and mechanisms of Patrinia scabiosifolia are unclear. Consequently, it has emerged as a potential target for combating bacterial and drug-resistant infections. Therefore, this study is designed to screen active components of Patrinia scabiosifolia targeting AD and further investigate the underlying antibacterial mechanisms.

METHODS: To validate the hypothesis, we constructed an arcA deletion strain (ΔarcA), in which the arcA gene encoding the AD protein was deleted via homologous recombination. Antimicrobial experiments in vitro and in vivo confirmed it as one target of Patrinia scabiosifolia. Furthermore, the active components of Patrinia scabiosifolia with anti-MRSE activity were screened by molecular docking and molecular dynamics (MD) simulations, using AD as the molecular target. In addition, the in vitro and in vivo antibacterial activities of the active components from Patrinia scabiosifolia were investigated. At last, the regulatory effect of the active components of Patrinia scabiosifolia on AD was determined by measuring the expression of the metabolite arcA gene and ornithine; Meanwhile, the binding effect of the active components of Patrinia scabiosifolia on AD was confirmed via determining the binding of AD to these components by microscale thermophoresis (MST), as well as identifying the binding sites through molecular simulation. Finally, nitric oxide (NO) and catalase (CAT) were determined, which further illustrates the antibacterial mechanism of the active components of Patrinia scabiosifolia targeting AD.

RESULTS: This study successfully constructed an MRSE arcA-deletion strain (ΔarcA), whose arcA gene encodes the AD protein, confirming that the deletion strain exhibited no growth defects. The minimum inhibitory concentration (MIC) of Patrinia scabiosifolia against MRSE was 5 mg/mL, whereas its MIC against ΔarcA was 10 mg/mL. Through MD simulations, 21 active components with anti-MRSE activity were screened from Patrinia scabiosifolia, among which hexadecanal demonstrated high antibacterial activity-showing a MIC of 7.81 μg/mL and a minimum bactericidal concentration (MBC) of 125 μg/mL against MRSE, and a MIC of 31.25 μg/mL against ΔarcA with no detectable MBC. Measurements of arcA gene expression and ornithine levels in MRSE treated with or without hexadecanal indicated that hexadecanal significantly downregulated arcA gene expression and reduced ornithine levels. MST and molecular simulation experiments further confirmed that hexadecanal binds to AD with high affinity, and identified Phe148 as its key binding site. In-depth studies revealed that hexadecanal targets AD, leading to marked reductions in NO and CAT levels.

CONCLUSIONS: This study elucidates that hexadecanal serves as the active components of Patrinia scabiosifolia targeting AD, and clarifies its antibacterial mechanism via binding to AD to regulate AD function and subsequently initiates oxidative stress.