The evolution and spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant hidden risk to human public health. The majority of antibiotics used clinically have become mostly ineffective, and so the development of novel anti-infection strategies is urgently required. Since Staphylococcus aureus (S. aureus) cysteine transpeptidase sortase A (SrtA) mediates the surface-anchoring of proteins to its surface, compounds that inhibit SrtA are considered potential antivirulence treatments. Herein, we report on the efficacy of the potent SrtA inhibitor taxifolin (Tax), a flavonoid compound isolated from Chinese herbs. It was able to reversibly block the activity of SrtA with an IC50 of 24.53 ± 0.42 μM. Tax did not display toxicity toward mammalian cells or S. aureus at a concentration of 200 μM. In addition, Tax attenuated the virulence-related phenotype of SrtA in vitro by decreasing the adherence of S. aureus, reducing the formation of a biofilm, and anchoring of S. aureus protein A on its cell wall. The mechanism of the SrtA-Tax interaction was determined using a localized surface plasmon resonance assay. Subsequent mechanistic studies confirmed that Asp-170 and Gln-172 were the principal sites on SrtA with which it binds to Tax. Importantly, in vivo experiments demonstrated that Tax protects mice against pneumonia induced by lethal doses of MRSA, significantly improving their survival rate and reducing the number of viable S. aureus in the lung tissue. The present study indicates that Tax is a useful pioneer compound for the development of novel agents against S. aureus infections.

Recurrent epidemics of methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) have illustrated that the effectiveness of antibiotics in clinical application is rapidly fading. A feasible approach is to combine natural products with existing antibiotics to achieve an antibacterial effect. In this molecular docking study, we found that theaflavin (TF) preferentially binds the allosteric site of penicillin-binding protein 2a (PBP2a), inducing the PBP2a active site to open, which is convenient for β-lactam antibiotics to treat MRSA infection, instead of directly exerting antibacterial activity at the active site. Subsequent TMT-labeled proteomics analysis showed that TF treatment did not significantly change the landscape of the S. aureus USA300 proteome. Checkerboard dilution tests and kill curve assays were performed to validate the synergistic effect of TF and ceftiofur, and the fractional inhibitory concentration index (FICI) was 0.1875. The antibacterial effect of TF combined with ceftiofur was better than that of single-drug treatment in vitro. In addition, TF effectively enhanced the activity of ceftiofur in a mouse model of MRSA-induced pneumonia. Our findings provide a potential therapeutic strategy to combine existing antibiotics with natural products to resolve the prevalent infections of multidrug-resistant pathogens.

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Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Novel antimicrobial agents against MRSA are urgently being developed.

Due to powerful drug resistance and fatal toxicity of methicillin-resistant Staphylococcus aureus (MRSA), therapeutic strategies against virulence factors present obvious advantages since no evolutionary pressure will induce bacterial resistance. Alpha-hemolysin (Hla) is an extracellular toxin secreted by Staphylococcus aureus and contributes to bacterial pathogenicity. Herein, we identified a natural product 2,3-dehydrokievitone (2,3-DHKV) for inhibiting Hla activity of MRSA strain USA300 but not affecting bacteria growth. 2,3-DHKV significantly decreased hemolysin expression in a dose-dependent manner, but it did not potently neutralize hemolysin activity. Subsequently, cellular thermal shift and heptamer formation assays confirmed that 2,3-DHK affects hemolytic activity through indirect binding to Hla. RT-qPCR and western blot revealed that 2,3-DHKV suppressed Hla expression at the mRNA and protein levels, and further decreased accessory gene regulator A (agrA) transcription levels. We also observed that 2,3-DHK significantly attenuated the damage of A549 cells by S. aureus and reduced the release of lactate dehydrogenase (LDH). Moreover, in the MRSA-induced pneumonia mouse model, 2,3-DHK treatment prolonged the life span of mice and reduced the bacterial load in the lungs, which significantly alleviated the damage to the lungs. In summary, this study proved that 2,3-DHK as a Hla inhibitor is a potential antivirulence agent against MRSA infection.