The virulence of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), depends on the expression of toxins and virulence factors controlled by the quorum-sensing (QS) system, encoded on the virulence accessory gene regulator (agr) locus. The aim of this study was to identify a phytochemical that inhibits Agr-QS function and to elucidate its mechanism. We screened 577 compounds and identified physalin H, physalin B, and isophysalin B—–phytochemicals belonging to physalins found in plants of the Solanaceae family—–as novel Agr-QS modulators. Biological analyses and in vitro protein–DNA binding assays suggested that these physalins suppress gene expression related to the Agr-QS system by inhibiting binding of the key response regulator AgrA to the agr promoters, reducing the function of hemolytic toxins downstream of these genes in MRSA. Furthermore, although physalin F suppressed gene expression in the Agr-QS system, its anti-hemolytic activity was lower than that of physalins H, B, and isophysalin B. Conversely, five physalins isolated from the same plant with the ability to suppress Agr-QS did not reduce bacterial Agr-QS activity but inhibited AgrA binding to DNA in vitro. A docking simulation revealed that physalin interacts with the DNA-binding site of AgrA in three docking states. The carbonyl oxygens at C-1 and C-18 of physalins, which can suppress Agr-QS, were directed to residues N201 and R198 of AgrA, respectively, whereas these carbonyl oxygens of physalins, without Agr-QS suppression activity, were oriented in different directions. Next, 100-ns molecular dynamics simulations revealed that the hydrogen bond formed between the carbonyl oxygen at C-15 of physalins and L186 of AgrA functions as an anchor, sustaining the interaction between the carbonyl oxygen at C-1 of physalins and N201 of AgrA. Thus, these results suggest that physalin H, physalin B, and isophysalin B inhibit the interaction of AgrA with the agr promoters by binding to the DNA-binding site of AgrA, suppressing the Agr-QS function of S. aureus. Physalins that suppress the Agr-QS function are proposed as potential lead compounds in the anti-virulence strategy for MRSA infections.

中文翻译：

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Physalin H、Physalin B 和 isophysalin B 通过与 AgrA 结合来抑制金黄色葡萄球菌的群体感应功能


金黄色葡萄球菌（包括耐甲氧西林金黄色葡萄球菌 （MRSA））的毒力取决于由群体感应 （QS） 系统控制的毒素和毒力因子的表达，编码在毒力辅助基因调节因子 （agr） 基因座上。本研究的目的是确定一种抑制 Agr-QS 功能的植物化学物质并阐明其机制。我们筛选了 577 种化合物，并确定了 physalin H、physalin B 和 isophysalin B——在茄科植物中发现的属于 physalins 的植物化学物质——作为新的 Agr-QS 调节剂。生物学分析和体外蛋白质-DNA 结合测定表明，这些植酸盐蛋白通过抑制关键反应调节因子 AgrA 与 agr 启动子的结合来抑制与 Agr-QS 系统相关的基因表达，从而降低 MRSA 中这些基因下游溶血毒素的功能。此外，虽然植酸菌素 F 抑制了 Agr-QS 系统中的基因表达，但其抗溶血活性低于植酸菌素 H、B 和异藻菌素 B。相反，从同一植物中分离的 5 种具有抑制 Agr-QS 能力的植酸菌素并没有降低细菌 Agr-QS 活性，但在体外抑制了 AgrA 与 DNA 的结合。对接模拟显示，藻类素以三种对接状态与 AgrA 的 DNA 结合位点相互作用。植酸素的 C-1 和 C-18 位点的羰基氧可以抑制 Agr-QS，分别指向 AgrA 的残基 N201 和 R198，而植酸素的这些羰基氧没有 Agr-QS 抑制活性，则指向不同的方向。 接下来，100 ns 分子动力学模拟表明，植酸菌素 C-15 的羰基氧与 AgrA 的 L186 之间形成的氢键起锚的作用，维持植酸菌素 C-1 的羰基氧与 AgrA 的 N201 之间的相互作用。因此，这些结果表明，植酸菌素 H、植酸菌素 B 和异藻菌素 B 通过与 AgrA 的 DNA 结合位点结合来抑制 AgrA 与 agr 启动子的相互作用，从而抑制金黄色葡萄球菌的 Agr-QS 功能。抑制 Agr-QS 功能的 Physalins 被提议作为 MRSA 感染抗毒力策略中的潜在先导化合物。