Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China.
PLoS Comput Biol. 2013;9(1):e1002869. doi: 10.1371/journal.pcbi.1002869. Epub 2013 Jan 17.
Alpha-hemolysin (α-HL) is a self-assembling, channel-forming toxin produced by most Staphylococcus aureus strains as a 33.2-kDa soluble monomer. Upon binding to a susceptible cell membrane, the monomer self-assembles to form a 232.4-kDa heptamer that ultimately causes host cell lysis and death. Consequently, α-HL plays a significant role in the pathogenesis of S. aureus infections, such as pneumonia, mastitis, keratitis and arthritis. In this paper, experimental studies show that oroxylin A (ORO), a natural compound without anti-S. aureus activity, can inhibit the hemolytic activity of α-HL. Molecular dynamics simulations, free energy calculations, and mutagenesis assays were performed to understand the formation of the α-HL-ORO complex. This combined approach revealed that the catalytic mechanism of inhibition involves the direct binding of ORO to α-HL, which blocks the conformational transition of the critical "Loop" region of the α-HL protein thereby inhibiting its hemolytic activity. This mechanism was confirmed by experimental data obtained from a deoxycholate-induced oligomerization assay. It was also found that, in a co-culture system with S. aureus and human alveolar epithelial (A549) cells, ORO could protect against α-HL-mediated injury. These findings indicate that ORO hinders the lytic activity of α-HL through a novel mechanism, which should facilitate the design of new and more effective antibacterial agents against S. aureus.
α-溶血素（α-HL）是一种自组装的通道形成毒素，由大多数金黄色葡萄球菌菌株作为 33.2 kDa 的可溶性单体产生。在与易感细胞膜结合后，单体自组装形成 232.4 kDa 的七聚体，最终导致宿主细胞裂解和死亡。因此，α-HL 在金黄色葡萄球菌感染的发病机制中起着重要作用，如肺炎、乳腺炎、角膜炎和关节炎。在本文中，实验研究表明，小檗碱（ORO）是一种没有抗金黄色葡萄球菌活性的天然化合物，可抑制α-HL 的溶血活性。进行了分子动力学模拟、自由能计算和突变体测定实验，以了解α-HL-ORO 复合物的形成。这种综合方法表明，抑制的催化机制涉及 ORO 与 α-HL 的直接结合，从而阻止了α-HL 蛋白关键“Loop”区域的构象转变，从而抑制其溶血活性。这一机制得到了去氧胆酸盐诱导的寡聚化测定实验获得的实验数据的证实。还发现，在金黄色葡萄球菌和人肺泡上皮（A549）细胞的共培养系统中，ORO 可以防止 α-HL 介导的损伤。这些发现表明，ORO 通过一种新的机制阻碍了α-HL 的裂解活性，这应该有助于设计针对金黄色葡萄球菌的新型和更有效的抗菌剂。
