槲皮素对金黄色葡萄球菌凝固酶的抑制作用及机制

Staphylococcus aureus (S. aureus) is a zoonotic pathogenic Gram-positive bacterium, causing purulent infections and posing a serious threat to human health. Many antibiotics can be used for the treatment of these infections. However the antibiotic-resistant strains were isolated rapidly due to the massive unregulated use of antibiotics. Drugs targeting virulence is an alternative approach to treat infections due to bacteria. Clinical isolates of S. aureus secrete coagulases, polypeptides that bind to and activate prothrombin, thereby converting fibrinogen to fibrin and promoting the clotting of plasma or blood. Two staphylococcal products, the canonical coagulase (Coa) as well as the recently identified von Willebrand factor binding protein (vWbp), which leads to the formation of abscesses and bacterial persistence in host tissues and also enables the pathogen to cause lethal sepsis. Circumstantial evidence suggests that the product of coagulase activity, staphylococci captured within a fibrin meshwork, enable this pathogen to disseminate as thromboembolic lesions and to resist opsonophagocytic clearance by host immune cells. Preclinical evidence suggests that inactivation or neutralization of coagulases may prevent the pathogenesis of staphylococcal infections, a strategy that could be used to combat the current epidemic of hospital acquired infections with drug-resistant S. aureus isolates. This project takes quercetin as the object of study. At first, the effective inhibitory dose against coagulases in S. aureus was determined by in vitro experiments, and then S. aureus infection animal model with coagulases as the main virulence factors was made. With the help of these models we investigate the protective and therapeutic activity of quercetin on S. aureus infection. Furthermore, molecular docking and dynamics simulation was used to find the binding site of quercetin and coagulases ,and to explore the mechanism of quercetin in treatment of S. aureus infections by inhibiting coagulases enzyme activity. This work will provide theoretical foundation and scientific basis for the development of drug against novel target of S. aureus.

金黄色葡萄球菌是一种重要的人兽共患病原菌，滥用抗生素导致其对各种抗生素耐药率逐年上升，迫切需要开拓新的抗细菌感染策略。金葡菌可以分泌两种凝固酶-凝固酶(Coa)和von-Wilkbrand因子结合蛋白(vWbp)，能够结合并激活凝血酶原，将纤维蛋白原转化为纤维蛋白，有助于金葡菌抵抗吞噬细胞的吞噬，促使感染局限化和血栓形成，最终导致脓肿的形成进而引发致死性败血症。临床前证据表明，凝固酶的失活可能会阻止金葡菌感染后的发病。我们通过对大量抗感染天然药物的筛选，发现槲皮素能够明显抑制血液凝固反应。本项目以槲皮素为研究对象，首先通过体外实验确定其有效剂量，建立以凝固酶为主要毒力因子的金葡菌感染动物模型，考察药物对金葡菌感染的保护和治疗作用。然后通过分子对接和动力学模拟的方法确定槲皮素与凝固酶相互作用的结合位点，阐明槲皮苷通过抑制凝固酶活性治疗感染的机制，为以凝固酶为靶点的新型抗菌药研发奠定理论基础。

金黄色葡萄球菌（Staphylococcus aureus，简称金葡菌），是一种常见的医院和社区获得性病原菌，严重威胁着人类的健康。然而随着抗菌药的过度使用，细菌耐药性愈加严重，使我们临床可供选择的抗菌药越来越少。因此，迫切需要寻找新型的抗菌策略以减少细菌耐药性的产生。金葡菌凝固酶Coa是导致金葡菌感染一种重要的毒力因子。Coa可结合并激活凝血酶原，将纤维蛋白原转化为纤维蛋白。形成的纤维蛋白网络可保护金葡菌逃避宿主免疫吞噬。因此，以Coa作为靶点，筛选其抑制剂对抗金葡菌感染有重要意义。. 本研究采用试管凝血法筛选能够延长Coa凝血时间的中药单体，从而筛选出槲皮素作为研究对象，研究槲皮素对体外构建的Coa蛋白的凝血活性抑制作用及机制，并进一步观察其对体内外金葡菌导管相关性感染的治疗作用。研究通过试管凝血法和琼脂糖血平板凝血法验证发现，槲皮素能够延长Coa凝血时间和降低Coa凝血区域；Thermal Shift Assay发现，槲皮素作用下，Coa的Tm值与没有加药组相比，升高6℃；进一步通过计算机分子对接和分子动力学模拟阐述了结合模式，又通过点突变和荧光淬灭分析发现突变蛋白Pro-227和His-228结合常数和结合位点数较Coa明显降低；最终通过构建体内外金葡菌导管相关性感染模型，发现槲皮素显著降低了金葡菌在体外导管表面的黏着，显著降低了导管和肾脏了载菌量，并减轻了肾脓肿。. 综上，槲皮素对金葡菌Coa活性有抑制作用；槲皮素和Coa发生相互作用，提高Coa的热稳定性；槲皮素以一个紧凑的构象结合在Coa结构弯曲处的“中心腔”上，推测作用位点是残基Pro-227和His-228；槲皮素可通过抑制金葡菌Coa活性而抗金葡菌导管相关性感染，为抗金葡菌感染新药设计研发提供了理论依据和实验证据。