金黄色葡萄球菌粘附素SraP糖基化的研究

It is predicted that Asp1、GtfA and GtfB function as glycosyltransferases in staphylococcus aureus by Protein Homology/analogY Recognition Engine (PHYRE) program.Gene neigborhood predicts that GtfA binds GtfB, and Asp1 interacts Asp3，etc..Primary study showed:(1)SraP was glycosylated by GtfA coexpressing with GtfB;(2)GtfA interacting with GtfB was demonstrated using pull-down assays;(3)Asp1 binding to Asp3 was identified using affinity purification.Therefore it is suspected that Asp1、GtfA,GtfB and their interactive partners form enzyme complexs to regulate glycosylation of Srap.In future,gene replacement and mass spectrometer will be used to demonstrate further that GtfA,GtfB and Asp1 of staphylococcus aureus functions as a glycosyltransferase,and Co-IP will be used to confirm enzyme complexes GtfAB and Asp13 in staphylococcus aureus. Then GtfA△I mutant and Asp1△I mutant（△I means deletion of interactive domain） will be constructed to analyse that whether the separation of enzyme complexes GtfAB and Asp13 has an effect on the glycosylation of SraP,influences on the ability which staphylococcus aureus adhere platelet and cause infective endocarditis.Therefore,the mechanism for regulating SraP glycosylation will be disclosed in this study,and moreover,scientific evidences will be provided for inhibiting adhesion and pathogenosis from staphylococcus aureus.

PHYRE软件预测金葡菌的Asp1、GtfA和GtfB具有糖基化转移酶功能，基因邻居法推测GtfB与GtfA，Asp1与Asp3等蛋白相互作用。预实验显示：（1）SraP在GtfA和GtfB共存时能被糖基化；（2）Pull-down实验证实GtfA和GtfB相互作用；（3）亲和纯化发现Asp1与Asp3相互作用，故GtfA、GtfB和Asp1等应通过相互作用组成酶复合物调控Srap的糖基化。未来将采用基因置换与质谱分析等方法进一步证实GtfA、GtfB和Asp1等具有糖基化转移酶功能，并通过Co-IP证实金葡菌体内存在酶复合物GtfAB和Asp13，然后构建GtfA△I和Asp1△I突变株（△I表示无相互作用结构域）,分析酶复合物解离对Srap糖基化、金葡菌粘附血小板以及其致感染性心内膜炎能力的影响。因此本研究不仅可明确Srap的糖基化调控机制，而且为抑制金葡菌的粘附与致病提供科学依据。

Srap,一种O-糖基化的糖蛋白，是金黄色葡萄球菌最重要的粘附素致病因子。在本研究中，（1）我们首先采用基因同源重组技术获得金黄色葡萄球菌N315的GtfA和GtfB突变株，sWGA凝集素印迹试验显示N315的GtfA和GtfB突变株均不能糖基化Srap，同时构建了大肠杆菌SraP的糖基化模型，发现SraP只有在GtfA和GtfB共存的情况下才能被糖基化，进一步支持GtfA和GtfB为糖基化转移酶；（2）由于N315的GtfA和GtfB突变株具有相同表型，因此我们采用酵母双杂交技术、GST PULL-DOWN技术以及Co-IP技术检测GtfA和GtfB的相互作用，3种方法均证实GtfA和GtfB存在相互作用，而且GtfA的DUF1975未其相互作用的重要结构域；（3）酶复合物GtfAB解离不能检测到糖基化的SraP，说明GtfAB的完整是SraP糖基化的重要保障；（4）酶复合物GtfAB解离导致金葡菌粘附能力下降明显；（5）酶复合物GtfAB解离不影响体外金葡菌生物膜的形成与其生长能力。