Characterization of the importance of post-translational protein glycosylation in the pathogenesis of Staphylococcus aureus infections

迄今为止鉴定的大多数细菌糖蛋白是致病菌的毒力因子，即粘附素和侵入素。然而，蛋白糖基化对人类主要病原体金黄色葡萄球菌的影响在很大程度上仍然未知。我们发现纤溶酶敏感表面蛋白Pls是一种翻译后修饰的糖蛋白，并鉴定了参与糖转移的糖基转移酶（Gtfs）。先前，pls基因已被证明是小鼠脓毒性关节炎的毒力因子。糖基化发生在Pls丝氨酸-天冬氨酸(SD)-重复区域的丝氨酸残基上。功能表征表明，Pls糖基残基通过刺激细胞间粘附直接参与生物膜的形成。此外，最近我们发现另一种金黄色葡萄球菌SD-repeat表面糖蛋白SdrE在糖基化后也能促进生物膜的形成。在这个项目中，我们打算准确地定义由糖修饰介导的生物膜形成增加的分子机制。为此，将产生表达糖化或非糖化的表面糖蛋白Pls、SdrE、SdrC和SdrD的金黄色葡萄球菌菌株，并使用共聚焦激光扫描显微镜对其进行表征。为了鉴定相邻葡萄球菌细胞上各自的糖基残基特异性相互作用伙伴，将进行下拉试验和elisa。为了生产下拉试验中的诱饵，将在大肠杆菌中克隆pls、sdrE、sdrC和sdrD基因，纯化各自的蛋白并在体外糖基化。我们也有兴趣阐明糖基化机制，因此在体外糖基化试验中将使用不同的Gtfs和活化糖前体组合，随后，糖基化蛋白将通过质谱分析。这将确定修饰糖的位置和组成，这可能对表面糖蛋白的功能和抗原性有重要影响。此外，还计划进行不同的检测，以表征Pls、SdrE、SdrC和SdrD的修饰糖基残基在适应性和先天免疫功能中的潜在参与。糖基化表面蛋白Pls和SdrE的x射线晶体学有望阐明碳水化合物修饰介导的细胞间粘附和生物膜形成的机制。我们还打算分析修饰糖基残基在人宿主细胞定植和内化宿主组织中的作用。当碳水化合物与人类宿主细胞的粘附、定植和/或内化有关时，我们将进行下拉试验以确定假定的宿主细胞受体。最后，修饰表面糖蛋白的糖基残基在体内的重要性将在我们研究所最近开发的生物膜形成的小鼠模型中进行表征。

Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains largely unknown. We found that the plasmin-sensitive surface protein Pls is a post-translationally modified glycoprotein and identified the glycosyltransferases (Gtfs) involved in the sugar transfer. Previously, the pls gene has been demonstrated to be a virulence factor in mouse septic arthritis. Glycosylation occurs at serine residues in the Pls serine-aspartate (SD)-repeat region. Functional characterization revealed that Pls glycosyl residues are directly involved in biofilm formation via stimulation of intercellular adherence. Moreover recently, we found that another S. aureus SD-repeat surface glycoprotein, SdrE, also promotes biofilm formation, when glycosylated. In this project, we intend to exactly define the molecular mechanisms underlying increased biofilm formation mediated by the sugar modifications. For this, S. aureus strains will be generated that express glycosylated or non-glycosylated versions of the surface glycoproteins Pls, SdrE, SdrC, and SdrD and characterized using confocal laser scanning microscopy. To identify the respective glycosyl residue-specific interaction partner(s) on the neighbouring staphylococcal cell, pull-down assays and ELISAs will be performed. To produce the bait in the pull-down assays, the pls, sdrE, sdrC, and sdrD genes will be cloned in Escherichia coli, the respective proteins will be purified and in-vitro glycosylated. We are also interested to elucidate the glycosylation machinery, therefore different combinations of Gtfs and activated sugar precursors will be used in the in-vitro glycosylation assays and subsequently, glycosylated proteins will be analysed by mass spectrometry. This will define the positions and compositions of the modifying sugars, which may have an important impact on the function and antigenicity of surface glycoproteins. Additionally, different assays are planned to characterize a potential involvement of the modifying glycosyl residues of Pls, SdrE, SdrC, and SdrD in the adaptive and innate immune functions. X-ray crystallographie of glycosylated surface proteins Pls and SdrE is expected to clarify the mechanisms involved in intercellular adherence and biofilm formation mediated by the carbohydrate modifications. We also intend to analyse the role of the modifying glycosyl residues in host tissue colonization and internalization by human host cells. When the carbohydrates are involved in the adherence to human host cells, colonization, and/or internalization, we will perform pull-down assays to identify the putative host cell receptor(s). Finally, the in vivo importance of the modifying glycosyl residues of surface glycoproteins will be characterized in a mouse model of biofilm formation recently developed at our institute.