Protein engineering of enterobactin-binding human NGAL for high-affinity scavenging of novel virulence-associated siderophores

在宿主感染期间，病原体分泌许多与毒力相关的小分子，这些小分子通常不是获得性免疫系统的靶标。其中一类重要的毒力因子是铁载体，在严重缺铁的哺乳动物宿主环境中，铁载体对于铁的获取是必不可少的。天然防御蛋白NGAL(中性粒细胞明胶酶相关Lipocalin)对于通过高亲和力结合和随后的肾脏清除来中和细菌铁载体(主要是肠动蛋白)是至关重要的。然而，一些病原体，如肠沙门氏菌和炭疽杆菌，通过铁载体修饰获得了对NGAL的耐药性，包括糖基化(Salmochelin)或单羟化(Petrobactin)。因此，该项目的目的是对人类NGAL进行定向进化，以开发新的高亲和力变异体来清除毒力相关的铁载体。为此，应使用NGAL结合口袋的随机突变来创建突变库，该库将用于通过噬菌体展示来选择新的NGAL变异体。所选突变体的新结合特性将通过结构-功能分析进行表征，其抑制效果将通过后续的体外和体内研究进行评估。利用这种方法，将在分子水平上研究NGAL介导的铁载体结合的机制和生理作用，并进一步建立基于蛋白质定向进化的毒力特异性病原菌控制的体内模型系统。

During host infection, pathogens secrete a number of virulence-associated small molecules that are generally not targeted by the acquired immune system. An important class of these virulence factors are siderophores, which are essential for iron acquisition in the severely iron-limited milieu of mammalian hosts. The innate defense protein NGAL (Neutrophil Gelatinase Associated Lipocalin) is crucial for neutralization of bacterial siderophores (mainly enterobactin) by high-affinity binding and subsequent renal clearance. However, a number of pathogens such as Salmonella enterica and Bacillus anthracis gained resistance against NGAL by siderophore modifications including glycosylations (salmochelins) or unusal hydroxylations (petrobactin). Thus, the aim of this project is the directed evolution of human NGAL to develop novel high-affinity variants for scavenging virulence-relevant siderophores. For this purpose, random mutagenesis of the NGAL binding pocket shall be used to create a mutant library, which will serve for the selection of new NGAL variants by phage display. The novel binding properties of selected variants shall be characterized by structure-function analysis, and their inhibitory efficacy will be evaluated by subsequent in vitro and in vivo studies. With this approach, both the mechanism and the physiological role of NGAL-mediated siderophore-binding shall be studied at the molecular level and, furthermore, an in vivo model system for virulence-specific pathogen control based on directed protein evolution shall be established.