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

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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.