Stabilization and regulation of a Bacillus anthracis spore lytic enzyme

炭疽芽孢杆菌等致病细菌的脱除 受污染的场地或材料具有挑战性，因为孢子对 常规的抗菌治疗。炭疽杆菌孢子皮质肽多糖的活化 裂解机械会导致孢子休眠和抗性迅速丧失。这 因此，机械可以作为萌发激活和孢子杀灭处理的目标。这个 皮层裂解酶Slib以非活性和高度稳定的形式储存在休眠孢子中，并 在萌发过程中很早就活跃起来。YpeB蛋白是 将Slib结合到休眠孢子中，在萌发过程中， YpeB释放Slib来解聚皮质。拟议工作的目标1包括 生物化学、遗传学和结构生物学方法揭示蛋白质-蛋白质接触形成 YpeB和Sleb在休眠的孢子中。含有YpeB或SERB的蛋白质复合体将是 从孢子中提纯的蛋白质和共提纯的蛋白质将得到鉴定。YpeB的更改形式将是 在体内表达以确定该蛋白参与功能和蛋白质的关键区域 互动。将使用X射线结晶学来确定YpeB的结构，以便 了解突变的影响。这项工作将阐明滑板稳定的机理， 它可适用于生产稳定的蛋白质药物、疫苗和 其他工业产品。目标2是鉴定处理YpeB和YpeB的蛋白酶 从而激活了sRb。对这种蛋白水解酶及其调控的了解将为我们提供方法 通过YpeB和Slib激活萌发。拟议的工作将有助于两个主要方面 翻译目标：激活孢子萌发，以改善去污和 对极端蛋白质稳定机制的理解。

Elimination of spores of Bacillus anthracis and other disease-causing bacteria from contaminated sites or materials is challenging due to the extreme resistance of spores to conventional antimicrobial treatments. Activation of the B. anthracis spore cortex peptidoglycan lytic machinery results in rapid loss of spore dormancy and resistance properties. This machinery can thus be targeted for germination-activating and spore-killing treatments. The cortex lytic enzyme SleB is stored in an inactive and highly stable form in the dormant spore and becomes active early during the germination process. The YpeB protein is required for incorporation of SleB into the dormant spore, and during germination, proteolytic cleavage of YpeB releases SleB to depolymerize the cortex. Aim 1 of the proposed work includes biochemical, genetic, and structural biology methods to reveal protein-protein contacts formed by YpeB and SleB in the dormant spore. Protein complexes containing YpeB or SleB will be purified from spores and co-purified proteins will be identified. Altered forms of YpeB will be expressed in vivo to identify key regions of the protein involved in function and protein interaction. The YpeB structure will be determined using X-ray crystallography in order to understand the effects of mutations. This work will clarify the mechanism of SleB stabilization, which may be adaptable to the production of stable protein pharmaceuticals, vaccines, and other industrial products. Aim 2 is the identification of the protease that processes YpeB and thus activates SleB. Knowledge of this protease and its regulation will suggest methods for the activation of germination via YpeB and SleB. The proposed work will contribute to two major translational goals: Activation of spore germination in order to improve decontamination and understanding of a mechanism of extreme protein stabilization.