Determining the role of autophagy in regulating the Staphylococcus aureus a-toxin receptor ADAM10

项目摘要 金黄色葡萄球菌是社区和医疗机构大量感染的罪魁祸首。尤其是作为 抗生素耐药菌株的数量和发病率继续上升，需要采取替代干预措施 方法正变得越来越重要。开发新疗法的一个策略是识别和阻止宿主 被病原体利用来致病的途径。自噬就是这样一种途径。自噬是一种高度的 保守的，普遍存在的细胞过程，其中双膜自噬小体吞噬受损的胞浆 成分和溶酶体降解的靶点；然而，最近的研究表明它的关键 在病原体耐受性和清除中的作用。 卡德韦尔实验室和托雷斯实验室合作进行的实验证明了 对于ATG16L1，一种在金黄色葡萄球菌耐药中介导自噬小体形成的蛋白质。使用体内模型 ATG16L1几乎完全被取消的自噬丧失，增加了对致死性的敏感性 挑战被发现依赖于金黄色葡萄球菌毒力因子-毒素的产生。在进一步 实验表明，缺乏ATG16L1的内皮细胞表现出较高的血浆水平 膜毒素受体ADAM10.这些数据表明，自噬对 ADAM10.然而，目前尚不清楚自噬(一种胞质降解途径)是如何调节这些水平的。 膜结合型ADAM10。 我们的目标是确定宿主因子、机制和/或途径，这些因素、机制和/或途径是由 影响ADAM10水平的自噬。使用我们为此开发的体外系统，我们最初的 数据表明，ATG16L1独立于溶酶体或蛋白酶体调节ADAM10。相反，细胞 缺乏ATG16L1显示含有ADAM10的细胞外小泡产生减少。我们计划继续 准确测试ATG16L1和自噬如何影响ADAM10的本地化；特别是通过 包装成囊泡，用于细胞外释放、运输到质膜和内吞 内部化。此外，我们的目标是确定ADAM10的氨基酸基序或结构元件 这赋予了它依赖于自噬的监管。这些策略中的每一种都是为了更好地理解 自噬调节ADAM10的生物学途径及其底物可作为替代途径 治疗金黄色葡萄球菌感染和其他涉及内皮屏障的疾病的目标。

Project Summary S. aureus is responsible for a large number of infections in the community and healthcare setting. Especially as the number and incidence of antibiotic resistant strains continue to rise, the need for alternative intervention methods is becoming increasingly critical. A strategy to develop novel therapies is to identify and block host pathways exploited by pathogens to cause disease. Autophagy is one such pathway. Autophagy is a highly conserved, ubiquitous cellular process in which a double membrane autophagosome engulfs damaged cytosolic components and targets them for lysosomal degradation; however, recent research has demonstrated its critical role in pathogen tolerance and clearance. Experiments performed in collaboration between the Cadwell and Torres labs demonstrated a vital role for ATG16L1, a protein that mediates autophagosome formation, in S. aureus tolerance. Using an in vivo model of autophagy loss where ATG16L1 is almost completely abolished, the increased susceptibility to lethal challenge was found to be dependent on the production of the S. aureus virulence factor, -toxin. Upon further experimentation, it was shown that endothelial cells lacking ATG16L1 display higher levels of the plasma membrane -toxin receptor ADAM10. These data suggest autophagy plays a negative regulatory role on ADAM10. However, it is remains unclear how autophagy, a cytosolic degradation pathway, regulates the levels of membrane bound ADAM10. Our aim is to identify host factors, mechanisms, and/or pathways that are differentially regulated by autophagy that affect ADAM10 levels. Using an in vitro system that we developed for this purpose, our initial data suggests that ATG16L1 regulates ADAM10 independently of the lysosome or proteasome. Instead, cells lacking ATG16L1 show decreased production of extracellular vesicles containing ADAM10. We plan to continue to test precisely how ATG16L1 and autophagy influence native localization of ADAM10; particularly through packaging into vesicles meant for extracellular release, trafficking to the plasma membrane, and endocytic internalization. Additionally, our goal is to determine the amino acid motifs or structural elements of ADAM10 that confer its autophagy dependent regulation. Each one of these strategies is an attempt to better understand the biology of ADAM10 regulation by autophagy as this pathway and its substrates may serve as alternative targets for treatment of S. aureus infections and other conditions involving the endothelial barrier.