ATG16L1 AND MOLECULAR REGULATION OF BACTERIAL PERSISTENCE

描述（由申请方提供）：病原体已经进化出多种破坏宿主防御武器库的方式，以长期存在并诱导复发性感染。我们以前已经证明，尿路致病性E。大肠杆菌（UPEC）作为膀胱壁内的膜结合隔室中的静止储库持续存在，并导致频繁和复发的尿路感染（UTI）。这些储库如何形成和持续存在尚不清楚;这种知识的缺乏阻碍了我们开发治疗方法以预防复发性UTI的能力。动物用来控制细胞内病原体感染的一种机制是自噬，这是一种进化上保守的过程，在饥饿或应激下被激活，通过将营养物质和受损的膜递送到溶酶体进行降解来回收营养物质和受损的膜。我们最近发现，自噬基因Atg 16 L1在UTI发病机制中起着关键作用：Atg 16 L1基因的突变限制了UPEC持续存在并导致复发性UTI的能力，并与尿路上皮结构缺陷相关。这些缺陷导致膀胱正常功能和UPEC发病机制所需的浅表尿路上皮细胞中复杂膜再循环事件的改变。本申请的目的是确定UPEC劫持膀胱上皮细胞的正常囊泡运输以形成持久储库的机制。测试的中心假设是UPEC通过占用自噬途径避免被尿路上皮细胞中的溶酶体破坏，并且Atg 16 L1缺陷将细菌重新路由到它们不能持续存在的细胞内隔室。我们建议测试这一假设如下：在目的1中，我们将系统地破坏尿路上皮细胞的结构，通过使用新建立的尿路上皮特异性cre小鼠驱动Atg 16 L1和其他自噬途径蛋白的功能丧失，以确定细胞内UPEC生态位的性质和自噬在UPEC持久性中的作用。在目标2中，我们将使用尿路上皮细胞培养模型来阐明Atg 16 L1的调节如何改变UPEC侵袭、细胞内存活进入尿路上皮细胞和从细胞中排出的过程。我们预计，我们的工作将为UTI期间自噬途径和UPEC之间的遗传和分子相互作用提供新的见解，并将为开发治疗干预措施以对抗复发性感染提供细胞靶点。鉴于尿路感染是常见且昂贵的，并且耐药性病原体正变得越来越普遍，这种知识有助于开发新的治疗方案以限制或根除复发性UTI的来源的潜力可能是至关重要的。

DESCRIPTION (provided by applicant): Pathogens have evolved a number of ways to subvert host defense arsenals to persist long term and induce recurrent infections. We have previously shown that uropathogenic E. coli (UPEC) persists as quiescent reservoirs in membrane-bound compartments within the bladder wall and causes frequent and recurrent urinary tract infections (UTIs). How these reservoirs form and persist is not known; this lack of knowledge impedes our ability to develop treatments to prevent recurrent UTIs. One mechanism used by animals to control infection by intracellular pathogens is autophagy, an evolutionarily conserved process that is activated under starvation or stress to recycle nutrients and damaged membranes by delivering them to the lysosome for degradation. We recently showed that an autophagy gene, Atg16L1, plays a key role in UTI pathogenesis: a mutation in the Atg16L1 gene limits the ability of UPEC to persist and cause recurrent UTIs and is associated with urothelial architectural defects. These defects lead to alterations in complex membrane recycling events in superficial urothelial cells required for both the normal function of the bladder and UPEC pathogenesis. The objective of this application is to determine the mechanisms by which UPEC hijack the normal vesicle trafficking of the bladder epithelial cells to form persistent reservoirs. The centrl hypothesis tested is that UPEC avoid destruction by lysosomes in urothelial cells by appropriating the autophagy pathway, and that Atg16L1 deficiency re-routes bacteria to an intracellular compartment where they cannot persist. We propose to test this hypothesis as follows: in Aim 1, we will systematically disrupt urothelial cell architecture by using newly established urothelial-specific cre mice driving loss of function of Atg16L1 and other autophagy pathway proteins to determine both the nature of the intracellular UPEC niche in vivo and the role of autophagy in UPEC persistence. In Aim 2, we will use a urothelial cell culture model to elucidate how modulation of Atg16L1 alters the process of UPEC invasion, intracellular survival into urothelial cells, and egress out of cells. We anticipate that our work will provide new insighs into the genetic and molecular interplay between the autophagy pathway and UPEC during a UTI and will provide cellular targets for development of therapeutic interventions to combat recurrent infections. Given that urinary tract infections are common and costly and that antibiotic-resistant pathogens are becoming increasingly prevalent, the potential of this knowledge to contribute to development of new treatment regimens to limit or eradicate sources of recurrent UTI could be vital.