ATG16L1 AND MOLECULAR REGULATION OF BACTERIAL PERSISTENCE

描述（由申请人提供）：病原体已经进化出许多方法来破坏宿主的防御库，以长期存在并诱导复发性感染。我们之前的研究表明，尿路致病性大肠杆菌（UPEC）在膀胱壁的膜结合腔室中作为静止的储存库持续存在，并引起频繁和反复的尿路感染（uti）。这些储层是如何形成和持续存在的尚不清楚；这种知识的缺乏阻碍了我们开发预防尿路感染复发的治疗方法的能力。动物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膜传递给溶酶体进行降解，从而回收营养物质和受损膜。我们最近发现自噬基因Atg16L1在UTI发病机制中起着关键作用：Atg16L1基因的突变限制了UPEC持续存在和引起复发性UTI的能力，并与尿路上皮结构缺陷有关。这些缺陷导致膀胱正常功能和UPEC发病机制所必需的浅表尿路上皮细胞复杂膜循环事件的改变。本应用的目的是确定UPEC劫持膀胱上皮细胞正常囊泡运输形成持久储存库的机制。经检验的中心假设是，UPEC通过利用自噬途径避免尿路上皮细胞中溶酶体的破坏，而Atg16L1缺陷将细菌重新引导到细胞内腔室，在那里它们不能持续存在。我们建议通过以下方法来验证这一假设：在Aim 1中，我们将使用新建立的尿路上皮特异性cre小鼠，通过驱动Atg16L1和其他自噬途径蛋白的功能丧失，系统地破坏尿路上皮细胞结构，以确定体内细胞内UPEC生态位的性质以及自噬在UPEC持续性中的作用。在Aim 2中，我们将使用尿路上皮细胞培养模型来阐明Atg16L1的调节如何改变upc侵袭、细胞内存活到尿路上皮细胞和细胞外的过程。我们预计，我们的工作将为UTI期间自噬途径和upc之间的遗传和分子相互作用提供新的见解，并将为开发治疗性干预措施提供细胞靶点，以对抗复发性感染。鉴于尿路感染很常见且费用高昂，而且耐抗生素病原体正变得越来越普遍，这方面的知识有助于开发新的治疗方案，以限制或根除复发性尿路感染的来源，这可能是至关重要的。

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.