Two-component system interactions as uropathogenic Escherichia coli drug targets

描述（由申请人提供）：尿路感染（uti）是人类最常见的细菌感染之一，并且具有很高的复发率。随着对一线抗生素的耐药性不断上升，迫切需要制定旨在预防和/或治疗尿路感染的有针对性战略。尿路致病性大肠杆菌（UPEC）占尿路感染的大多数，可以形成坚固的细胞外生物膜，以及生物膜样细胞内细菌群落（IBCs）。影响IBC形成的因素之一是QseC传感器激酶，其破坏可减少IBC的形成并减弱upc的毒力。我们已经证明，QseC是其同源反应调节因子QseB的去磷酸化和失活所必需的，而QseB在缺乏QseC的情况下会被组成性激活。组成型活性QseB导致bbb500基因失调，干扰核心代谢过程，下调毒力基因表达。在缺乏QseC的情况下，研究QseB磷酸化的来源，发现PmrAB双组分系统（TCS）是QseB的主要激活剂。我们进一步证明，在野生型UPEC中，PmrB的信号接收也可以激活非同源伴侣QseB，这表明QseBC和PmrAB信号之间可能存在重叠。这些结果导致两种假设：1)在UPEC发病过程中，非同源TCS伴侣之间存在强大的串扰，这在生理上具有重要意义；2)我们可以将QseC功能作为一种减轻毒力的手段。本提案将探讨QseBC和PmrAB之间的调控相互作用及其在UTI发病机制中的作用的基本问题，并努力利用这些信息来治疗/预防UTI。目的1和2将阐明UPEC QseBC和PmrAB之间串扰的分子机制。目的3将探讨感染过程中QseBC-PmrAB相互作用的作用，并将确定靶向QseC磷酸酶功能的化合物，和/或偏置QseB和PmrB之间的串扰。我们已经开发了一系列广泛的分子工具，我们正在研究中使用，包括非极性缺失突变体，以及非活性或组成活性点突变体。结合我们正在使用的成熟的尿路感染小鼠模型，我们的显微镜追踪膀胱感染的能力，以及范德比尔特大学令人印象深刻的设施和资源，我们相信我们将利用信息来阐明宿主内外的QseBC-PmrAB分子相互作用。我们的长期目标是利用本文提出的研究结果来开发更好的治疗UPEC感染的方法。

DESCRIPTION (provided by applicant): Urinary tract infections (UTIs) are among the most frequent bacterial infections afflicting humans and have a high degree of recurrence. With rising resistance to front-line antibiotics, there is a pressing need for the development of targeted strategies aimed at preventing and/or treating UTIs. Uropathogenic Escherichia coli (UPEC) account for the majority of UTIs and can form robust extracellular biofilms, as well as biofilm- like intracellular bacterial communities (IBCs). Among the factors impacting IBC formation is the QseC sensor kinase, the disruption of which diminishes IBC formation and attenuates UPEC virulence. We have shown that QseC is required for the de-phosphorylation and deactivation of its cognate response regulator QseB, which becomes constitutively activated in the absence of QseC. Constitutively active QseB leads to the dysregulation of >500 genes, interferes with core metabolic processes and downregulates virulence gene expression. Investigating the source of QseB phosphorylation in the absence of QseC identified the PmrAB two-component system (TCS) as the primary QseB activator. We went on to show that in wild-type UPEC, signal reception by PmrB can also activate the non-cognate partner QseB, indicating a potential overlap between QseBC and PmrAB signaling. These results lead to two hypotheses: 1) There is robust cross-talk between non-cognate TCS partners that is of physiological significance during UPEC pathogenesis and, 2) We can target QseC function as a means to attenuate virulence. This proposal will engage fundamental questions about the regulatory interactions between QseBC and PmrAB and their role in UTI pathogenesis, and endeavor to harness this information to treat/prevent UTIs. Aims 1 and 2 will elucidate the molecular mechanisms underlying the cross-talk between UPEC QseBC and PmrAB. Aim 3 will probe the role of QseBC-PmrAB interactions during infection and will identify compounds that target the QseC phosphatase function, and/or bias cross-talk between QseB and PmrB. We have developed an extensive array of molecular tools that we are using in our studies, including non-polar deletion mutants, as well as inactive or constitutively active point mutants. Combined with the well-established murine model of UTI we are using, our microscopy capabilities to track infection in the bladder, and the impressive facilities and resources of Vanderbilt University, we are confident that we will harness information that will elucidate the QseBC-PmrAB molecular interplay within and outside the host. Our long-term goal is to leverage the outcomes of the herein proposed studies to develop better therapies against UPEC infection.