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，复发率高。随着一线抗生素耐药性的增加，迫切需要开发旨在预防和/或治疗UTI的靶向策略。尿路致病性大肠杆菌（UPEC）占UTI的大多数，并且可以形成稳健的细胞外生物膜以及生物膜样细胞内细菌群落（IBC）。影响IBC形成的因素之一是QseC传感器激酶，其破坏减少IBC形成并减弱UPEC毒力。我们已经表明，QseC是必要的去磷酸化和失活的同源反应调节QseB，这成为组成性激活QseC的情况下。组成型活性QseB导致>500个基因的失调，干扰核心代谢过程并下调毒力基因表达。在不存在QseC的情况下研究QseB磷酸化的来源，鉴定了PmrAB双组分系统（TCS）作为主要的QseB激活剂。我们继续表明，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信号传导之间存在潜在的重叠。这些结果导致两个假设：1）在UPEC发病过程中具有生理意义的非同源TCS配偶体之间存在强烈的串扰，以及2）我们可以靶向QseC功能作为减弱毒力的手段。该提案将涉及有关QseBC和PmrAB之间的相互作用及其在UTI发病机制中的作用的基本问题，并奋进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将探索感染期间QseBC-PmrAB相互作用的作用，并将鉴定靶向QseC磷酸酶功能和/或偏向QseB和PmrB之间的串扰的化合物。我们已经开发了一系列广泛的分子工具，我们正在使用我们的研究，包括非极性缺失突变体，以及无活性或组成型活性点突变体。结合我们正在使用的成熟的UTI小鼠模型，我们跟踪膀胱感染的显微镜能力，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抗UPEC感染的疗法。

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.