Elucidating the contributions of c-di-GMP and PlzA to tick- and mammalian host-adaptation in Lyme disease spirochetes

项目摘要 莱姆病是由伯氏疏螺旋体引起的一种多系统感染性疾病。使用 据估计，每年有476,000例被诊断和治疗，LD很容易成为最常见的节肢动物传播的疾病 在美国的感染。Bb在载体和宿主之间的转运涉及复杂和糟糕的 了解基因表达与运动控制的协调。BB中差异基因表达的研究 在很大程度上由两个全球调控网络协调-RpoN/rpos途径和HK1/Rrp1两个 组件系统(TCS)。Rpos途径在传输过程中被血粉激活，并保持在 在哺乳动物感染的整个过程中，并在幼虫获取期间迅速关闭。HK1/Rrp1通路 合成的第二信使双-(3‘-5’)-环状二聚鸟苷一磷酸(c-di-GMP) 通过Rrp1，一种二鸟苷环化酶，对幼虫和若虫产生的未知分子做出反应 血餐。C-di-GMP对LD螺旋体具有多效性。缺少HK1或Rrp1的BB在扁虱中被破坏 在获取和传播过程中，但在小鼠中完全毒力。ΔRRP1 BB在体外表现出异常的运动能力， 表明c-di-GMP也影响鞭毛旋转。C-di-GMP依赖的反应通常是由 与具有Pilz结构域的蛋白质结合。因此，在BB中解开c-di-GMP信号的努力已经成为中心 关于Plza，它是大多数LD螺旋体中唯一的Pilz结构域蛋白。在饲养硬蜱中，ΔPlza BB表型ΔHK1和 Δrrp1突变体。矛盾的是，在HK1/Rrp1 TCS关闭的小鼠中，Plza缺乏症显著减弱 感染性，意味着在哺乳动物中对Plza具有c-di-GMP独立的功能。在其他细菌中，Pilz蛋白 (例如，YcgR)通过直接干扰鞭毛马达而减慢运动。令人惊讶的是，对BB运动的抑制 C-di-GMP似乎不依赖于Plza。这项建议旨在实现一个完整的概念和 C-di-GMP信号和Plza在整个地方病循环中发挥作用的机制框架。实验 在目标1中，旨在阐明Plza如何作为c-di-GMP生物传感器来促进tick和 伯氏疏螺旋体对哺乳动物宿主的适应。在Subaim 1.1中，我们将研究连接- 利用EMSA和DNA-蛋白质相互作用分析，Plza与c-di-GMP调节的启动子(GLP操纵子)结合， 并产生Plza点突变体，以识别负责DNA结合的残基。在Subaim 1.2中，我们使用Pull- 向下和定点突变以深入了解使去连接成为可能的蛋白质-蛋白质相互作用 Plza以完成其c-di-GMP不依赖于哺乳动物宿主的功能。AIM 2的设计目的是去卷曲 C-di-GMP对螺旋体运动的贡献(S)和在壁虱-哺乳动物界面的双相传播。 在Subaim 2.1中，我们将使用明胶基质作为体外蜱类中肠替代物来监测螺旋体在 对血餐的反应。在Subaim 2.2中，我们将确定是否需要降低c-di-GMP水平 以便BB在传输过程中转换回运动和/或退出中肠。

Project Summary Lyme disease (LD) is a multisystem infectious disorder caused by the spirochete Borrelia burgdorferi (Bb). With an estimated 476,000 cases diagnosed and treated annually, LD is easily the most prevalent arthropod-borne infection in the United States. Transit of Bb between vector and reservoir host involves an intricate and poorly understood coordination of gene expression with control of motility. Differential gene expression in Bb is orchestrated, in large part, by two global regulatory networks – the RpoN/RpoS pathway and the Hk1/Rrp1 two component system (TCS). The RpoS pathway is activated by the bloodmeal during transmission, remains ON throughout mammalian infection, and rapidly turns OFF during larval acquisition. The Hk1/Rrp1 pathway signals via the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) synthesized by Rrp1, a diguanylate cyclase, in response to unidentified molecules generated during the larval and nymphal blood meals. c-di-GMP exerts pleiotropic effects in LD spirochetes. Bb lacking Hk1 or Rrp1 are destroyed in ticks during acquisition and transmission but are fully virulent in mice. Δrrp1 Bb display aberrant motility in vitro, indicating that c-di-GMP also impacts flagellar rotation. c-di-GMP-dependent responses often are mediated by binding to proteins with a PilZ domain. Accordingly, efforts to unravel c-di-GMP signaling in Bb have centered about PlzA, the sole PilZ domain protein in most LD spirochetes. In feeding ticks, ΔplzA Bb phenocopy Δhk1 and Δrrp1 mutants. Paradoxically, in mice, where the Hk1/Rrp1 TCS is OFF, PlzA deficiency markedly attenuates infectivity, implying a c-di-GMP independent function for PlzA in mammals. In other bacteria, PilZ proteins (e.g., YcgR) slow motility by directly interfering with the flagellar motor. Surprisingly, inhibition of Bb motility by c-di-GMP appears to be PlzA-independent. This proposal is intended to achieve an integrated conceptual and mechanistic framework for c-di-GMP signaling and PlzA functions across the entire enzootic cycle. Experiments in Aim 1, are designed to elucidate how PlzA functions as a c-di-GMP biosensor to promote both tick and mammalian host-adaptation by Borrelia burgdorferi. In Subaim 1.1, we will investigate the ability of liganded- PlzA to bind to a c-di-GMP regulated promoter (glp operon) using EMSAs and DNA-protein interaction analysis, and generate PlzA point mutants to identify residues responsible for DNA-binding. In Subaim 1.2, we use pull- down and site-directed mutagenesis to gain insight into the protein-protein interactions that enable unliganded PlzA to fulfill its c-di-GMP-independent, mammalian host-specific functions. Aim 2 is designed to deconvolute the contribution(s) of c-di-GMP to spirochete motility and biphasic dissemination at the tick-mammal interface. In Subaim 2.1, we will use gelatin matrices as an ex vivo tick midgut surrogate to monitor spirochete motility in response to the blood meal. In Subaim 2.2, we will determine whether decreased levels of c-di-GMP are required for Bb to transition back to motility and/or exit the midgut during transmission.