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

阐明 c-di-GMP 和 PlzA 对莱姆病螺旋体蜱和哺乳动物宿主适应的贡献

基本信息

批准号：
10739945
负责人：
MELISSA J CAIMANO
金额：
$ 24.88万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10739945
关键词：
Address Adherence Affect Anabolism Arbovirus Infections Attenuated Back Bacteria Bacteria sigma factor KatF protein Basement membrane Binding Biological Biosensor Blood Borrelia burgdorferi Cell physiology Communicable Diseases Complex DNA Binding DNA-Protein Interaction Development Diagnosis Disease Divorce EMSA Epithelium Gelatin Gene Expression Gene Expression Regulation Genetic Transcription Guanosine Monophosphate Human In Vitro Infection Ixodes Larva Lead Ligands Lyme Disease Mammals Mediating Midgut Molecular Conformation Monitor Motor Mus Nature Nymph Operon Order Spirochaetales Organism Pathway interactions Periodicity Phase Phenocopy Phenotype Process Production Proteins Research Personnel Rodent Role Rotation Running Salivary Glands Second Messenger Systems Sigma Factor Signal Pathway Signal Transduction Site-Directed Mutagenesis Speed Structure System Tertiary Protein Structure Ticks Tissue-Specific Gene Expression Transcription Coactivator United States Virulent beta barrel cell motility cytosolic receptor design diguanylate cyclase dimer enzootic experience experimental study feeding insight migration mutant novel novel strategies pathogen pleiotropism posttranscriptional promoter protein protein interaction response tick feeding tick transmission transmission process vector virulence gene

项目摘要

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.

项目概要莱姆病 (LD) 是一种由伯氏疏螺旋体 (Bb) 螺旋体引起的多系统感染性疾病。据估计，每年诊断和治疗的病例有 476,000 例，LD 是最流行的节肢动物传播病例在美国，Bb 在媒介和储存宿主之间的传播涉及复杂且不良的过程。 Bb 中基因表达与运动控制的协调是已知的。很大程度上是由两个全球监管网络精心策划的——RpoN/RpoS 途径和 HK1/Rrp1 两个 RpoS 通路在传播过程中被血粉激活，保持开启状态。在整个哺乳动物感染过程中，Hk1/Rrp1 通路迅速关闭。通过合成的第二信使双-(3'-5')-环状二聚鸟苷单磷酸 (c-di-GMP) 发出信号 Rrp1（一种二鸟苷酸环化酶）响应幼虫和若虫期间产生的不明分子 c-di-GMP 对缺乏 Hk1 或 Rrp1 的 LD 螺旋体发挥多效作用。在获取和传播过程中，Δrrp1 Bb 在体外表现出异常的运动性，但在小鼠中具有完全的毒性。表明 c-di-GMP 也会影响 c-di-GMP 依赖性反应，通常是由 c-di-GMP 介导的。因此，解开 Bb 中的 c-di-GMP 信号转导的努力已成为焦点。关于 PlzA（大多数 LD 螺旋体中唯一的 PilZ 结构域蛋白）在摄食蜱中，ΔplzA Bb 表型 Δhk1 和矛盾的是，在 Hk1/Rrp1 TCS 关闭的小鼠中，PlzA 缺陷显着减弱。感染性，这意味着 PlzA 在哺乳动物中具有独立于 c-di-GMP 的功能，而在其他细菌中，PilZ 蛋白则具有独立的功能。（例如，YcgR）通过直接干扰鞭毛运动来减慢运动，令人惊讶的是，抑制 Bb 运动。由 c-di-GMP 看来是独立于 PlzA 的。该提案旨在实现一个综合的概念和概念。 c-di-GMP 信号传导和 PlzA 在整个地方性动物实验周期中发挥作用的机制框架。在目标 1 中，旨在阐明 PlzA 如何作为 c-di-GMP 生物传感器发挥作用，以促进蜱和在 Subaim 1.1 中，我们将研究伯氏疏螺旋体 (Borrelia burgdorferi) 的哺乳动物宿主适应能力。使用 EMSA 和 DNA-蛋白质相互作用分析，PlzA 与 c-di-GMP 调节启动子（glp 操纵子）结合，并生成 PlzA 点突变体来识别负责 DNA 结合的残基在 Subaim 1.2 中，我们使用 pull- 下调和定点诱变，以深入了解蛋白质-蛋白质相互作用，从而实现无配体 PlzA 旨在实现其不依赖于 c-di-GMP 的哺乳动物宿主特异性功能，目标 2 旨在解卷积。 c-di-GMP 对螺旋体运动和蜱-哺乳动物界面双相传播的贡献。在 Subaim 2.1 中，我们将使用明胶基质作为体外蜱中肠替代物来监测螺旋体运动在 Subaim 2.2 中，我们将确定是否需要降低 c-di-GMP 水平。让 Bb 在传输过程中转变回能动状态和/或退出中肠。