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.

项目摘要莱姆病（LD）是由螺旋体伯氏伯氏菌（BB）引起的多系统感染性疾病。和估计每年被诊断和治疗的476,000例病例很容易成为节肢动物传播美国感染。载体和储层宿主之间BB的过境涉及复杂且较差了解基因表达的协调能力控制运动。 BB中的差异基因表达为在很大程度上由两个全球监管网络精心策划 - RPON/RPOS途径和HK1/RRP1两个组件系统（TCS）。 RPOS途径在传播过程中被血液激活，仍在在整个哺乳动物感染中，并在幼虫获取期间迅速关闭。 HK1/RRP1途径通过第二信使bis-（3'-5'） - 循环二聚磷酸（C-DI-GMP）合成的信号由RRP1（二烷基酸酯环化酶）响应在幼虫和若虫期间产生的未鉴定分子血餐。 C-DI-GMP在LD螺旋体中发挥了多效性作用。缺少HK1或RRP1的BB被tick毁了在获取和传播过程中，但在小鼠中充满了毒气。 ΔRRP1BB在体外显示异常运动，表明C-DI-GMP也会影响鞭毛旋转。 C-DI-GMP依赖性响应通常是由与PILZ结构域结合蛋白质。彼此之间，揭开BB中C-DI-GMP信号传导的努力已集中关于PLZA，在大多数LD螺旋体中，唯一的PILZ结构蛋白。在进食tick中，Δplzabb表观ΔHK1和 ΔRRP1突变体。自相矛盾的是，在HK1/RRP1 TCS关闭的小鼠中，PLZA缺乏显着减弱感染性，意味着哺乳动物中PLZA的C-DI-GMP独立功能。在其他细菌中，PILZ蛋白（例如，YCGR）通过直接干扰鞭毛电动机来缓慢运动。令人惊讶的是，抑制BB运动性通过C-DI-GMP，似乎是与PLZA无关的。该建议旨在实现综合的概念和 C-DI-GMP信号传导和PLZA功能的机械框架在整个enzootic循环中的功能。实验在AIM 1中，旨在阐明PLZA作为C-DI-GMP生物传感器的功能，以促进TICK和 Borrelia Burgdorferi的哺乳动物宿主适应。在Subaim 1.1中，我们将研究配体的能力 - PLZA使用EMSA和DNA-蛋白质相互作用分析与C-DI-GMP调控启动子（GLP操纵子）结合，并产生PLZA点突变体以识别负责DNA结合的残差。在Subaim 1.2中，我们使用plup- 向下和定向诱变，以深入了解蛋白质 - 蛋白质相互作用 PLZA实现其C-DI-GMP独立的哺乳动物宿主特异性功能。 AIM 2旨在反应 C-DI-GMP对tick妈妈界面处的螺旋体运动和双相传播的贡献。在Subaim 2.1中，我们将使用明胶矩阵作为离体tick midgut代理人来监视螺旋体运动对血液的反应。在Subaim 2.2中，我们将确定是否需要降低C-DI-GMP的水平使BB过渡到运动和/或在传输过程中退出中肠。