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Pathogen-host interactions in Lyme disease interrogated at Single Cell Level

在单细胞水平上探讨莱姆病的病原体与宿主的相互作用

基本信息

批准号：
10388319
负责人：
Janakiram Seshu
金额：
$ 22.5万
依托单位：
UNIVERSITY OF TEXAS SAN ANTONIO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-08 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10388319
关键词：
Amino Acids Anabolism Anti-Bacterial Agents Antibodies Area Binding Bite Black-legged Tick Borrelia Borrelia burgdorferi Branched-Chain Amino Acids Carditis Cell Communication Cell Line Cell Wall Cell membrane Cells Centers for Disease Control and Prevention (U.S.)Complex Critical Pathways Cues Data Disease Eukaryotic Cell Event Genetic Genetic Transcription Glucosamine Goals Health Care Costs Heterogeneity Human Immune response Incidence Infection Infectious Agent Knowledge Life Cycle Stages Life Style Lipids Lipoproteins Lyme Arthritis Lyme Disease Mammalian Cell Masks Mediating Membrane Metabolic Pathway Methodology Methods Molecular Morbidity - disease rate Muramic Acid Mus N-acetylmuramic acid Nature Nucleotides Order Spirochaetales Ornithine Pathway interactions Patients Peptidoglycan Phase Phenotype Play Polymers Prevention Proteins Proteomics Research Role Signal Pathway Signal Transduction Site Skin Surface Testing Tick-Borne Diseases Ticks Toll-like receptors Vector-transmitted infectious disease antimicrobial arthropod-borne bacterial vector base cell envelope crosslink enzootic innovation mouse model mutant novel novel strategies pathogen pathogenic bacteria peptidoglycan recognition protein prevent public health relevance receptor receptor-mediated signaling response single cell analysis single-cell RNA sequencing tick bite tool transcriptome sequencing transcriptomics transmission process treatment choice vaccine access vector tick

项目摘要

Lyme disease is the most common arthropod-borne illness in the US. Currently, there are no vaccines available to prevent human Lyme disease and antibacterial therapy is the treatment of choice. Borrelia burgdorferi, the agent of Lyme disease, is maintained in nature through an enzootic cycle involving a tick vector and a variety of vertebrate hosts. B. burgdorferi undergoes extensive remodeling of its cell envelope in response to environmental cues prevalent in these highly divergent hosts. The molecular interactions between the borrelial envelope or its major determinants, such as peptidoglycan (PG) and surface-exposed lipoproteins, with host cells play a critical role in the survival and infectivity of B. burgdorferi during different stages of its infectious cycle. Moreover, B. burgdorferi is an extreme auxotroph lacking metabolic pathways for biosynthesis of essential biomolecules and is therefore a highly host-adapted pathogen. In addition, the peptidoglycan (PG) of B. burgdorferi has a unique composition with L-Ornithine as the branching amino acid in its pentapeptide cross-linking the N-acetyl glucosamine and N-acetyl muramic acid polymers, which is distinct from the pentapeptides present in the PG of Gram-negative and Gram-positive bacterial pathogens. Two mutants strains of B. burgdorferi lacking Borrelia host-adaptation Regulator (BadR) or Borrelia host-adaptation Protein (BadP) hyper- and hypo-express lipoproteins, respectively, serving as ideal genetic tools to interrogate pathogen-host cell interactions mediated by differences in levels of surface-exposed lipoproteins. The long- term objective of the proposed research is to disrupt the complex lifestyle of Lyme spirochetes within ticks and mammalian hosts to reduce incidence of Lyme disease. In this proposal, we will test the central hypothesis that remodeling of borrelial cell envelope alters host antimicrobial responses influencing survival of B. burgdorferi in different hosts. The central hypothesis will be tested with two specific aims: Specific Aim 1: To determine signaling effects of borrelial PG/lipoproteins on tick and mammalian cells using single cell RNA-Seq (scRNA- Seq) analysis. Large arrays of transcriptional data will be analyzed and validated to identify pathways and cellular phenotypes that distinguish response of single cells from transmission, reservoir and dead-end hosts exploiting the advantages of using small numbers of host cells, spirochetes and minuscule levels of purified borrelial determinants. Role of Peptidoglycan Recognition Proteins and Hippo signaling pathway in modulating Bb infection will be interrogated. Specific Aim 2: To define effects of intact spirochetes defective in PG/lipoprotein composition on primary host cells at single-cell level. Mutant spirochetes defective in PG stability/synthesis or with altered levels of lipoproteins will be used to define their interactions with host cells using RNA-Seq analysis and further validated for colonization capabilities using the experimental mouse models of Lyme disease. The impact of this innovative study of pathogen-host interactions at single-cell level will enable identification of novel signaling pathways critical for colonization/transmission of Lyme spirochetes.

莱姆病是美国最常见的节肢动物传播疾病。目前还没有疫苗可用抗菌疗法来预防人类莱姆病，是治疗的首选。氏疏莱姆病的病原体--伯氏莱姆病，通过一个涉及蜱虫的地方性流行周期在自然界中得以维持载体和各种脊椎动物宿主。B。burgdorferi经历了广泛的重塑其细胞包膜，对这些高度分化的宿主中普遍存在的环境线索的反应。之间的分子相互作用疏螺旋体包膜或其主要决定簇，如肽聚糖（PG）和表面暴露的脂蛋白，在B的存活和感染性中起关键作用。在不同的阶段，传染周期而且，B。burgdorferi是一种极端的营养缺陷型，缺乏生物合成的代谢途径因此是一种高度适应宿主的病原体。此外，肽聚糖（PG）的B。burgdorferi具有独特的组成，L-鸟氨酸作为其五肽中的分支氨基酸使N-乙酰基葡糖胺和N-乙酰基胞壁酸聚合物交联，这不同于使N-乙酰基葡糖胺和N-乙酰基胞壁酸聚合物交联。存在于革兰氏阴性和革兰氏阳性细菌病原体的PG中的五肽。两种突变体 B.缺乏疏螺旋体宿主适应调节因子（BadR）或疏螺旋体宿主适应蛋白的伯氏螺旋体（BadP）高表达和低表达脂蛋白，分别作为理想的遗传工具，询问病原体-宿主细胞相互作用介导的差异，在表面暴露的脂蛋白。很长的- 拟议研究的长期目标是破坏蜱虫内莱姆病螺旋体的复杂生活方式，哺乳动物宿主，以减少莱姆病的发病率。在本提案中，我们将检验中心假设，疏螺旋体细胞包膜的重塑改变了影响B存活的宿主抗微生物反应。布格多费里因不同的宿主中心假设将通过两个具体目标进行检验：具体目标1：使用单细胞RNA-Seq（scRNA-Seq）的疏螺旋体PG/脂蛋白对蜱和哺乳动物细胞的信号传导作用 Seq）分析。将分析和验证大量的转录数据，以确定途径，区分单细胞应答与传播、储存和死端宿主的细胞表型利用使用少量宿主细胞、螺旋体和微量纯化的疏螺旋体决定簇肽聚糖识别蛋白和Hippo信号通路在调节细胞凋亡中的作用将询问Bb感染。具体目标2：确定完整的螺旋体缺陷的影响，在单细胞水平上的原代宿主细胞上的PG/脂蛋白组成。PG缺陷型突变螺旋体稳定性/合成或改变脂蛋白水平的方法将用于定义它们与宿主细胞的相互作用使用RNA-Seq分析并使用实验小鼠进一步验证定殖能力莱姆病模型这一创新性研究在单细胞水平上对病原体-宿主相互作用的影响将能够识别对莱姆螺旋体的定殖/传播至关重要的新信号传导途径。