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

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 可用于预防人类莱姆病和抗菌治疗是首选的治疗方法。疏螺旋体 伯格多费氏菌是莱姆病的病原体，在自然界中是通过一只扁虱的地方性循环来维持的。 媒介和各种脊椎动物宿主。B.burgdorferi经历了其细胞膜的广泛重塑 对这些高度分化的宿主中普遍存在的环境线索的反应。分子间的相互作用 脆性包膜或其主要决定因素，如肽聚糖(PG)和表面暴露的脂蛋白， 宿主细胞在伯氏杆菌不同阶段的存活和感染性中起关键作用。 感染性循环。此外，伯氏杆菌是一种极端的营养缺陷型生物，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，肽多聚糖(PG) 伯氏杆菌具有独特的成分，其五肽中的支链氨基酸为L-鸟氨酸 N-乙酰氨基葡萄糖和N-乙酰胞壁酸聚合物的交联化，这与 革兰氏阴性和革兰氏阳性细菌的PG中存在五肽。两个突变体 缺乏疏螺旋体宿主适应调节剂(BADR)或疏螺旋体宿主适应蛋白的伯氏杆菌菌株 (BadP)高表达和低表达的脂蛋白分别是询问的理想遗传工具 病原体-宿主细胞之间的相互作用由表面暴露的脂蛋白水平的差异所介导。长的- 拟议研究的长期目标是扰乱莱姆螺旋体在扁虱体内的复杂生活方式 哺乳动物宿主可降低莱姆病的发病率。在这个提案中，我们将检验中心假设 伯氏杆菌囊膜重塑改变宿主抗菌反应影响伯氏杆菌存活 不同的主人。中心假设将通过两个具体目标进行检验：具体目标1：确定 利用单细胞RNA-Seq(scRNA-Seq)技术研究脆性PG/脂蛋白对森林和哺乳动物细胞的信号转导作用 SEQ)分析。将对大量转录数据进行分析和验证，以确定途径和 区分单细胞反应的细胞表型与传播宿主、储存宿主和死端宿主 利用宿主细胞数量少、螺旋体数量少、纯化程度低的优点 硼的决定因素。肽聚糖识别蛋白和河马信号通路在调控中的作用 将对BB感染进行讯问。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，原代宿主细胞上的PG/脂蛋白组成。突变螺旋体在PG中存在缺陷 稳定/合成或改变水平的脂蛋白将被用来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植能力 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植/传播至关重要的新的信号通路。

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.