Genetic basis for persistence of Borrelia burgdorferi

摘要 在脊椎动物宿主中建立持续感染所需的遗传因素是最少的之一。 了解伯氏疏螺旋体感染的各个方面。建立持久性的多个触发器 已经被提出了。然而，缺乏灵敏的血清学和分子检测在很大程度上阻碍了这一进程。 研究持续性螺旋体的生物学及其在治疗后莱姆病中的直接作用 综合征(PTLDS)。我们已经开发了两种分析方法，克服了这些主要限制，并可以促进 对持续性感染的研究。TBDCapSeq是下一代捕获测序分析，具有 灵敏度高于定量聚合酶链式反应，使伯氏杆菌基因组在宿主组织样本中组装成为可能。 待定血清芯片是一种多肽阵列，每次检测可以识别17万个线性多肽的抗体反应， 为B细胞对伯氏杆菌主要抗原的反应提供了前所未有的一瞥。在本建议书中 这些分析将被用于三个特定的目标，将检查一个人的分子和遗传足迹 持续的伯氏杆菌感染。在具体目标1中，我们将进行全面的血清学分析 血清和脑脊液来自一个特征良好的PTLDS患者队列。我们将确定所有主要的 两种标本类型的免疫反应表位。这些数据将与来自 其他非PTLDS莱姆病队列与PTLDS潜在的特定生物标记物有关。在具体目标2中， 我们将使用TBDCapSeq来识别PTLDS患者队列中伯氏杆菌的分子足迹。 TBDCapSeq的卓越灵敏度与高测序深度相结合，将提供 对伯氏杆菌和PTLDS之间联系的最可靠的分子分析。在具体目标3中，我们将 伯氏杆菌在C3H/HEN、BALB/c和Permyscus中三种不同基因型的动物模型的建立 白斑小鼠。每种伯氏杆菌基因型在有无持续状态下建立持久性的能力 将对抗生素治疗进行评估，并将使用TBDCapSeq来识别独特的遗传特征 持久的螺旋体。随着基因组分析，我们将进行第一次转录组研究 持久的螺旋体。这些目标结合在一起，将提供对慢性支气管炎患者免疫反应的洞察 PTLDS和确定螺旋体持续存在的机制触发因素。

ABSTRACT The genetic factors required to establish a persistent infection in vertebrate hosts is one of the least understood aspects of an infection with Borrelia burgdorferi. Multiple triggers for establishment of persistence have been proposed. However, the lack of sensitive serologic and molecular assays has substantially impeded work examining the biology of persistent spirochetes and their direct role in Post-Treatment Lyme Disease Syndrome (PTLDS). We have developed two assays that overcome these major limitations and can facilitate studies of persistent infections. The TBDCapSeq, a next generation capture sequencing assay, has a sensitivity superior to quantitative PCR and enables B. burgdorferi genome assembly in host tissue samples. The TBD-Serochip, a peptide array that can identify antibody responses to 170,000 linear peptides per assay, provides unprecedented glimpses into B cell responses to the major antigens of B. burgdorferi. In this proposal these assays will be employed in three Specific Aims that will examine molecular and genetic footprints of a persistent B. burgdorferi infection. In Specific Aim 1, we will perform a comprehensive serologic analysis of sera and cerebrospinal fluid from a well characterized PTLDS patient cohort. We will identify all major immunoreactive epitopes in both specimen types. These data will be differentiated with serology data from other non-PTLDS Lyme disease cohorts to implicate potential specific biomarkers of PTLDS. In Specific Aim 2, we will employ the TBDCapSeq to identify molecular footprints of B. burgdorferi in the PTLDS patient cohort. The combination of superior sensitivity of TBDCapSeq coupled with high sequencing depth will provide the most robust molecular analysis of the link between B. burgdorferi and PTLDS. In Specific Aim 3, we will establish an animal model for three distinct genotypes of B. burgdorferi in C3H/HeN, BALB/c and Peromyscus leucopus mice. The capacity of each B. burgdorferi genotype for establishing persistence with and without antibiotic treatment will be assessed and the TBDCapSeq will be used to identify genetic features unique to persistent spirochetes. Along with genomic analysis, we will perform the first transcriptome investigation of persistent spirochetes. Combined, these Aims will provide insight into the immune response of patients with PTLDS and identify mechanistic triggers of spirochete persistence.