Characterizing the Pan-genome of a Rickettsia Infecting the Eastern Black-legged Tick

项目总结 节肢动物疾病病媒的微生物居住者对其宿主的生物学产生了深远的影响，两者 积极的(例如，获得营养、防御病原体)和消极的(例如，生殖寄生)。这个 支撑这些微生物过程的机制是遗传变异和 通常由移动遗传元件(MGES)介导。即使这些驻留的微生物并不是已知的 在病原性方面，它们对寄主生物的影响可以影响媒介传播病原体的分布和传播。 鹿扁虱是一种广泛分布于美国东部的软体动物，是一种重要的传播者。 几种人类病原体，包括疏螺旋体(莱姆病)和无浆体吞噬细胞性 (无浆体病)。鹿虱还藏有一种普遍存在的细胞内细菌，布氏立克次体，这种细菌在 立克次体在几个方面：1)它是垂直遗传的，在鹿体内效率很高，但不会循环 2)令人惊讶的是，它是迄今发现的唯一立克次体物种 和3)它的基因组大量富含伪基因和携带有趣货物的MGES， 包括生物素合成、抗生素合成和耐药性以及非核糖体肽合成的基因。vt.给出 它在鹿体内的维持，以及它的潜在功能，布氏杆菌将对其产生重大影响 关于这一重要疾病媒介的生物学。 这项研究的长期目标是确定布氏杆菌和我之间的关系的性质。 肩胛骨。目前的工作旨在通过应对重大挑战来推进这一研究目标；即， 缺乏高质量的参考基因组，感染率和分布数据不一致，以及缺乏 关于菌株水平基因组变异的信息。该项目的中心假设是高分辨率 感染率和基因组亚群结构的数据将阐明布氏杆菌向专性种群的轨迹 内共生的生活方式。拟议的工作将通过以下方式衡量布氏罗非鱼特定物种创新的程度 使用1)长阅读测序产生闭合基因组，2)RNA-SEQ确认假基因预测，以及3) 系统基因组学，以确定布氏杆菌特有的基因和其他基因组元件(目标1)。海流 计划的工作还将通过使用定量聚合酶链式反应来评估其感染来表征布氏杆菌的泛基因组。 2)短读深度测序以确定R。 Buchhneri的遗传变异、等位基因和MGES，以及3)系统发育学以表征侧枝的程度(和起源)。 基因导入布氏罗非鱼(AIM 2)。阐明布氏管圆线虫基因组变异和壁虱感染频率将 深入了解它与鹿虱的关系，并最终为未来使用其MGES曲目的努力提供参考 作为传播因子的基因驱动工具，以对抗壁虱传播的疾病。

PROJECT SUMMARY Microbial residents of arthropod disease vectors engender profound effects on the biology of their host, both positive (e.g., nutrient acquisition, defense from pathogens) and negative (e.g., reproductive parasitism). The mechanisms that underpin these microbial processes arise as evolutionary consequences of genetic variation and are often mediated by mobile genetic elements (MGEs). Even when these resident microbes are not known to be pathogenic, their impact on host biology can influence the distribution and transmission of vector-borne pathogens. The deer tick, Ixodes scapularis, is widely distributed in the Eastern United States and is an important transmitter of several human pathogens, including Borrelia species (Lyme disease) and Anaplasma phagocytophilum (anaplasmosis). Deer ticks also harbor a prevalent intracellular bacterium, Rickettsia buchneri, that is unique among Rickettsia species in several ways: 1) it is vertically inherited with high efficiency in the deer tick, but does not cycle between ticks and vertebrates in nature; 2) it is, astonishingly, the only Rickettsia species that has ever been detected in I. scapularis; and 3) its genome is substantially enriched with pseudogenes and MGEs that carry intriguing cargo, including genes for biotin synthesis, antibiotic synthesis and resistance, and nonribosomal peptide synthesis. Given its maintenance in deer ticks, and its arsenal of potential functions, R. buchneri stands to exert significant influence on the biology of this important disease vector. The long-term goal of this research is to determine the nature of the relationship between R. buchneri and I. scapularis. The current work is designed to advance this research goal by addressing significant challenges; namely, the lack of a high-quality reference genome, inconsistencies in infection rate and distribution data, and a dearth of information regarding strain-level genomic variation. The central hypothesis of this project is that high-resolution data on infection rate and genomic population substructure will elucidate R. buchneri's trajectory toward an obligate endosymbiotic lifestyle. The proposed work will gauge the extent of species-specific innovation in R. buchneri by using 1) long-read sequencing to generate a closed genome, 2) RNA-seq to confirm pseudogene prediction, and 3) phylogenomics to characterize genes and other genomic elements unique to R. buchneri (AIM 1). The current proposal work will also characterize the R. buchneri pan-genome by using 1) quantitative PCR to assess its infection rate among natural populations of deer ticks, 2) short-read deep sequencing to determine the distribution of R. buchneri genetic variants, alleles, and MGEs, and 3) phylogenomics to characterize the extent (and origin) of lateral gene transfers into R. buchneri (AIM 2). Illuminating R. buchneri genomic variation and tick infection frequency will lead to insights into its relationship with the deer tick and ultimately inform future efforts to use its repertoire of MGEs as gene drive tools for spreading factors to combat tick-borne diseases.