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

项目摘要 节肢动物疾病媒介的微生物居民对其宿主的生物学产生深远的影响， 正的（例如，营养获取，防御病原体）和阴性（例如，生殖寄生）。的 支持这些微生物过程的机制是遗传变异的进化结果， 通常由移动的遗传元件（MGE）介导。即使不知道这些常驻微生物 虽然它们具有致病性，但它们对宿主生物学的影响可影响病媒病原体的分布和传播。 鹿蜱，肩胛硬蜱，广泛分布于美国东部，是一种重要的传播媒介， 几种人类病原体，包括疏螺旋体属（莱姆病）和嗜吞噬细胞无形体 （无形体病）。鹿蜱还携带一种流行的细胞内细菌，布氏立克次体，这是唯一的， 立克次体物种在几个方面：1）它是垂直遗传与高效率的鹿蜱，但不循环 自然界中蜱和脊椎动物之间的联系; 2）令人惊讶的是，它是唯一被发现的立克次体物种 在重症肩胛肌;和3）其基因组实质上富含携带有趣货物的假基因和MGE， 包括生物素合成、抗生素合成和抗性以及非核糖体肽合成的基因。给定 其在鹿蜱中的维持，以及其潜在功能，R.布赫奈里将发挥重要影响 这一重要疾病载体的生物学研究。 本研究的长期目标是确定R。布赫内里和我 肩胛肌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。该项目的核心假设是，高分辨率 感染率和基因组群体亚结构的数据将阐明R. Buchneri的轨道 内共生生活方式这项工作将衡量R.布赫内里比 使用1）长读序测序以产生封闭基因组，2）RNA-seq以确认假基因预测，以及3） 基因组学来表征基因和其他基因组元件独特的R。buchneri（AIM 1）。当前 建议工作也将表征R.布赫内氏菌泛基因组，1）定量PCR，以评估其感染 鹿蜱自然种群中的感染率，2）短读深测序以确定R. Buchneri遗传变异、等位基因和MGE，以及3）用于表征横向遗传变异的程度（和起源）的基因组学。 基因转移到R. buchneri（AIM 2）。照明河布赫内氏基因组变异和蜱虫感染频率将 导致深入了解其与鹿蜱的关系，并最终为未来使用其MGE曲目的努力提供信息 作为传播因子的基因驱动工具来对抗蜱传疾病。

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.