Identification of essential factors contributing to vector colonization and transmission of relapsing fever spirochetes

项目摘要 了解病原体在不同自然环境中的生命周期是一个进化缓慢的过程 传染病研究领域。壁虱传播的复发热(RF)螺旋体就是这样一种病原体 已经适应了节肢动物和哺乳动物的血液。当扁虱吸食传染性的血粉时，SPI- 轮虫进入中肠。在~2周内，一群人随后迁移并定居在唾液中 腺体。然而，促进媒介定植和随后传播到 哺乳动物的宿主仍不清楚。因此，描绘RF螺旋体生命周期的错综复杂是 尤其与公共卫生有关。 RF螺旋体基因组学、转录组学和遗传学方面的局限性已经通过开发- 建立了斑点疏螺旋体-鸟形目斑点斑病模型。利用功能基因组学方法，有两个重要的 据观察，这一建议建立在：1)经过220代后，斑点热菌丢失基因Es-1。 在载体定植和/或确定哺乳动物感染方面有意义；2)150kb线性巨质粒 (Lp150)可能对媒介的定植和传播很重要。在Plas的3‘端有一个36kb的基因座- MID包含一系列编码表面蛋白的基因，这些基因在TICK载体中上调 被感染的哺乳动物的血液。这一应用的中心假设是，存在斑疹病毒亚群 在中肠和唾液腺定植和建立早期哺乳动物感染中所必需的基因。那个人-- 下层目标是有目的的： 目的1：通过比较Ge-Ge基因的差异，找出在硬虱-哺乳动物传播周期中必不可少的基因。 Nomics，并定义了斑疹伤寒杆菌定位于 36kb的基因座。目的是量化病原体生活过程中36kb基因座上的基因表达。 在扁虱载体中循环，并将结果与蛋白质生产相一致。另外，比较基因组分析 野生型螺旋体和非定殖性/非感染性菌株之间的差异将识别可能的必要基因。 目的2：鉴定和鉴定突变基因在载体定植和传播中的作用。 SIS、成像、结构和生物物理分析。目标是生成和评估B.tu- Ricatae突变体，以确定扁虱介导的传播给小鼠的缺陷。这些基因在人类基因组中的意义 扁虱-哺乳动物的传播循环将进一步通过定点突变和结构和 生物物理分析。 随着RF螺旋体表面蛋白质组在发病机制中发挥重要作用，这些发现将 指导策略，以中断扁虱和哺乳动物中病原体的生命周期。其结果将广泛应用于-- 向旧大陆和新大陆物种发出电报，并解决NIAID更好地了解和预防疾病的使命。

Project Summary Understanding the life cycle of a pathogen within different natural environments is a poorly evolved area of infectious disease research. Tick-borne relapsing fever (RF) spirochetes are one such pathogen that have adapted to colonize arthropods and mammalian blood. When a tick imbibes an infectious bloodmeal, spi- rochetes enter the midgut. Within ~ 2 weeks a population subsequently migrates and colonizes the salivary glands. However, the molecular events facilitating vector colonization and subsequent transmission to the mammalian host remain unclear. Consequently, delineating the intricacies of the life cycle of RF spirochetes is particularly relevant to public health. Limitations in RF spirochete genomics, transcriptomics, and genetics have been addressed by develop- ing the Borrelia turicatae-Ornithodoros turicata model. Utilizing a functional genomic approach, two important observations were made that this proposal builds upon: 1) after 220 generations, B. turicatae losses genes es- sential in vector colonization and/or establishing mammalian infection; 2) the 150 kb linear megaplasmid (lp150) is likely important for vector colonization and transmission. A 36 kb locus toward the 3' end of the plas- mid contains a series of genes that coded for surface proteins that are up-regulated in the tick vector compared to infected mammalian blood. The central hypothesis of this application is that there are subsets of B. turicatae genes essential in midgut and salivary gland colonization and establishing early mammalian infection. The fol- lowing aims are purposed: Aim 1: Identify genes that are essential in the tick-mammalian transmission cycle by comparative ge- nomics, and define temporal expression and protein production of B. turicatae genes localized in the 36 kb locus. The objective is to quantify the expression of genes in the 36 kb locus during the pathogens life cycle in the tick vector, and coincide the findings with protein production. Also, a comparative genomic analysis between a wild type spirochetes and noncolonizing/noninfectious strains will identify likely essential genes. Aim 2: Identify and characterize genes essential in vector colonization and transmission by mutagene- sis, imaging, and structural and biophysical analyses. The objective is to generate and evaluate B. tu- ricatae mutants, to identify defects in tick-mediated transmission to mice. The significance of these genes in the tick-mammalian transmission cycle will be further verified by site directed mutagenesis and structural and biophysical analyses. With the surface proteome of RF spirochetes serving essential roles in pathogenesis, these findings will guide strategies to interrupt the pathogen's life cycle in the tick and mammal. The results will be broadly appli- cable to Old and New World species, and address NIAID's mission to better understand and prevent disease.