Development of Paratransgenic Ticks for Disease Control

在这一修订后的竞争性续期申请中，我们建议在前一个阶段的关键进展的基础上， 授予期，即，培养分离斑点热组（SFG）蜱共生体;两种蜱的稳定转化 SFG立克次体蜱共生体表达GFP，以及它们作为模式生物体追踪 蜱内的SFG立克次体;在这些立克次体中鉴定与 R.猫质粒，以及在其他立克次体分离株中发现质粒，这与 立克次体不具有染色体外DNA;肩胛肌免疫反应。 我们现在建议通过基因组比较、分析 全球基因表达和改进的转化技术的发展。为了补充这些 通过努力，我们将测试立克次体转化体定殖蜱的能力，产生副转基因蜱。的 拟议研究的目标是开发基因组和转化技术，使我们能够 批判性地评价作为分析载体-微生物相互作用的策略的副转化范例。 我们建议通过基因组比较，全球性的分析，系统地探讨立克次体共生生物学。 基因表达和改进的转化技术的开发。我们的主要假设是：（1）A 共生体和病原体基因组的详细比较和关键基因的功能分析将a）揭示 关于共生体为蜱提供血液中缺乏的营养物质的能力的重要细节 膳食; B）确定病原体传播给脊椎动物宿主并诱发疾病的可能性。（二） 表达报告基因的转基因立克次体可用于建立蜱类副转殖模型 使我们能够检查和识别导致蜱虫成功定植的生物学过程， 立克次体，在蜱中建立立克次体转化体的先决条件。我们有三个目标：（1） 孔雀立克次体共生体基因组鸟枪法测序及全局基因探针芯片 表达分析并与立克次体进行比较，以鉴定这些基因及其 产生共生体的产物与产生病原体的产物。结果将提供一个直接 共生立克次体和致病立克次体入侵和存活机制的比较2）我们提出 为SFG立克次体开发新的转化工具，即，用于立克次体转化的穿梭载体 基于我们在摩纳哥立克次体中鉴定的质粒pRM。此外，我们还将优化 Himar 1转座酶系统，我们已经成功地应用于转化蜱传无形体 SFG立克次体的嗜吞噬细胞菌。这将解决最近发现的富含GC区域的Tn 5偏倚。第三章 最后，在目标3中，我们建议对表达荧光蛋白的立克次体进行实时成像， 分析立克次体在蜱中的运动，以探索蜱定殖的机制，这是建立 共生体和病原体。

In this revised competing application for renewal, we propose to build on key advances during the previous grant period, i.e., culture isolation of spotted fever group (SFG) tick symbionts; stable transformation of two SFG rickettsial tick symbionts to express GFP, and their use as model organisms to track routes of dispersal of SFG rickettsiae within ticks; identification of a plasmid in these rickettsiae having sequence homology with the R. felis plasmids, and discovery of plasmids in other rickettsial isolates, which is contrary to the paradigm that rickettsiae do not possess extrachromosomal DNA; and characterization of I. scapularis immune responses. We now propose to systematically probe rickettsial symbiont biology by genomic comparison, analysis of global gene expression, and development of improved transformation technology. To complement these efforts, we will test the ability of rickettsial transformants to colonize ticks, generating paratransgenic ticks. The goal of the proposed research is to develop genomic and transformation technologies that will enable us to critically evaluate the paratransgenesis paradigm as a strategy for analysis of the vector - microbe interaction. We propose to systematically probe rickettsial symbiont biology by genomic comparison, analysis of global gene expression, and development of improved transformation technology. Our central hypotheses are 1) A detailed comparison of symbiont and pathogen genomes and a functional analysis of key genes will a) reveal important details about the ability of symbionts to provide ticks with nutrients that are absent from the blood meal; b) define the potential of pathogens to be transmitted to vertebrate hosts and induce illness. 2) transgenic rickettsiae expressing reporter genes can be used to create a tick paratransgenesis model that will enable us to examine and identify the biological processes that lead to successful colonization of ticks by rickettsiae, a prerequisite for establishment of rickettsial transformants in ticks. There are 3 aims: 1) We will shotgun-sequence the Rickettsia peacockii symbiont genome and probe tiling arrays for global gene expression analysis and comparison with Rickettsia rickettsii in an effort to identify those genes and their products that make a symbiont versus those that make a pathogen. The results will provide a direct comparison of invasion and survival mechanisms used by symbiotic and pathogenic rickettsiae 2) We propose to develop novel transformation tools for SFG rickettsiae, i.e., a shuttle vector for transformation of rickettsiae based on the plasmid, pRM, that we have identified in Rickettsia monacensis. In addition, we will optimize the Himar1 transposase system that we have successfully applied to transformation of tick-borne Anaplasma phagocytophilum, for SFG rickettsiae. This will address the recently found bias of Tn5 for GC rich regions. 3) Finally, in aim 3, we propose to carry out real-time imaging of fluorescent protein expressing rickettsiae and analyze rickettsial movement in ticks to probe mechanisms of tick colonization, a prerequisite for establishment of both symbionts and pathogens.