Development of Paratransgenic Ticks for Disease Control

在这份修订的竞争性续期申请中，我们建议在上一次 授权期，即斑点热群(SFG)硬蜱共生体的培养分离；两个 SFG立克次体共生体表达绿色荧光蛋白及其作为模式生物追踪病毒传播途径的研究 壁虱内的SFG立克次体；在这些立克次体中鉴定与序列同源的质粒 费氏立克次体，以及在其他立克次体分离株中发现的质粒，这与 立克次体不具有染色体外DNA；以及肩袖立克次体免疫反应的特征。 我们现在建议通过基因组比较、分析来系统地探索立克次体共生生物学 全球基因表达，以及改进的转化技术的发展。为了补充这些内容 通过努力，我们将测试立克次体转化子定植硬蜱的能力，从而产生副转基因硬蜱。这个 拟议研究的目标是开发基因组和转化技术，使我们能够 批判性地评估副转基因范式作为分析载体-微生物相互作用的策略。 我们建议通过对全球立克次体的基因组比较、分析，系统地探索立克次体共生生物学。 基因表达，以及改良转化技术的发展。我们的中心假设是1)A 共生菌和病原菌基因组的详细比较以及关键基因的功能分析将揭示 关于共生体为扁虱提供血液中没有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引发疾病的可能性。2) 表达报告基因的转基因立克次体可以用来建立扁虱异育模型，它将 使我们能够通过以下方式检查和确定导致硬蜱成功定居的生物过程 立克次体，这是在硬体中建立立克次体转化子的先决条件。有三个目标：1)我们将 孔雀立克次体共生体基因组的鸟枪测序和全球基因的探针拼接阵列 与立克次体的表达分析和比较，以努力确定这些基因和它们的 制造共生体的产品与制造病原体的产品。结果将提供一个直接的 共生立克次体和致病性立克次体侵袭和存活机制的比较2)我们提出 开发SFG立克次体新型转化工具，即穿梭载体转化立克次体 根据我们在莫纳克次体中鉴定的质粒PrM。此外，我们还将优化 我们已成功应用的Himar1转座酶系统转化蜱传无浆体 吞噬细胞，对SFG立克次体。这将解决最近发现的TN5对GC富集区的偏见。3) 最后，在目标3中，我们提出了对表达立克次体和 分析硬体内立克次体的运动，探讨硬体定植的机制，是建立立克次体的前提 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.