Development of Paratransgenic Ticks for Disease Control

描述（由申请人提供）：在这一修订后的竞争性续期申请中，我们建议在上一个赠款期的关键进展的基础上，即，斑点热群（SFG）蜱共生体的培养分离;两种SFG立克次体蜱共生体的稳定转化以表达GFP，以及它们作为模式生物以追踪SFG立克次体在蜱内的传播途径的用途;鉴定这些立克次体中与R.猫立克次体质粒，并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的范式相反;肩胛肌免疫反应。我们现在建议通过基因组比较、全球基因表达分析和改进转化技术的发展来系统地探测立克次体共生体生物学。为了补充这些努力，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。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。我们建议通过基因组比较、全球基因表达分析和改进转化技术的发展来系统地探测立克次体共生体生物学。我们的中心假设是1）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）揭示共生体为蜱提供血餐中缺乏的营养物质的能力的重要细节; B）确定病原体传播到脊椎动物宿主并诱发疾病的潜力。2)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，使我们能够检查和鉴定导致立克次体成功定殖蜱类的生物学过程，这是在蜱类中建立立克次体转化体的先决条件。有三个目标：1）我们将对孔雀立克次体共生体基因组进行鸟枪测序，并将探针平铺阵列用于全球基因表达分析，并与立克次体进行比较，以努力识别那些构成共生体的基因及其产物与那些构成病原体的基因及其产物。结果将提供共生和致病性立克次体所使用的入侵和存活机制的直接比较2）我们建议开发SFG立克次体的新的转化工具，即，一种基于我们在摩纳哥立克次体中鉴定的质粒pRM的用于立克次体转化的穿梭载体。此外，我们将优化Himar 1转座酶系统，我们已经成功地应用于转化蜱传的嗜吞噬细胞无形体，SFG立克次体。这将解决最近发现的富含GC区域的Tn 5偏倚。3)最后，在目标3中，我们建议对表达立克次体的荧光蛋白进行实时成像，并分析立克次体在蜱中的运动，以探测蜱定殖的机制，这是建立共生体和病原体的先决条件。蜱携带大量的微生物，导致哺乳动物的疾病，包括病毒，立克次体，其他细菌和原生动物。其中许多是新出现或重新出现的病原体，如落基山斑疹热立克次体。此外，许多不会引起人类疾病的微生物与蜱虫共生，并干扰蜱虫种群中立克次体病病原体的维持。我们的研究将确定如何利用这些共生体来减少蜱传疾病病原体向人类的传播。

DESCRIPTION (provided by applicant): 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 extra-chromosomal 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. PUBLIC HEALTH RELEVANCE Ticks carry a vast array of microbes that cause diseases in mammals, including viruses, rickettsiae, other bacteria and protozoa. Many of these are emerging or re-emerging pathogens such as Rocky Mountain spotted fever rickettsiae. In addition, numerous microbes that do not cause human disease live symbiotically within ticks, and interfere with maintenance of rickettsial disease agents in tick populations. Our research will identify ways in which these symbionts can be exploited to reduce transmission of tick-borne disease agents to humans.