Development of Paratransgenic Ticks for Disease Control

描述（由申请人提供）：在这份修订后的竞争性续期申请中，我们建议以上一个资助期的关键进展为基础，即斑点热群（SFG）蜱虫共生体的培养分离；两种SFG立克次体蜱共生体表达GFP的稳定转化，以及它们作为模式生物在蜱内追踪SFG立克次体传播途径的研究；在这些立克次体中鉴定出一种质粒，其序列与felis的质粒具有同源性，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；肩胛棘球蚴免疫反应的表征。现在，我们建议通过基因组比较、全局基因表达分析和改进转化技术的发展，系统地探索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。提出的研究目标是开发基因组和转化技术，使我们能够批判性地评估作为载体-微生物相互作用分析策略的副转化范式。我们建议通过基因组比较、全局基因表达分析和改进转化技术的发展，系统地探索立克次体共生体生物学。我们的中心假设是：1)对共生体和病原体基因组的详细比较以及对关键基因的功能分析将揭示共生体为蜱虫提供血液中缺乏的营养物质的能力的重要细节；B)确定病原体传播给脊椎动物宿主并引起疾病的可能性。2)表达报告基因的转基因立克次体可用于建立蜱副突变模型，这将使我们能够检查和鉴定导致立克次体成功定植蜱的生物学过程，这是在蜱中建立立克次体转化体的先决条件。主要有3个目的：1)对立克次体的共生基因组进行散枪测序，对立克次体的整体基因表达进行分析，并与立克次体的立克次体进行探针排列，以确定产生共生与病原体的基因及其产物。研究结果将为共生立克次体和致病性立克次体的入侵和生存机制提供直接的比较。2)我们建议为SFG立克次体开发新的转化工具，即基于我们在monacensis立克次体中鉴定的质粒pRM的立克次体转化穿梭载体。此外，我们将优化已经成功应用于蜱传嗜吞噬细胞无原体转化SFG立克次体的Himar1转座酶系统。这将解决最近发现的富含GC区域的Tn5偏差。3)最后，在目标3中，我们提出对表达立克次体的荧光蛋白进行实时成像，分析立克次体在蜱中的运动，探讨蜱定植机制，这是建立共生体和病原体的前提。蜱虫携带大量能引起哺乳动物疾病的微生物，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在蜱虫体内共生，并干扰蜱虫种群中立克次体病病原体的维持。我们的研究将确定利用这些共生体减少蜱传疾病媒介向人类传播的方法。

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.