The Role of Plasmids in Rickettsia Biology

描述(申请人提供)：立克次体含有重要的病原体，由在真核宿主细胞内复制的小革兰氏阴性细菌组成。许多立克次体物种与食血节肢动物(扁虱、虱子、跳蚤和螨类)有关，这些节肢动物是它们传播给脊椎动物宿主的媒介。各种立克次体是人类斑疹伤寒和斑点热病的病原体，但其他种类显然仅限于节肢动物宿主作为内共生体。立克次体成员是已知的线粒体的最近的微生物近亲，它们具有基因组高度减少和与宿主细胞密切的代谢相互作用的特征。从历史上看，立克次体缓慢而专属的细胞内生长及其对基因操纵的抵抗力给他们的研究带来了巨大的技术挑战。我们的实验室在最近开发的对立克次体进行遗传操作的能力和发现立克次体质粒方面一直处于领先地位，立克次体是染色体外自主复制遗传元素的载体。病原菌中的质粒通常与涉及特定代谢能力或抗生素耐药性的毒力功能有关，是遗传多样性的重要来源，因为它们可能是可移动的，或者通过相同或不同物种的细菌之间的接合而在基因上传播。我们推测，质粒参与立克次体寄主适应/毒力和遗传可塑性。我们建议全面分析与食血节肢动物相关的立克次体的分布、系统发育及其在立克次体生物学中的作用。我们将使用脉冲场凝胶电泳和Southern印迹技术来确定该属内的质粒分布。我们将从立克次体物种中物理分离和克隆从非致病节肢动物内共生体到人类病原体的质粒。克隆的质粒将用高通量焦化测序技术(454生命科学公司)进行测序。并进行系统发育分析，以确定该属内质粒的可能起源(S)和进化历史，并获得其潜在的寄主适应功能的线索。我们将通过质粒固化和表型分析以及质粒接合实验来验证我们的假设，即质粒赋予立克次体内宿主适应功能和遗传多样性。这项拟议的研究将描绘出质粒的分布，并测试它们在宿主适应/毒力中的作用，这些细菌具有重要的医学意义，直到最近，人们还不认为这些细菌含有质粒。

DESCRIPTION (provided by applicant): The genus Rickettsia contains important pathogens and consists of small gram-negative bacteria that replicate within eukaryotic host cells. Many Rickettsia species are associated with blood-feeding arthropods (ticks, lice, fleas and mites) that serve as vectors for their transmission to vertebrate hosts. Various Rickettsia species are the etiologic agents of typhus and spotted fevers in humans but others are apparently confined to arthropod hosts as endosymbionts. Members of the Rickettsia are the closest known microbial relatives of mitochondria with which they share the characteristics of highly reduced genomes and intimate metabolic interactions with their host cells. Historically, the slow and obligate intracellular growth of rickettsiae and their resistance to genetic manipulation presented substantial technical challenges to their study. Our laboratory has been at the forefront of both the recently developed capability to genetically manipulate rickettsiae and the discovery of rickettsial plasmids, which are extra-chromosomal autonomously replicating genetic elements. Plasmids in pathogenic bacteria are often associated with virulence functions involving particular metabolic capabilities or antibiotic resistance and are an important source of genetic diversity because they may be mobile, or genetically transmissible, via conjugation between bacteria of the same or different species. We hypothesize that plasmids are involved in rickettsial host adaptation/virulence and genetic plasticity. We propose a comprehensive analysis of the distribution, phylogeny and role of plasmids in the biology of rickettsiae associated with blood-feeding arthropods. We will use pulsed field gel electrophoresis and Southern blot techniques to define the distribution of plasmids within the genus. We will physically isolate and clone plasmids from Rickettsia species that range from non-pathogenic arthropod endosymbionts to human pathogens. The cloned plasmids will be sequenced with high throughput pyro-sequencing technology (454 Life Science Corp.) and subjected to phylogenetic analysis to determine the probable origin(s) and evolutionary history of plasmids within the genus, and obtain clues to their potential host adaptive functions. We will test our hypotheses that plasmids confer host adaptive functions and genetic diversity within rickettsiae through plasmid curing and phenotype analyses and plasmid conjugation experiments. The proposed research will delineate the distribution of plasmids and test their role in host adaptation/virulence in a group of medically important obligate intracellular bacteria that, until recently, were not believed to harbor plasmids.