The Role of Plasmids in Rickettsia Biology

描述（由申请人提供）：立克次体属含有重要的病原体，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血食性节肢动物（蜱、虱子、跳蚤和螨虫）有关，这些节肢动物是它们向脊椎动物宿主传播的媒介。各种立克次体是人类斑疹伤寒和斑疹热的病原，但其他立克次体显然局限于节肢动物宿主作为内共生体。立克次体成员是已知的与线粒体最接近的微生物亲戚，它们具有高度减少的基因组和与其宿主细胞密切的代谢相互作用的特征。从历史上看，立克次体的缓慢和专性细胞内生长及其对基因操作的抵抗力对其研究提出了实质性的技术挑战。我们的实验室一直处于最近发展的遗传操纵立克次体的能力和立克次体质粒的发现的前沿，这是染色体外自主复制的遗传元件。致病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，并且是遗传多样性的重要来源，因为它们可以通过相同或不同物种的细菌之间的结合而移动或遗传传播。我们假设质粒参与了立克次体宿主的适应/毒力和遗传可塑性。我们建议全面分析与血食性节肢动物相关的立克次体的分布、系统发育和质粒在生物学中的作用。我们将使用脉冲场凝胶电泳和Southern blot技术来确定属内质粒的分布。我们将物理分离和克隆立克次体物种的质粒，范围从非致病性节肢动物内共生体到人类病原体。克隆的质粒将使用高通量焦磷酸测序技术（454 Life Science Corp.）进行测序，并进行系统发育分析，以确定该属质粒的可能起源和进化历史，并获得其潜在宿主适应功能的线索。我们将通过质粒固化、表型分析和质粒偶联实验来验证我们的假设，即质粒赋予立克次体宿主适应功能和遗传多样性。拟议的研究将描述质粒的分布，并测试它们在宿主适应/毒力中的作用，在一组医学上重要的专性细胞内细菌中，直到最近才被认为含有质粒。

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.