Symbiotic Rickettsia Species as a Model System for the Study of Folate Biosynthes

描述（由申请人提供）：非致病性提供营养的细菌内共生体已被证明通过向其宿主提供必需维生素和氨基酸而有助于其宿主的适应性。蜱类内共生体之间共生关系的代谢基础尚不清楚。我们实验室最近的代谢重建、PCR和DNA测序结果表明，太平洋硬蜱中的一种立克次体携带有辅助因子和维生素的合成基因。叶酸（维生素B 9）的生物合成途径特别令人感兴趣，因为我们实验室先前的生物信息学代谢重建表明该途径完全存在于立克次体物种的基因组中。该项目的总体目标是研究细菌维生素生物合成基因，这些基因是与蜱宿主共生关系所必需的。我们假设内共生立克次体物种合成叶酸，维生素被蜱宿主利用。本论文的主要工作包括三个方面：1）利用PCR、克隆和测序技术对太平洋硬蜱立克次体叶酸代谢途径基因进行了研究。2)使用定量逆转录酶PCR（qRT-PCR）表征太平洋硬蜱中立克次体物种叶酸途径中基因的基因表达模式。3)通过互补作用和体外酶法测定太平洋硬蜱立克次体叶酸生物合成途径中基因产物的功能。该研究将增加我们对蜱中叶酸代谢的理解，以及共生立克次体与太平洋硬蜱之间共生相互作用的代谢基础。描述生物合成基因的功能，并定义的能力的维生素贡献的立克次体物种在太平洋硬蜱，将提供重要的见解，内共生关系的分子机制，在太平洋硬蜱。 公共卫生相关性：莱姆病和无形体病是美国最常见的蜱传疾病，预防这些疾病已成为当前蜱研究的重点。生活在真核细胞中的细菌让它们的宿主 获得代谢能力，否则无法获得。这些细菌伴侣通过提供维生素和氨基酸等必需营养素来促进宿主的健康。主要或完全以血液为食的节肢动物实际上是这些类型的繁殖地 因为脊椎动物的血液很少含有足够数量的必需B族维生素和氨基酸。因此，食血节肢动物的饮食补充细菌合成的维生素（2，5，76）。大多数蜱的唯一食物是脊椎动物的血液， 但太平洋硬蜱的维生素B来源不明。最近对肩突硬蜱基因组的测序揭示了这种蜱的细菌伴侣的基因组序列。使用免费提供的计算机软件，以及我们实验室编写的计算机算法，我们已经发现了参与维生素B 9生物合成途径的所有六个基因。该途径来自太平洋硬蜱（Ixodes pacificus）中的一种立克次体，太平洋硬蜱是肩突硬蜱的西海岸亲戚。在这个建议中，我们假设立克次体物种，生活在太平洋硬蜱细胞，产生必需的B 9维生素，而不是由太平洋硬蜱合成。我们将确定立克次体B 9维生素途径中的所有基因是否在太平洋硬蜱蜱中活跃转录，以及这些细菌基因是否编码合成维生素B 9的蛋白质。拟议的研究将提供一个理想的模型系统，研究细菌内共生体对蜱代谢的影响，更具体地说，立克次体内共生体对太平洋硬蜱健身的影响。

DESCRIPTION (provided by applicant): Nonpathogenic nutrient-providing bacterial endosymbionts have been shown to contribute to their hosts' fitness by supplying them with essential vitamins and amino acids. The metabolic basis for the symbiotic relationship of endosymbionts in ticks has been unknown. Recent metabolic reconstruction, PCR, and DNA sequencing results in our lab have shown that a Rickettsia species in Ixodes pacificus carries genes for the anabolism of cofactors and vitamins. The biosynthetic pathway of folate (vitamin B9) is of particular interest because previous bioinformatic metabolic reconstruction in our lab indicates that the pathway exists in its entirety in the genome of the Rickettsia species. The overall objective of the project is to study bacterial vitamin biosynthetic genes that are essentia for the symbiotic relationship with the tick host. We hypothesize that the endosymbiotic Rickettsia species synthesizes folate, and that the vitamin is utilized by the tick host. The proposed work has three major aspects: 1) Characterization of genes in the folate pathway of the Rickettsia species in Ixodes pacificus using PCR, cloning, and sequencing. 2) Characterization of gene expression patterns of genes in the folate pathway of the Rickettsia species in Ixodes pacificus using quantitative reverse transcriptase PCR (qRT-PCR). 3) Characterization of function of gene products in the folate biosynthetic pathway of the Rickettsia species in Ixodes pacificus by complementation and in vitro enzymatic assay. The research will increase our understanding of the metabolism of folate in ticks, as well as the metabolic basis of symbiotic interactions between the symbiotic Rickettsia species and Ixodes pacificus ticks. Describing the functions of the biosynthetic genes and defining the capacity of the vitamins contributed by the Rickettsia species in Ixodes pacificus, will provide important insights into molecular mechanisms of the endosymbiotic relationship in Ixodes pacificus. PUBLIC HEALTH RELEVANCE: Lyme borreliosis and anaplasmosis are the most common tick-borne diseases in the United States, and prevention of these diseases has become the focus of current tick research. Bacteria that live within eukaryotic cells have allowed their hosts to acquire metabolic capabilities otherwise unavailable. These bacterial partners contribute to their hosts' fitness by providing essential nutrients such as vitamins and amino acids. Arthropods that feed primarily or entirely on blood are virtually a breeding ground for these types of partnerships because vertebrate blood rarely contains sufficient quantities of essential B-vitamins and amino acids. Therefore, the diet of blood feeding arthropods is complemented with bacterially synthesized vitamins (2, 5, 76). The sole diet of most ticks is blood from vertebrates, but the sources of B-vitamins for Ixodes pacificus ticks are unknown. The recent sequencing of the Ixodes scapularis genome revealed genomic sequences of the tick's bacterial partner. Using freely available computer software, as well as computer algorithms written in our lab, we have discovered all six genes involved in the vitamin B9 biosynthetic pathway. The pathway is from a Rickettsia species in Ixodes pacificus, the West Coast relative of Ixodes scapularis. In this proposal, we hypothesize that the Rickettsia species, living within Ixodes pacificus cells, produces essential B9 vitamin that is not synthesized by Ixodes pacificus. We will determine if all genes in the B9 vitamin pathway of the Rickettsia species are actively transcribed in Ixodes pacificus ticks, and if these bacterial genes encode proteins that synthesize vitamin B9. The proposed studies will offer an ideal model system to study the impact of bacterial endosymbionts on the metabolism of ticks and more specifically, the effect of the Rickettsia endosymbiont on Ixodes pacificus fitness.