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宿主提供必需的维生素和氨基酸来促进其健康。蜱内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，太平洋冰蚊中的立克次体携带辅助因子和维生素合成代谢的基因。叶酸（维生素B9）的生物合成途径特别有趣，因为我们实验室先前的生物信息学代谢重建表明，该途径完整地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，这些基因对于与蜱虫宿主的共生关系至关重要。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。主要包括三个方面的工作：1)利用PCR、克隆和测序技术对太平洋冰蚊立克次体叶酸途径基因进行鉴定。2)利用定量逆转录酶PCR （qRT-PCR）研究太平洋伊蚊立克次体叶酸途径基因表达模式。3)利用互补和体外酶法研究太平洋冰蚊立克次体叶酸生物合成途径中基因产物的功能。本研究将增加我们对蜱体内叶酸代谢的认识，以及共生立克次体与太平洋冰蜱共生相互作用的代谢基础。描述生物合成基因的功能和确定立克次体物种在太平洋冰蚊中贡献的维生素的能力，将为了解太平洋冰蚊内共生关系的分子机制提供重要的见解。

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.