MOLECULAR CHARACTERIZATION OF T. GONDII MEROZOITES TO DEVELOP A CULTURE SYSTEM

描述（由申请人提供）：细胞内寄生虫是全世界人类疾病负担的重要组成部分。顶复体弓形虫是比较成功的寄生虫之一，估计有三分之一的人口被感染。在美国，每年大约有150万新的感染病例，它是今天食源性疾病死亡的第二大原因。弓形虫的生命周期有两个方面使它如此普遍，一是感染大量中间宿主的能力，二是通过一次感染猫（最终宿主）产生数百万个具有环境抗性的卵囊的能力。在了解中间阶段，速殖子和慢殖子的生物学方面已经进行了大量的工作，但到目前为止，对慢殖子以外的阶段，分殖子和有性阶段的培养方法还没有发展起来，这阻碍了研究寄生虫生命周期的大部分的能力。这个项目描述了一个策略

DESCRIPTION (provided by applicant): Intracellular parasites represent a significant portion of human disease burden throughout the world. The Apicomplexan parasite Toxoplasma gondii is one of the more successful where it is estimated up to a third of the human population has been infected. With approximately 1.5 million new infections in the U.S. per year it is the second leading cause of death by foodborne illness today. There are two aspects of the Toxoplasma life cycle that allow it to be so prevalent, the ability to infect a vast number of intermediate hosts ad the ability to produce millions of environmentally resistant oocysts through a single infection of cat, the definitive host. Much work has been carried out to understand the biology of the intermediate stages, the tachyzoite and bradyzoite, but as of yet, culturing methods for stages beyond the bradyzoite, the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasites life cycle. This project describes a strategy that will begin to unravel the molecular aspects of the merozoite stage and plans to use this information to design a forward selection strategy that will allow us to observe merozoite differentiation in tissue culture. We have harvested merozoite parasites and hybridized mRNA to the Affymetrix Toxoplasma Gene Chip. This microarray data of Toxoplasma merozoites represents the first global gene expression study for this stage, as well as the first for any intraepithelial enteric stage of the parasite, and provides a global profile of merozoite-specific information that will be critical to unlocking how the parasite senses and responds to the felid gut environment. Transgenic parasites that express drug selectable markers only at the merozoite stage will be used in a forward selection strategy to screen for tissue culture conditions that are favorable to merozoite growth. In addition to having tagged transgenic parasites for following merozoite development, we will develop merozoite-specific antibodies for several of the candidate merozoite genes. Creating an intestinal cell culture from the definitive host will provide the needed host component in further understanding the definitive host-parasite relationship that comprises the sexual stage of the parasite. Advances in the understanding of intestinal cell biology have led to the creation of in vitro culture methods for intestinal crypt stem cells. These methods are applicable to both mouse and human cells and hold promise that they can be applied to felid intestinal stem cells. Given the correct conditions, transgenic parasites containing drug selection/reporters driven by merozoite promoters should provide the indication that they have converted to the merozoite stage. By quantifying the number of parasites that differentiate to merozoites across a number of different conditions we will be able to determine those conditions most favorable to inducing the parasite into this developmental switch. Observing this in tissue culture would be a first, and will provide a foundation for the study of what is a relatively unknown portion of the Toxoplasma gondii life cycle. In working on this project I hope to continue research where I have concentrated upon parasite gene expression and control, and host parasite interactions. This research will increase our understanding of how this parasite is able to transmit so pervasively and may be applicable to other host-parasite relationships between parasites and their definitive hosts.