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

描述(由申请人提供)：细胞内寄生虫在全世界人类疾病负担中占很大比例。弓形虫寄生虫是比较成功的寄生虫之一，估计有三分之一的人口感染了弓形虫。在美国，每年约有150万新感染病例，它是当今食源性疾病导致的第二大死亡原因。弓形虫生命周期的两个方面使其如此流行，一是感染大量中间宿主的能力，二是通过感染最终宿主CAT产生数百万个环境抗性卵囊的能力。人们已经做了很多工作来了解中间阶段，即速殖子和缓殖子的生物学，但到目前为止，还没有开发出除缓殖子、裂殖子和有性阶段之外的阶段的培养方法，这阻碍了对寄生虫生活史很大一部分的研究。该项目描述了一种战略 这将开始揭开裂殖子阶段的分子方面，并计划利用这些信息设计一种向前选择策略，使我们能够在组织培养中观察裂殖子的分化。我们采集了裂殖子寄生虫，并将其与Affymetrix弓形虫基因芯片杂交。这种弓形虫裂殖子微阵列数据代表了这一阶段的第一个全球基因表达研究，也是该寄生虫任何上皮内肠道阶段的第一个，并提供了裂殖子特定信息的全球概况，这将是解开寄生虫如何感知和响应猫科肠道环境的关键。只在裂殖子阶段表达药物选择标记的转基因寄生虫将被用于正向选择策略，以筛选有利于裂殖子生长的组织培养条件。除了标记转基因寄生虫以跟踪裂殖子发育外，我们还将开发针对几个候选裂殖子基因的裂殖子特异性抗体。从最终宿主创建肠道细胞培养将为进一步了解构成寄生虫有性阶段的最终宿主-寄生虫关系提供所需的宿主成分。随着对肠道细胞生物学认识的进步，肠隐窝干细胞的体外培养方法应运而生。这些方法既适用于老鼠细胞，也适用于人类细胞，并有望应用于猫科动物肠道干细胞。在正确的条件下， 由裂殖子启动子驱动的含有药物选择的转基因寄生虫/记者应提供它们已转换到裂殖子阶段的迹象。通过量化在许多不同条件下分化为裂殖子的寄生虫的数量，我们将能够确定哪些条件最有利于诱导寄生虫进入这种发育转换。在组织培养中观察到这一点将是第一次，并将为研究弓形虫生活史中相对未知的部分提供基础。在这个项目的工作中，我希望继续研究寄生虫的基因表达和控制，以及宿主寄生虫的相互作用。这项研究将增加我们对这种寄生虫如何能够如此普遍地传播的理解，并可能适用于寄生虫与其最终宿主之间的其他宿主-寄生虫关系。

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.