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

弓形虫裂殖子的分子表征以开发培养系统

基本信息

批准号：
9198852
负责人：
Michael Sean Behnke
金额：
$ 10.8万
依托单位：
LOUISIANA STATE UNIV A&M COL BATON ROUGE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-01 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9198852
关键词：
Animal Model Antibodies Antibody Formation Automobile Driving Biological Assay Biology Cause of Death Cell Culture System Cell Culture Techniques Cell Line Cells Cellular biology Cyst Data Development Enteral Environment Epithelial Cells Ethics Family Felidae Feces Felis catus Fibroblasts Foundations Gene Chips Gene Expression Genes Growth Harvest High-Throughput Nucleotide Sequencing Host-Parasite Relations Human Hybrids In Vitro Infection Intestines Knowledge Life Cycle Stages Medical Messenger RNA Methods Molecular Morphology Mus Oocysts Oxygen Parasites Pharmaceutical Preparations Plasmids Platelet Factor 4 Population Proteins RNA-Binding Proteins Reagent Recombinants Reporter Reporter Genes Research Resistance Serum Stem cells Supplementation System Taurine Testing Time Tissues Toxoplasma Toxoplasma gondii Transcription Factor AP-2 Alpha Transgenic Organisms Trypanosoma brucei brucei United States Work base burden of illness design feeding foodborne illness human disease in vitro testing induced pluripotent stem cell intestinal crypt intraepithelial promoter tissue culture tool

项目摘要

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.

描述（由申请方提供）：细胞内寄生虫是全世界人类疾病负担的重要组成部分。顶复门寄生虫弓形虫是其中一种较为成功的寄生虫，据估计，多达三分之一的人口已被感染。在美国，每年约有150万新感染者，它是今天食源性疾病死亡的第二大原因。弓形虫生命周期的两个方面使其如此流行，即感染大量中间宿主的能力和通过猫（最终宿主）的单次感染产生数百万个环境抗性卵囊的能力。已经进行了大量的工作来了解中间阶段，速殖子和缓殖子的生物学，但是到目前为止，还没有开发出用于缓殖子以外的阶段，裂殖子和有性阶段的培养方法，这阻碍了研究寄生虫生命周期的大部分的能力。该项目描述了一种战略这将开始解开裂殖子阶段的分子方面，并计划使用这些信息来设计正向选择策略，使我们能够观察裂殖子在组织培养中的分化。我们收获了裂殖子寄生虫，并将mRNA与Affyssin弓形虫基因芯片杂交。弓形虫裂殖子的微阵列数据代表了该阶段的第一个全球基因表达研究，以及寄生虫的任何上皮内肠道阶段的第一个，并提供了裂殖子特异性信息的全球概况，这对于解开寄生虫如何感知和响应猫科动物肠道环境至关重要。仅在裂殖子阶段表达药物选择标记的转基因寄生虫将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用于后续裂殖子发育外，我们还将针对几个候选裂殖子基因开发裂殖子特异性抗体。从确定的主机创建肠细胞培养物将提供所需的主机组件在进一步了解确定的主机-寄生虫的关系，包括寄生虫的性阶段。对肠细胞生物学的理解的进展导致了肠隐窝干细胞体外培养方法的建立。这些方法适用于小鼠和人类细胞，并有望应用于猫肠道干细胞。在适当的条件下，含有由裂殖子启动子驱动的药物选择/报道基因的转基因寄生虫应该提供它们已经转化到裂殖子阶段的指示。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，我们将能够确定最有利于诱导寄生虫进入这种发育转换的那些条件。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命周期中相对未知的部分提供基础。在这个项目的工作中，我希望继续研究我专注于寄生虫基因表达和控制，以及宿主寄生虫相互作用。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。