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（由申请人提供）：细胞内寄生虫代表了全世界人类疾病负担的很大一部分。 Apicomplexan寄生虫弓形虫弓形虫是最成功的毒素，其中估计已被感染到三分之一的人口。每年在美国大约有150万种新感染，这是当今食源性疾病的第二大死亡原因。弓形虫生命周期有两个方面，使其如此普遍，能够感染大量中间宿主AD AD的能力，能够通过单一的猫（最终宿主）产生数百万个具有环境的卵囊。已经开展了许多工作来了解中间阶段的生物学，tachyzoite和bradyzoite，但到目前为止，尚未发展出培养阶段的培养方法，梅罗龙夫人和性阶段，尚未开发出来，阻碍了研究寄生虫生命周期的大部分地区的能力。该项目描述了策略这将开始揭示梅罗佐伊特阶段的分子方面，并计划使用此信息来设计一种前向选择策略，这将使我们能够观察到组织培养中的Merozoite分化。我们已经收集了梅罗洛伊特寄生虫，并将mRNA与Affymetrix弓形虫基因芯片杂交。弓形虫蛋白酶的微阵列数据代表了该阶段的首个全球基因表达研究，也代表了寄生虫的任何上皮内肠肠阶段的第一个研究，并提供了梅罗寄生虫特异性信息的全球概况，这对于释放寄生虫的感觉和响应对Felid gut gut环境至关重要。仅在梅罗唑群体阶段表达药物可选标记物的转基因寄生虫将用于远期选择策略，以筛选有利于梅罗唑群体生长的组织培养条件。除了具有标记的转基因寄生虫以遵循梅罗洛济岩的发育外，我们还将开发几种候选蛋白酶基因的梅罗寄生虫特异性抗体。从确定的宿主创建肠道细胞培养将提供所需的宿主成分，以进一步理解构成寄生虫性阶段的确定宿主 - 寄生虫关系。理解肠细胞生物学的进步导致创造了肠道隐窝干细胞的体外培养方法。这些方法适用于小鼠和人类细胞，并承诺可以将其应用于Felid肠干细胞。考虑到正确的条件，含有梅罗唑人物启动子驱动的药物选择/报告者的转基因寄生虫应提供迹象表明它们已转化为梅罗洛罗兹矿阶段。通过量化在许多不同条件下分化为梅罗寄生的寄生虫的数量，我们将能够确定最有利于诱导寄生虫进入此发育开关的条件。在组织培养中观察这将是第一个，并将为研究弓形虫生命周期的相对未知部分的研究提供基础。在从事该项目的过程中，我希望继续研究我专注于寄生虫基因表达和控制以及寄生寄生虫相互作用。这项研究将提高我们对这种寄生虫如何如此普遍传播的理解，并可能适用于寄生虫及其确定宿主之间的其他宿主寄生虫关系。