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

• 批准号: 8616949
• 负责人: Michael Sean Behnke
• 金额: $ 16.2万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 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/8616949
• 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; In Vitro; Infection; Intestines; Knowledge; Learning; Life Cycle Stages; Messenger RNA; Methods; Molecular; Mus; Oocysts; Oxygen; Parasites; Pharmaceutical Preparations; Plasmids; Platelet Factor 4; Population; Proteins; RNA-Binding Proteins; Reagent; Recombinants; Reporter; Reporter Genes; Research; Resistance; Serum; Staging; 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; hatching; human disease; in vitro testing; induced pluripotent stem cell; intestinal crypt; intraepithelial; promoter; tissue culture; tool

Project Summary/Abstract 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 and the ability to produce millions of environmentally resistant oocysts through a single infection of a 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 GeneChip. 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.

项目摘要/摘要 在全世界，细胞内寄生虫占人类疾病负担的很大一部分。 弓形虫寄生虫是比较成功的寄生虫之一，估计有三分之一。 的人口已经被感染。美国每年约有150万新感染者 它是今天由食源性疾病导致的第二大死亡原因。弓形虫有两个方面 使其如此流行的生命周期，感染大量中间宿主的能力和能力 通过感染一只猫(最终宿主)产生数百万个对环境有抵抗力的卵囊。 已经开展了许多工作来了解中间阶段的生物学，速殖子和 缓殖子，但到目前为止，培养方法超出了缓殖子、裂殖子和有性阶段， 还没有被开发出来，阻碍了研究很大一部分寄生虫生命周期的能力。这个项目 描述了一种将开始解开裂殖子阶段的分子方面的策略，并计划使用 这些信息来设计一种正向选择策略，使我们能够观察到裂殖子在 组织培养。我们收集了裂殖子寄生虫，并将其与弓形虫杂交。 基因芯片。这一弓形虫裂殖子微阵列数据代表了首个全球基因表达研究 对于这一阶段，以及第一次为任何上皮内肠道阶段的寄生虫，并提供了全球 裂殖子特异性信息的概况，这些信息对解锁寄生虫的感知和反应方式至关重要 到猫科动物的肠道环境。仅在裂殖子上表达药物选择标记的转基因寄生虫 将在正向选择策略中使用阶段来筛选有利于 裂殖子生长。除了标记转基因寄生虫以跟踪裂殖子发育外，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。创造一种肠道 最终宿主的细胞培养将提供所需的宿主成分，以进一步了解 确定的宿主-寄生虫关系，包括寄生虫的有性阶段。国内外最新研究进展 对肠道细胞生物学的了解导致了肠隐窝体外培养方法的建立。 干细胞。这些方法既适用于老鼠细胞，也适用于人类细胞，并承诺它们可以 应用于猫科动物肠道干细胞。在正确的条件下，含有药物的转基因寄生虫 由裂殖子启动子驱动的选择/记者应提供他们已转化为 裂殖子期。通过量化在许多地区分化为裂殖子的寄生虫的数量 不同的条件，我们将能够确定哪些条件最有利于诱导寄生虫进入 这种发育的转变。在组织培养中观察到这一点将是第一次，并将为 对弓形虫生命周期中相对未知的部分的研究。在这个项目中工作 我希望继续我专注于寄生虫基因表达和控制的研究，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传播的了解 可能适用于其他寄主-寄生虫之间的关系及其最终的 主持人。