Sexual Development of Toxoplasma in Feline Intestinal Organoids

猫肠道类器官中弓形虫的性发育

• 批准号: 10318931
• 负责人: LAURA J KNOLL
• 金额: $ 38.21万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-15 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318931
• 关键词: Affect; Apical; Arachidonic Acids; Biomedical Engineering; Brain; Cell Culture Techniques; Cell Line; Cells; Chromosome Segregation; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Data; Development; Devices; Diet; Family Felidae; Feces; Felis catus; Generations; Genetic; Genetic Crosses; Genome; Goals; Individual; Infection; Intestines; Knock-out; Lead; Life Cycle Stages; Linoleic Acids; Lipids; Lipoxygenase; Maps; Mass Spectrum Analysis; Methods; Michigan; Microinjections; Modeling; Molecular; Molecular Analysis; Mus; Nutritional; Oleic Acids; Oocysts; Organoids; Oxygen; Parasites; Polyunsaturated Fatty Acids; Process; Production; Quantitative Trait Loci; Rivers; Saturated Fatty Acids; Sexual Development; Sexual Reproduction; Side; Signal Pathway; Signal Transduction; Signaling Molecule; Small Intestines; Species Specificity; Supplementation; Techniques; Test Result; Testing; Toxoplasma; Toxoplasma gondii; Transcript; Tube; Universities; Up-Regulation; Vaccines; Virulence; asexual; comparative; feeding; fungus; linoleoyl-CoA desaturase; mouse model; nutrition; octadecadienoic acid; overexpression; pi bond; promoter; quorum sensing; three dimensional cell culture; three-dimensional modeling; tissue culture; tool; transcriptome sequencing; transcriptomics

The sexual cycle of Toxoplasma gondii is limited to the feline intestine where millions of oocysts are formed and subsequently excreted in their feces. The goal of our previous R21 was to recapitulate the T. gondii sexual cycle in tissue culture. We achieved this goal by generating cat intestinal organoids in collaboration with Jason Spence at the University of Michigan and supplementing the T. gondii infected organoids with saturated fatty acids, leading us to hypothesize that saturated fatty acids are used as a signaling molecules and not for nutrition. Polyunsaturated fatty acids are often processed to signaling molecules by oxygenation of their double bonds through the action of lipoxygenases. The T. gondii genome encodes four lipoxygenases, one of which, annotation TGME49_315970 called TgLOX1, is 600-fold upregulated in cat intestine compared to asexual stages. We hypothesize that TgLOX1 oxygenates linoleic acid to signal sexual development. One of the goals of this R01 is to test this hypothesis. Another goal of this R01 is to test the hypothesis that 3D culture and low oxygen conditions will maximize oocyst production. While we can generate sporulated oocysts that are infectious to mice, the overall yield is low because most of the oocysts in the culture do not sporulate. Currently, we plate our feline intestinal cells on snapwell inserts so that the apical side of the intestine is easy to infect, but the cells are in 2D, which may affect T. gondii development. To model 3D intestines, we will test two methods in this R01, 1) microinjection of the whole intestinal organoid, 2) a simple biomedical engineering device that creates intestinal tubes. These new 3D models as well as lowering the oxygen concentration will more closely model the cat small intestine and likely produce more mature oocysts that sporulate with greater efficiency. The final goal for this R01 is to determine the species specificity for T. gondii sexual development. The sexual cycle of T. gondii is restricted to the cat, but it is unclear why. Overall, creation of cell culture sexual development conditions will allow classical genetic crosses to become a routine technique for the T. gondii field. It will also allow for a molecular analysis of the complete life cycle of a protozoan parasite, as T. gondii asexual development can already be performed in tissue culture.

弓形虫的性周期仅限于猫的肠道，在那里形成数百万个卵囊，随后通过粪便排出。我们以前的R21的目标是概括T。组织培养中的弓形虫性周期。我们通过与密歇根大学的Jason Spence合作产生猫肠道类器官并补充T。弓形虫用饱和脂肪酸感染类器官，使我们假设饱和脂肪酸被用作信号分子，而不是营养。多不饱和脂肪酸通常通过脂氧合酶的作用使其双键氧化而加工成信号分子。霸王弓形虫基因组编码四种脂氧合酶，其中之一，注释TGME49_315970称为TgLOX 1，与无性阶段相比，在猫肠中上调600倍。我们假设TgLOX 1氧化亚油酸信号性发育。本R01的目标之一就是检验这一假设。R01的另一个目标是测试3D培养和低氧条件将最大化卵囊产量的假设。虽然我们可以产生对小鼠具有感染性的孢子化卵囊，但总产量较低，因为培养物中的大多数卵囊不形成孢子。目前，我们将猫的肠道细胞接种在snapwell插入物上，这样肠道的顶侧容易感染，但细胞是二维的，这可能会影响T。弓形虫发育为了建立3D肠道模型，我们将在R01中测试两种方法，1）整个肠道类器官的显微注射，2）一种简单的生物医学工程设备，可以创建肠道。这些新的3D模型以及降低氧气浓度将更接近猫小肠的模型，并可能产生更成熟的卵囊，以更高的效率形成孢子。本R01的最终目标是确定T的种属特异性。弓形虫性发育T.弓形虫仅限于猫，但原因尚不清楚。总的来说，细胞培养性发育条件的建立将使经典的遗传杂交成为T。弓形虫病场它还将允许对原生动物寄生虫的完整生命周期进行分子分析，如T。弓形虫无性发育已经可以在组织培养中进行。