Sexual Development of Toxoplasma in Feline Intestinal Organoids

刚地弓形虫的性循环仅限于猫的肠道，在那里形成数百万个卵囊，随后随粪便排出体外。我们之前的R21的目标是在组织培养中重现弓形虫的性循环。我们通过与密歇根大学的Jason Spence合作，生成猫肠道类器官，并向感染弓形虫的类器官补充饱和脂肪酸，从而实现了这一目标，这使我们假设饱和脂肪酸被用作信号分子，而不是用于营养。多不饱和脂肪酸通常通过脂加氧酶的作用，通过双键的氧化作用加工成信号分子。弓形虫基因组编码四种脂氧合酶，其中一种名为TGME49_315970的脂氧合酶被称为TgLOX1，与无性繁殖阶段相比，它在猫肠道中的表达上调了600倍。我们假设TgLOX1氧合亚油酸是性发育的信号。这个R01的目标之一就是检验这个假设。本R01的另一个目标是测试3D培养和低氧条件将最大限度地提高卵囊产量的假设。虽然我们可以产生对小鼠具有传染性的有孢子的卵囊，但总体产量很低，因为培养中的大多数卵囊不产孢子。目前，我们将猫肠细胞移植在snapwell插入物上，使肠顶端容易感染，但细胞是二维的，可能会影响弓形虫的发育。为了建立三维肠道模型，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)一个简单的生物医学工程设备来创建肠道。这些新的3D模型以及降低氧浓度将更接近猫小肠的模型，并有可能产生更成熟的卵囊，以更高的效率产孢。该R01的最终目标是确定弓形虫性发育的物种特异性。弓形虫的性循环仅限于猫，但原因尚不清楚。总的来说，创造细胞培养性发育条件将使经典遗传杂交成为弓形虫领域的常规技术。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，因为弓形虫的无性发育已经可以在组织培养中进行。

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.