Modulation of the host cell cholesterol metabolism by Eimeria bovis

牛艾美耳球虫是一种牛的尖端复合体内寄生虫，是引起奶牛严重出血性腹泻的重要原因。以往的数据表明，牛嗜血杆菌清除宿主细胞胆固醇以保证其在细胞内发育成大单体，其特征是受感染的宿主细胞内寄生虫的巨大放大和&GT的产生；与其他球虫(如弓形虫)相比，牛E.bovis感染同时上调胆固醇的合成和低密度脂蛋白的摄取，以保证寄生虫的成功发育，并不仅通过乙酰辅酶A-乙酰基转移酶(ACAT)调节胆固醇的酯化，而且还通过胆固醇-25-羟基酶(CH25H)调节胆固醇的加工，这表明氧固醇也可能参与了牛弓形虫的复制过程。本研究的目的是比较修饰的低密度脂蛋白(乙酰化和氧化的低密度脂蛋白)与经典的低密度脂蛋白和相应的宿主细胞受体(低密度脂蛋白受体)的影响OLE1)在内皮宿主细胞中成功复制牛E.bovis。此外，我们打算通过功能分析来分析CH25H和氧化甾醇在大单体形成中的作用。除了植物甾醇外，对细胞前体、代谢物和最终产物的生化分析将分别阐明细胞从头合成和细胞外摄取胆固醇在感染宿主细胞中的实际作用。此外，还将进行生化实验，分析细胞裂解物中胆固醇酯化的等级，以评估ACAT活性对寄生虫增殖的影响。使用荧光胆固醇类似物在细胞内差异运输，从而导致ACAT的差异可及性，将使我们能够分析感染细胞中胆固醇的储存，并获得关于游离和酯化胆固醇对寄生虫复制的可用性的信息。为了获得更多关于牛埃希氏菌引发的宿主细胞胆固醇代谢调控的新见解，我们最终打算对牛埃希氏菌感染的内皮细胞中胆固醇的运输进行初步实验。

Eimeria bovis is an intracellular apicomplexan parasite of cattle and represents an important cause of bovine coccidiosis leading to severe haemorrhagic diarrhea in calves. Out previous data indicate that E. bovis scavenges host cell cholesterol to guarantee its intracellular development into macromeronts that is characterized by an enormous enlargement of the parasite within infected host cells and the production of > 120.000 merozoites per meront I. In contrast to other coccidians, such as Toxoplasma gondii, E. bovis infections simultaneously up-regulates both, de novo synthesis of cholesterol and uptake of low densitiy lipoprotein (LDL) in infected cells to guarantee successful development of the parasite and modulates not only cholesterol esterification via acetyl-CoA-acetyltransferase (ACAT) but additionally enhances cholesterol procession by cholesterol-25-hydroxylase (CH25H) indicating that oxysterols may also be involved in the replication process of E. bovis.The aim of this study is to investigate the impact of modified LDL (acetylated and oxidized LDL) in comparison to classical LDL and of respective host cell receptors (LDLR, OLR1) on successful replication of E. bovis in endothelial host cells. Furthermore, we intend to analyze the role of CH25H and of oxysterols in macromeront formation via functional assays. Biochemical analyses on the cell contents of precursors, metabolites and end-products in addition to phytosterols will elucidate the actual role of cellular de novo synthesis and extracellular up-take of cholesterol in infected host cells, respectively. In addition, biochemical experiments will be conducted to analyze the grade of cholesterol esterification in cell lysates in order to estimate the impact of ACAT activity on parasite proliferation. The use of fluorescent cholesterol analogs that are differentially transported in cells thus leading to differential accessibility for ACAT will allow us to analyze cholesterol storage in infected cells and to get information on the usability of free and esterified cholesterol for parasite replication. To get more new insights into E. bovis-triggered modulation of host cell cholesterol metabolism, we finally intend to conduct first experiments on the cholesterol transport in E. bovis-infected endothelial cells.