Modulation of the host cell cholesterol metabolism by Eimeria bovis

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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.