Differential inhibition of host cell cholesterol de novo biosynthesis and processing abrogates Eimeria bovis intracellular development.

Abstract

Eimeria bovis macromeront formation in bovine endothelial host cells is an energy- and nutrient-demanding process. Obligate intracellular replicating coccidians are generally considered as auxotrophic for cholesterol synthesis and scavenge cholesterol from the host cell by either enhancing the uptake of extracellular cholesterol sources or by upregulating the host cellular de novo biosynthesis. We here focused on the latter mechanism and analyzed the effects of several inhibitors targeting the host cellular mevalonate biosynthesis pathway and cholesterol processing. The following inhibitors were used: lovastatin, squalestatin, CI976 and C75 targeting HMG-CoA reductase, squalene synthase, acyl-CoA:cholesterol acyltransferase, and fatty acid synthase, respectively. In summary, all inhibitors significantly interfered with E. bovis meront formation and merozoite production in a dose-dependent manner. Dose effect responses identified lovastatin as the most effective compound, followed by CI976, C75, and squalestatin, respectively. Overall, merozoite production was inhibited by 99.6, 99.7, 84.6, and 70.2% via lovastatin (1 μM), CI976, C75, and squalestatin (all 5 μM) treatments, respectively. Concerning macromeront formation, both the rate and size of developing meronts were affected by inhibitor treatments. The effects were characterized by developmental arrest and meront degradation. In the case of CI976 treatment, we additionally observed detrimental effects on host cellular lipid droplet formation leading to meront developmental arrest irrespective of the time point of treatment onset. These analyses clearly indicate that successful E. bovis intracellular development strictly depends on the host cellular de novo biosynthesis of cholesterol and on the adequate subsequent processing thereof.