Novel Insights Into Sterol Uptake and Intracellular Cholesterol Trafficking During Eimeria bovis Macromeront Formation.

Liliana M R Silva,Anja Taubert,Zahady D Velásquez,Carlos Hermosilla,Sara López-Osorio

doi:10.3389/fcimb.2022.809606

Abstract

Apicomplexan parasites are considered as defective in cholesterol synthesis. Consequently, they need to scavenge cholesterol from the host cell by either enhancing the uptake of extracellular cholesterol sources or by upregulating host cellular de-novo biosynthesis. Given that Eimeria bovis macromeront formation in bovine lymphatic endothelial host cells in vivo is a highly cholesterol-demanding process, we here examined host parasite interactions based on host cellular uptake of different low-density lipoprotein (LDL) types, i.e., of non-modified (LDL), oxidized (oxLDL), and acetylated LDL (acLDL). Furthermore, the expression of lipoprotein-oxidized receptor 1 (LOX-1), which mediates acLDL and oxLDL internalization, was monitored throughout first merogony, in vitro and ex vivo. Moreover, the effects of inhibitors blocking exogenous sterol uptake or intracellular transport were studied during E. bovis macromeront formation in vitro. Hence, E. bovis-infected primary bovine umbilical vein endothelial cells (BUVEC) were treated with inhibitors of sterol uptake (ezetimibe, poly-C, poly-I, sucrose) and of intracellular sterol transport and release from endosomes (progesterone, U18666A). As a read-out system, the size and number of macromeronts as well as merozoite I production were estimated. Overall, the internalization of all LDL modifications (LDL, oxLDL, acLDL) was observed in E. bovis-infected BUVEC but to different extents. Supplementation with oxLDL and acLDL at lower concentrations (5 and 10 µg/ml, respectively) resulted in a slight increase of both macromeront numbers and size; however, at higher concentrations (25–50 µg/ml), merozoite I production was diminished. LOX-1 expression was enhanced in E. bovis-infected BUVEC, especially toward the end of merogony. As an interesting finding, ezetimibe treatments led to a highly significant blockage of macromeront development and merozoite I production confirming the relevance of sterol uptake for intracellular parasite development. Less prominent effects were induced by non-specific inhibition of LDL internalization via sucrose, poly-I, and poly-C. In addition, blockage of cholesterol transport via progesterone and U18666A treatments resulted in significant inhibition of parasite development. Overall, current data underline the relevance of exogenous sterol uptake and intracellular cholesterol transport for adequate E. bovis macromeront development, unfolding new perspectives for novel drug targets against E. bovis.

Highlights

Apicomplexans are obligate intracellular protozoan parasites, which cause important diseases in humans and animals
An upregulation of molecules associated with both de-novo biosynthesis pathway and low-density lipoprotein (LDL)-mediated cholesterol uptake was reported for E. bovis-infected host cells at the transcriptional level (Taubert et al, 2010; Hamid et al, 2014; Hamid et al, 2015)
Coccidian parasites are considered defective for de-novo cholesterol biosynthesis and, as such, must scavenge cholesterol from their infected host cells by either enhancing uptake of cholesterol from extracellular sources or by upregulating host cell de-novo biosynthesis (Bansal et al, 2005; Labaied et al, 2011; Coppens, 2013; Ehrenman et al, 2013; Hamid et al, 2014)

Summary

Introduction

Apicomplexans are obligate intracellular protozoan parasites, which cause important diseases in humans and animals. Successful replication of these parasites entirely relies on scavenging cholesterol from their host cells, either by enhancing host cellular endogenous de-novo synthesis or by upregulating low-density lipoprotein (LDL)-mediated cholesterol uptake from extracellular sources. Plasmodium spp. hepatic stages accomplish cholesterol acquisition by both pathways, but they seem not essential for parasite replication (Labaied et al, 2011). In this context, few studies are currently available on slow developing coccidia, but these indicate distinct cholesterol acquisition pathways. An upregulation of molecules associated with both de-novo biosynthesis pathway and LDL-mediated cholesterol uptake was reported for E. bovis-infected host cells at the transcriptional level (Taubert et al, 2010; Hamid et al, 2014; Hamid et al, 2015). An increased LDL receptor surface abundance was shown for E. bovis-infected endothelial host cells and excess LDL (10 μg/ml) supply boosted parasite proliferation (Hamid et al, 2015)

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