Ezetimibe blocks Toxoplasma gondii-, Neospora caninum- and Besnoitia besnoiti-tachyzoite infectivity and replication in primary bovine endothelial host cells.

Liliana M. R. Silva,Anja Taubert,Camilo Larrazabal,Carlos Hermosilla

doi:10.1017/s0031182021000822

Abstract

Coccidia are obligate apicomplexan parasites that affect humans and animals. In fast replicating species, in vitro merogony takes only 24–48 h. In this context, successful parasite proliferation requires nutrients and other building blocks. Coccidian parasites are auxotrophic for cholesterol, so they need to obtain this molecule from host cells. In humans, ezetimibe has been applied successfully as hypolipidaemic compound, since it reduces intestinal cholesterol absorption via blockage of Niemann−Pick C-1 like-1 protein (NPC1L1), a transmembrane protein expressed in enterocytes. To date, few data are available on its potential anti-parasitic effects in primary host cells infected with apicomplexan parasites of human and veterinary importance, such as Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. Current inhibition experiments show that ezetimibe effectively blocks T. gondii, B. besnoiti and N. caninum tachyzoite infectivity and replication in primary bovine endothelial host cells. Thus, 20 μm ezetimibe blocked parasite proliferation by 73.1−99.2%, via marked reduction of the number of tachyzoites per meront, confirmed by 3D-holotomographic analyses. The effects were parasitostatic since withdrawal of the compound led to parasite recovery with resumed proliferation. Ezetimibe-glucuronide, the in vivo most effective metabolite, failed to affect parasite proliferation in vitro, thereby suggesting that ezetimibe effects might be NPC1L1-independent.

Highlights

Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti are cyst-forming species belonging to the Apicomplexa phylum, which consists of a large group of obligatory intracellular protozoan parasites that affect both humans and animals
Ezetimibe treatments markedly reduced the number of tachyzoites per meront in all three parasite species, the strongest effect was observed for B. besnoiti, with a reduction of 68.2% on the mean number of tachyzoites per meront, followed by T. gondii and N. caninum showing more than 50% reduction (56.5% and 50.2%, respectively)
Niemann−Pick C-1 like-1 protein (NPC1L1) is a trans-membrane protein highly expressed in enterocytes that mediates sterol internalization via clathrin-coated vesicles and it has widely been accepted as main target of the lipid-lowering drug ezetimibe (Altmann et al, 2004; Garcia-Calvo et al, 2005; Betters and Yu, 2010)

Summary

Introduction

Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti are cyst-forming species belonging to the Apicomplexa phylum, which consists of a large group of obligatory intracellular protozoan parasites that affect both humans and animals. Despite morphological similarities between coccidian species, host specificity and clinical consequences greatly differ among them In this context, T. gondii is considered a major public health problem and an abortive agent especially in ovines (Benavides et al, 2017) and humans (Nayeri et al, 2020). During the acute stage of infection, coccidian parasites undergo asexual replication within host cells In this context, host endothelial cells have shown high permissiveness for tachyzoite infection and proliferation in vivo (Alvarez-Garcia et al, 2013; Konradt et al, 2016). Primary bovine endothelial cells have consistently been reported as suitable for in vitro replication of T. gondii, N. caninum and B. besnoiti (Taubert et al, 2006, 2016; Silva et al, 2019; Velásquez et al, 2019), allowing high tachyzoite proliferation rates in an experimental set up close to the in vivo scenario. LDL-mediated cholesterol incorporation was described as pivotal, but not exclusive mechanism to fulfil cholesterol requirement during fast replicating coccidia proliferation (Nolan et al, 2015; Silva et al, 2019)

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