P-Glycoprotein Inhibitors Differently Affect Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti Proliferation in Bovine Primary Endothelial Cells

Abstract

Apicomplexan parasites are obligatory intracellular protozoa. In the case of Toxoplasma gondii, Neospora caninum or Besnoitia besnoiti, to ensure proper tachyzoite production, they need nutrients and cell building blocks. However, apicomplexans are auxotrophic for cholesterol, which is required for membrane biosynthesis. P-glycoprotein (P-gp) is a transmembrane transporter involved in xenobiotic efflux. However, the physiological role of P-gp in cholesterol metabolism is unclear. Here, we analyzed its impact on parasite proliferation in T. gondii-, N. caninum- and B. besnoiti-infected primary endothelial cells by applying different generations of P-gp inhibitors. Host cell treatment with verapamil and valspodar significantly diminished tachyzoite production in all three parasite species, whereas tariquidar treatment affected proliferation only in B. besnoiti. 3D-holotomographic analyses illustrated impaired meront development driven by valspodar treatment being accompanied by swollen parasitophorous vacuoles in the case of T. gondii. Tachyzoite and host cell pre-treatment with valspodar affected infection rates in all parasites. Flow cytometric analyses revealed verapamil treatment to induce neutral lipid accumulation. The absence of a pronounced anti-parasitic impact of tariquidar, which represents here the most selective P-gp inhibitor, suggests that the observed effects of verapamil and valspodar are associated with mechanisms independent of P-gp. Out of the three species tested here, this compound affected only B. besnoiti proliferation and its effect was much milder as compared to verapamil and valspodar.

Thiosemicarbazone Copper Chelator BLT-1 Blocks Apicomplexan Parasite Replication by Selective Inhibition of Scavenger Receptor B Type 1 (SR-BI)

“…These findings are in line with the function of SR-BI in endothelial cell cholesterol efflux [27], being a consequence of SR-BI blockage, thereby impairing its capacity for HDL-dependent cholesterol efflux and finally resulting in cellular neutral lipid accumulation [52]. Interestingly, we recently reported that verapamil treatments also reduced T. gondii-, N. caninumand B. besnoiti tachyzoite replication, which was accompanied by enhanced neutral lipid accumulation [15]. It seems, therefore, plausible to speculate that cholesterol efflux impairment may generally affect obligate intracellular coccidian development.…”

“…For LD visualization, BUVEC were seeded into 35 mm tissue culture µ-dishes (Ibidi ® ) and treated with the vehicle and BLT-1 for 48 h as described above. Then, the treated cells were loaded with BODIPY 493/503 (2 µg/mL, 1 h, 37 • C, 5% CO 2 ; Cayman Chemical, Ann Arbor, MI, USA) as described elsewhere [15]. Cellular LDs in the BLT-1-treated cells and non-treated controls (n = 50 per condition) were manually counted based on morphology and BODIPY 493/503-driven signal accumulation in endothelial cells [39] and expressed as the number of LDs per cell.…”

“…Afterwards, cells were washed twice in 1× PBS (600× g; 5 min) and samples were analyzed with a BD Accuri C6 ® FACS cell analyzer (Becton-Dickinson, Heidelberg, Germany). The cells were gated according to their size and granularity, while BODIPY 493/503-derived signals were assessed in the FL-1 channel as described elsewhere [15].…”

“…In this context, primary endothelial cells were proven to be appropriate for the development of several coccidian species. This in vitro system, which is closely related to the in vivo scenario, allows intracellular replication [12][13][14][15][16] and consequently delivers a methodological bridge for analysing these divergent families in the same host cell type, thereby avoiding host cell type-driven variations.…”

Eimeria bovis Macromeront Formation Induces Glycolytic Responses and Mitochondrial Changes in Primary Host Endothelial Cells

“…Remarkably, the host cells survive and support this development by an intense cytoskeletal re-organization (Hermosilla et al, 2008). Full macromeront formation takes up to 25 days and thereby considerably exceeds the merogonies of other closely related apicomplexans, such as T. gondii, Neospora caninum, or B. besnoiti (Conejeros et al, 2019;Velaśquez et al, 2019;Velaśquez et al, 2020a;Velaśquez et al, 2020b;Larrazabal et al, 2021). Besides cytoskeleton, E. bovis modulates several pathways and cellular functions, such as apoptosis, cholesterol biosynthesis, immune responses, or cell cycle progression to complete its intracellular development (Lang et al, 2009;Hamid et al, 2015;Velaśquez et al, 2020b).…”