New insights into the role of cholesterol-related pathways in apicomplexan parasite infections

Abstract

Coccidia are a large family of obligate intracellular parasites that belong to the apicomplexan phylum and are responsible for diseases in human and animal populations. In this context, veterinary-relevant coccidian species are grouped into certain families, such as Sarcocistidae (i. e. Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti) and Eimeriidae (i. e. Eimeria bovis and Eimeria arloingi) which largely differ in terms of life cycle and host specificity. It is well-known that coccidian parasites highly rely on host cell metabolism and their capacity to hijack their host cell to fulfill their metabolic requirements during intracellular development. In that context, complex molecules like cholesterol are scavenged from host cells to sustain proliferative requirements. Physiologically, cells obtain cholesterol either by de novo biosynthesis or by extracellular uptake. The latter is largely driven by LDL internalization in mammalian cells, which is accepted as pivotal route to be exploited by apicomplexan parasites. Nevertheless, since coccidian biology largely differs between species, the overall knowledge on cholesterol acquisition by coccidia has been oversimplified. As such, alternative cholesterol-related acquisition routes driven by NPC1L1, P-pg and SR-BI have scarcely been considered, so far. Related to these topics, the following results were achieved in the current work: GC-MS-based profiling of cholesterol-related sterols in B. besnoiti-infected primary endothelial cells showed an enhancement of several cholesterol precursors. Together with the finding of statin-mediated inhibition of B. besnoiti replication, these data strongly suggest a key role of cholesterol de novo biosynthesis in infected host cells. Furthermore, beneficial effects of LDL-supplementation on parasite proliferation proved acLDL as pivotal extracellular cholesterol source for B. besnoiti replication, thereby indicating that this coccidian parasite exploits alternative routes to sustain its cholesterol requirements. Moreover, in the case of E. bovis first merogony, lipidomic profiling revealed an enhancement of phytosterols over time indicating that this coccidian parasite significantly relies on extracellular sources for cholesterol acquisition. Interestingly, analysis of downstream cholesterol metabolites additionally documented an accumulation of cholesteryl esters and oxysterols (especially 25 hydroxycholesterol) throughout first merogony. Considering that exogenous oxysterols treatments impeded E. bovis macromeront development, these molecules may also result from anti-parasitic responses of infected-host cells. Regarding the participation of alternative cholesterol-related routes, the use of ezetimibe as NPC1L1 blocker revealed strong parasitostatic effects on T. gondii, N. caninum and B. besnoiti tachzoite proliferation. However, the absence of anti-coccidian effects driven by the in vivo active metabolite (glucoronated ezetimibe), in addition to the inconsistent gene expression of NPC1L1 in infected cells suggest that ezetimibe might affect parasite replication by an NPC1L1-independent mechanism. Additionally, chemical blockage of the ABC transporter P-gp by different generations of inhibitors (verapamil, valspodar, tariquidar) revealed differences in terms of anti-coccidian efficacies. Specifically, treatments with verapamil consistently reduced T. gondii, N. caninum and B. besnoiti replication and generated neutral lipid accumulation in host cells. Likewise, valspodar treatments induced singnificant anti-invasive and anti-proliferative effects in these three parasite species but failed to influence cellular neutral lipid abundance. In contrast, the most specific P-gp inhibitor tariquidar exclusively diminished B. besnoiti invasion and replication but failed to affect T. gondii or N. caninum, thereby suggesting parasite-specific reactions. Interestingly, blockage of the scavenger receptor SR-BI by BLT-1 treatments induced significant antireplicative effects not only in the fast-replicating coccidia T. gondii, N. caninum and B. besnoiti, but also in the pathogenic Eimeria species E. bovis and E. arloingi, thereby evidencing potentially conserved SR-BI-related mechanisms as key events for coccidian replication. However, BLT-1 treatments of free sporozoites and tachyzoites exclusively affected host cell invasion capacity in the latter group, being paralleled with a sustained Ca++ flux over time. Finally, studies on N. caninum host cell egress by live cell imaging revealed an infectiondriven Ca++ redistribution leading to increased Ca++ signals within intracellular N. caninum meronts. Furthermore, analyses on ionophore-induced N. caninum egress proved egress performance to be mainly influenced by meront maturity and showed no differences in Ca++ redistribution between time points of infection (24 or 42 h p. i.).