Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii.

As an obligate intracellular parasite, Toxoplasma gondii must import essential nutrients from the host cell into the parasitophorous vacuole. We previously reported that the parasite scavenges cholesterol from host endocytic organelles for incorporation into membranes and storage as cholesteryl esters in lipid droplets. In this study, we have investigated whether Toxoplasma utilizes cholesterol as a precursor for the synthesis of metabolites, such as steroids. In mammalian cells, steroidogenesis occurs in mitochondria and involves membrane-bound type I cytochrome P450 oxidases that are activated through interaction with heme-binding proteins containing a cytochrome b5 domain, such as members of the membrane-associated progesterone receptor (MAPR) family. Our LC-MS targeted lipidomics detect selective classes of hormone steroids in Toxoplasma, with a predominance for anti-inflammatory hydroxypregnenolone species, deoxycorticosterone and dehydroepiandrosterone. The genome of Toxoplasma contains homologs encoding a single type I CYP450 enzyme (we named TgCYP450mt) and a single MAPR (we named TgMAPR). We showed that TgMAPR is a hemoprotein with conserved residues in a heme-binding cytochrome b5 domain. Both TgCYP450 and TgMAPR localize to the mitochondrion and show interactions in in situ proximity ligation assays. Genetic ablation of cyp450mt is not tolerated by Toxoplasma; we therefore engineered a conditional knockout strain and showed that iΔTgCYP450mt parasites exhibit growth impairment in cultured cells. Parasite strains deficient for mapr could be generated; however, ΔTgMAPR parasites suffer from poor global fitness, loss of plasma membrane integrity, aberrant mitochondrial cristae, and an abnormally long S-phase in their cell cycle. Compared to wild-type parasites, iΔTgCYP450mt and ΔTgMAPR lost virulence in mice and metabolomics studies reveal that both mutants have reduced levels of steroids. These observations point to a steroidogenic pathway operational in the mitochondrion of a protozoan that involves an evolutionary conserved TgCYP450mt enzyme and its binding partner TgMAPR. Author summary: In addition to controlling membrane fluidity, cholesterol serves as a substrate for the synthesis of important biomolecules, in particular steroid hormones. Steroidogenesis involves mitochondrial cholesterol-metabolizing cytochrome P450s activated by membrane-associated progesterone receptor (MAPR) hemoproteins. The intravacuolar parasite Toxoplasma scavenges cholesterol from the host and our targeted lipidomics reveal the presence of pregnenolone and selected steroid hormones in the parasite. We investigated a potential steroidogenic pathway in Toxoplasma by characterizing the single CYP450 (TgCYP450mt) and the MAPR homolog (TgMAPR) in Toxoplama. Both TgCYP450mt and TgMAPR are expressed in the mitochondrion and interact with each other. Parasites lacking mapr are viable in vitro unlike ΔTgCYP450mt parasites. Conditional TgCYP450mt knock-out parasites and ΔTgMAPR suffer from developmental defects, are poorly virulent and have reduced levels of steroids compared to WT parasites. These data suggest that Toxoplasma has the ability to synthesize some steroids in the mitochondrion, using a cholesterol-metabolizing cytochrome P450 and reveal that steroidogenesis is an ancient pathway, conserved in eukaryotic lineages. [ABSTRACT FROM AUTHOR]