Toxoplasma ceramide synthases: a curious case of gene duplication, divergence and key functionality

Abstract

AbstractToxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause foetal damage and abortion in both animals and humans. Sphingolipids have indispensable functions as signaling molecules and are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Ceramide is the precursor for all sphingolipids, and here we report the identification, localisation and analyses of the Toxoplasma ceramide synthases TgCerS1 and TgCerS2 and, using a conditional gene regulation approach, establish their roles in pathogenicity and parasite fitness. Interestingly, we observed that whilst TgCerS1 was a fully functional orthologue of the yeast Lag1p capable of catalysing the conversion of sphinganine to ceramide, in contrast TgCerS2 was catalytically inactive. Furthermore, genomic deletion of TgCerS1 using CRISPR/Cas-9 led to viable but slow growing parasites indicating its importance but not indispensability. In contrast, genomic knock out of TgCerS2 was only accessible utilising the rapamycin-inducible Cre recombinase system. Surprisingly, the results demonstrated that this ‘pseudo’ ceramide synthase, TgCerS2, has an even greater role in parasite fitness than its catalytically active orthologue (TgCerS1). Phylogenetic analyses indicated that, as in humans and plants, the ceramide synthase isoforms found in Toxoplasma and other Apicomplexa arose through gene duplication. However, in the Apicomplexa the duplicated copy subsequently evolved into a non-functional ‘pseudo’ ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the unusual biology that characterize these protozoan parasites, a feature that could potentially be exploited in the development of new antiprotozoals.Author SummarySphingolipids, essential and ubiquitous lipids in the Eukaryota, are both synthesized and scavenged by the parasitic apicomplexan protozoa, including Toxoplasma gondii. Ceramide is the precursor for all sphingolipids and here we report the identification, localisation and analyses of the Toxoplasma ceramide synthases TgCerS1 and TgCerS2. Surprisingly, whilst TgCerS1 was fully functional, catalysing the conversion of sphinganine to ceramide, TgCerS2 was catalytically inactive. However, we demonstrated that this ‘pseudo’ ceramide synthase has an even greater role in parasite fitness than the catalytically active TgCerS1. Phylogenetic analyses indicated that these isoforms arose through gene duplication and the duplicated copy subsequently evolved into the ‘pseudo’ ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the highly unusual biology that characterizes these protozoan parasites, a feature that could potentially be exploited for the development of new antiprotozoals.