Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Abstract

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

Coordinated action of multiple transporters in the acquisition of essential cationic amino acids by the intracellular parasite Toxoplasma gondii

“…Of the 17 amino acids detected by GC-MS, only the uptake of 13 C-Lys was significantly reduced when TgApiAT6-1 expression was knocked down. These findings were replicated in a recent study that adopted a similar approach in measuring the uptake of 13 Clabelled amino acids in parasites depleted in TgApiAT6-1 [34]. These data suggested that TgA-piAT6-1 may be a Lys transporter, although it could also mediate the uptake of other amino acids not detected under the transport conditions of the experiments, or not detected by GC-MS, such as Arg.…”

An Uninvited Seat at the Dinner Table: How Apicomplexan Parasites Scavenge Nutrients from the Host

“…This hypothesis was supported in a recent publication in which TgApiAT6-1 was identified as the transporter that mediates TgApiAT1-independent L-arginine uptake in T. gondii [75]. Specifically, two parallel studies on TgApiAT6-1 have demonstrated that this protein is a general cationic amino acid transporter that mediates both the high-affinity uptake of L-lysine and the low-affinity uptake of L-arginine [75,76]. Furthermore, TgApiAT6-1 is the sole L-lysine transporter in T. gondii, explaining the indispensability reported in the corresponding gene [76].…”

“…Specifically, two parallel studies on TgApiAT6-1 have demonstrated that this protein is a general cationic amino acid transporter that mediates both the high-affinity uptake of L-lysine and the low-affinity uptake of L-arginine [75,76]. Furthermore, TgApiAT6-1 is the sole L-lysine transporter in T. gondii, explaining the indispensability reported in the corresponding gene [76]. TgApiAT6-1 has a broad specificity for many cationic and large neutral amino acids and L-arginine metabolites.…”

Toxoplasma metabolic flexibility in different growth conditions

“…In the acute infection stage, amino acid scavenging is important for rapid tachyzoite growth evidenced by very recent discoveries on the mechanisms of amino acid scavenging [8,9,[66][67][68][69]. The first such transporter identified was an amino acid transporter TgNPT1 (later renamed as apicomplexan amino acid transporter TgApiAT1) which primarily transports arginine from the host (Figure 1) [8].…”