Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii.

Joachim Kloehn,Emmanuel Varesio,David Dubois,Matteo Lunghi,Dominique Soldati-Favre

doi:10.3390/metabo11080476

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.

Highlights

The apicomplexans form a large phylum comprising thousands of obligate intracellular parasites, some of which infect humans and cause devastating diseases, including malaria, cryptosporidiosis and toxoplasmosis
major facilitator superfamily (MFS) transporters are found within the plasma membrane as well as within the membranes of parasite organelles, including the essential transporter facilitator protein 1 (TFP1), which localizes to the micronemes and is required for the maturation of this secretory organelle critical for motility, invasion and egress from host cells [16]
The requirements for amino acids and uptake of other metabolites likely differ between tachyzoites and the encysted bradyzoites, but these differences are poorly defined to date [35]

Summary

Introduction

The apicomplexans form a large phylum comprising thousands of obligate intracellular parasites, some of which infect humans and cause devastating diseases, including malaria, cryptosporidiosis and toxoplasmosis. The few characterized transporters for the import of vital metabolites include the parasite’s glucose transporters [9], a transporter for adenosine [10], the essential amino acids tyrosine and arginine [11,12], as well as folate [13]. MFS transporters are found within the plasma membrane as well as within the membranes of parasite organelles, including the essential transporter facilitator protein 1 (TFP1), which localizes to the micronemes and is required for the maturation of this secretory organelle critical for motility, invasion and egress from host cells [16]. We identified an essential plasma membrane transporter and employed untargeted metabolomics to uncover the Apicomplexan Amino Acid Transporter 6-1 (TgApiAT6-1, in the following referred to as AT6-1) [11] as the essential lysine transporter in T. gondii tachyzoites

Methods

Results

Conclusion