Characterization of an essential arginine transporter in the apicomplexan parasite toxoplasma gondii

Abstract

Parasites by their very nature must efficiently scavenge nutrients from the host that they infect. Transporters are proteins that facilitate the uptake of nutrients across biological membranes. Apicomplexans are a phylum of intracellular parasites that include Plasmodium spp, the causative agents of malaria, and Toxoplasma gondii, the causative agent of toxoplasmosis. Despite the critical role that transporters must play in the biology of these parasites, only a small number have been characterized thus far. A genome-wide search for transporter proteins in 2005 by Martin and colleagues revealed a large number of putative transporters in Plasmodium falciparum. One group termed the 'novel putative transporters' (NPTs) is a family of proteins unique to apicomplexans. NPTs have structural characteristics of membrane transport proteins but no predicted function. The T. gondii genome encodes 16 NPT proteins, one of which (TgNPT1) is the focus of this study. Genetic 'knockdown' of TgNPT1 resulted in impaired growth of T. gondii parasites in a conventional growth medium, consistent with a critical role of TgNPT1 in parasite survival. However the TgNPT1-knockdown parasite grew normally in a medium containing increased arginine and decreased lysine levels. The data were consistent with TgNPT1 being an arginine transporter and playing a role in the uptake of this amino acid into the parasite. This hypothesis was tested using two different approaches. First, TgNPT1 was expressed in Xenopus laevis oocytes and the uptake of arginine measured using both a radiolabelled form of the amino acid and electrophysiological techniques. Second, radiolabeled arginine uptake assays were performed in parasites lacking TgNPT1. These parasites showed reduced arginine uptake relative to parental controls. The data from these different systems were consistent with TgNPT1 being an arginine transporter. TgNPT1 is the first amino acid transporter to be described at the molecular level in apicomplexan parasites. Although TgNPT1 is critical for parasite growth in standard culture media, the data indicate the presence of an additional arginine transporter(s) in T. gondii. This second arginine transporter also transports lysine, and displays a lower affinity for arginine than TgNPT1. The virulence of T. gondii parasites lacking TgNPT1 was tested in the mouse model of T. gondii infection. When challenged with the wild type T. gondii parasites, all mice showed clinical signs of infection and succumbed to the infection. By contrast, mice challenged with the same dose of T. gondii parasites lacking TgNPT1 showed 100% survival. These data indicate that TgNPT1 is essential in vivo, and are consistent with TgNPT1 being the only physiologically relevant arginine transporter in T. gondii. The findings of this study highlight the importance of arginine uptake for T. gondii growth and identify the first amino acid transporter in apicomplexan parasites. Show more