Abstract
Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.
Highlights
Toxoplasma gondii is probably one of the most successful parasites in the world and it is thought to be potentially infective for all warm-blooded land and sea animals
Transporters belonging to Nucleoside-Cation Symporter 1 (NCS1) and Nucleobase-Ascorbate Transporter (NAT, known as NCS2), both belonging to the Amino acid-Polyamine-Organocation (APC)
Members of the Concentrative Nucleoside Transporter (CNT) family formed a separate branch close to the NCS1 transporters as did AzgA-like transporters, recent analyses have proposed AzgA-like transporters are associated with the NAT family [34,35]
Summary
Toxoplasma gondii is probably one of the most successful parasites in the world and it is thought to be potentially infective for all warm-blooded land and sea animals. The infection occurs predominantly by ingestion of: meat containing tissue cysts harboring bradyzoites; milk containing tachyzoites; or water or sward contaminated with sporulated oocysts. Infection by T. gondii can be lethal for some species of marsupials and sea mammals [3], but in most hosts it is effectively controlled by the immune response and the presence of the parasite is restrained to latent cysts formed in muscle and nervous tissues [1,2,4,5]. Livestock infections very much contribute to human infections as the consumption of meat containing T. gondii tissue cysts likely represents the most important source for horizontal transmission to human subjects [2]
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