Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche

Campagnaro, G. D., de Freitas Nascimento, J., Girard, R. B.M., Silber, A. M. and de Koning, H. P. (2018) Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche. Biochimica et Biophysica Acta: General Subjects, 1862(12), pp. 2750-2763. (doi:10.1016/j.bbagen.2018.08.015) (PMID:30251664)

Campagnaro, G. D., de Freitas Nascimento, J., Girard, R. B.M., Silber, A. M. and de Koning, H. P. (2018) Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche. Biochimica et Biophysica Acta: General Subjects, 1862(12), pp. 2750-2763. (doi:10.1016/j.bbagen.2018.08.015) (PMID:30251664)

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Abstract

Background: Trypanosoma cruzi, the causative agent of Chagas' disease is unable to synthesise its own purines and relies on salvage from the host. In other protozoa, purine uptake has been shown to be mediated by Equilibrative Nucleoside Transporters (ENTs). Methods: To investigate the functionality of T. cruzi-encoded ENT transporters, its four putative ENT genes (TcrNB1, TcrNB2, TcrNT1 and TcrNT2) were cloned and expressed in genetically adapted Trypanosoma brucei procyclic cells from which the nucleobase transporter locus was deleted. Results: TcrNB1 displayed very high affinity for hypoxanthine (Km 93.8 ± 4.7 nM for) and guanine, and moderate affinity for adenine. TcrNT1 was found to be a high-affinity guanosine/inosine transporter (inosine Km is 1.0 ± 0.03 μM; guanosine Ki is 0.92 ± 0.2 μM). TcrNT2 encoded a high-affinity thymidine transporter (Km = 223.5 ± 7.1 nM) with a clear preference for 2’-deoxypyrimidines. TcrNB2, whose activity could not be determined in our system, could be a low-affinity purine nucleobase transporter, given its sequence and predicted structural similarities to Leishmania major NT4. All 4 transporter genes were highly expressed in the amastigote forms, with much lower expression in the non-dividing stages. Conclusions: The data appear to show that, surprisingly, T. cruzi has a preference for oxopurines over aminopurines and efficiently transports 2′-deoxypyrimidines. The T. cruzi ENTs display exceptionally high substrate affinity as an adaptation to their intracellular localisation. General significance: This study reports the first cloning of T. cruzi purine and pyrimidine transporters, including the first gene encoding a pyrimidine-selective protozoan transporter.

Item Type:Articles
Keywords:Equilibrative Nucleoside Transporter, heterologous expression, pyrimidine-specific uptake, thymidine transporter, Trypanosoma brucei, Trypanosoma cruzi.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Campagnaro, Gustavo and De Koning, Professor Harry
Authors: Campagnaro, G. D., de Freitas Nascimento, J., Girard, R. B.M., Silber, A. M., and de Koning, H. P.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Biochimica et Biophysica Acta: General Subjects
Publisher:Elsevier
ISSN:0304-4165
ISSN (Online):1872-8006
Published Online:24 August 2018
Copyright Holders:Copyright © 2018 Elsevier B.V.
First Published:First published in Biochimica et Biophysica Acta: General Subjects 1862(12): 2750-2763
Publisher Policy:Reproduced in accordance with the publisher copyright policy

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