Aldfer, M. M. , AlSiari, T. A., Elati, H. A.A. , Natto, M. J., Alfayez, I. A., Campagnaro, G. D., Sani, B., Burchmore, R. J. S. , Diallinas, G. and De Koning, H. (2022) Nucleoside transport and nucleobase uptake null mutants in Leishmania mexicana for the routine expression and characterization of purine and pyrimidine transporters. International Journal of Molecular Sciences, 23(15), 8139. (doi: 10.3390/ijms23158139) (PMID:35897714) (PMCID:PMC9331716)
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Abstract
The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells’ ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.
| Item Type: | Articles |
|---|---|
| Additional Information: | This research was funded by a Wellcome Trust Institutional Strategic Support Fund (ISSF) Award (ref 317019); by studentships from the Libyan Government to M.M.A.; by a fellowship from the Government of Saudi Arabia to M.J.N.; by studentship from the Saudi Arabian Government to T.A.A. and I.A.A.; and by Science Without Borders with a scholarship to G.D.C. (206385/2014-5, CNPq, Brazil). |
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Burchmore, Dr Richard and Campagnaro, Gustavo and De Koning, Professor Harry and Aldfer, Mustafa Mohamed and Elati, Hamza Ali Abd and ALSiari, Tahani Abdulaziz A and Natto, Dr Manal and ALFAYEZ, IBRAHIM ABDULLAH M |
| Creator Roles: | Aldfer, M. M.Investigation ALSiari, T. A. A.Investigation Elati, H. A. A.Investigation Natto, M.Methodology, Investigation ALFAYEZ, I. A. M.Investigation Campagnaro, G.Investigation Burchmore, R.Methodology, Resources, Writing – review and editing De Koning, H.Methodology, Formal analysis, Writing – original draft |
| Authors: | Aldfer, M. M., AlSiari, T. A., Elati, H. A.A., Natto, M. J., Alfayez, I. A., Campagnaro, G. D., Sani, B., Burchmore, R. J. S., Diallinas, G., and De Koning, H. |
| College/School: | College of Medical Veterinary and Life Sciences College of Medical Veterinary and Life Sciences > School of Infection & Immunity |
| Journal Name: | International Journal of Molecular Sciences |
| Publisher: | MDPI |
| ISSN: | 1661-6596 |
| ISSN (Online): | 1422-0067 |
| Published Online: | 23 July 2022 |
| Copyright Holders: | Copyright © 2022 The Authors |
| First Published: | First published in International Journal of Molecular Sciences 23(15):8139 |
| Publisher Policy: | Reproduced under a Creative Commons License |
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