Authors
M Natto1; M Aldfer1; H Elati1; H P De Koning1; 1 Institute of Infection, Immunity and Inflammation, College of Veterinary, Medical and Life Sciences, University of Glasgow, United Kingdom, UK Discussion
Background:Equilibrative nucleoside transporters (ENTs) are essential to the life cycle of Leishmania mexicana, allowing for the salvage of nutrients needed for survival of the parasite. To date, five ENT genes have been identified for L. mexicana, only three of which (NT1.1, NT1.2, NT2) are known to be involved in nucleoside salvage; NT3 and NT4 are nucleobase transporters of promastigotes and amastigotes, respectively. Deletion of the NT1.1, NT1.2 and NT2 genes should provide a favourable expression system for the heterologous expression and characterise the activity of (protozoan) nucleoside and nucleobase transporters.
Method: This study aimed to create a new ‘super-knockout’ cell line (SUPKO) by generating a null background cell line that does not take up any purine or pyrimidine nucleosides, surviving through the salvage of purine nucleobases and biosynthesis of pyrimidines. Using CRISPR-Cas9 technology, four different L. mexicana cell lines were generated: NT1-sKO, NT1-dKO, NT2-sKO and NT2-dKO. The NT1-sKO cell line was generated by deleting the NT1.1/NT1.2 locus and replacing it with a blasticidin resistance cassette; the double knockout used the cytotoxic adenosine analogue tubercidin to select L. mexicana NT1.1/NT1.2 null parasites. The first NT2 allele was deleted using a puromycin-resistant cassette transfected with Cas9 guide RNAs and the second allele was removed by transfecting with the same puromycin-resistant cassette, with added selection pressure from inosine analogue formycin B.
Results: The SUPKO cell line was confirmed by PCR for the deleted genes, for which it was negative, using cas9 parental cells as control. Exhaustive transport assays showed that [3H]-adenosine uptake was ~90% lower in these cells, [3H]-guanosine uptake was reduced by >95%, [3H]-Uridine transport was almost undetectable, and [3H]-thymidine uptake was also reduced by >90%. Residual uptake rates were very low and appeared to reflect low affinity uptake through the NT3 nucleobase transporter as it could be inhibited by hypoxanthine. Surprisingly, the rate of transport for [3H]-hypoxanthine was ~30% lower in SUPKO than in cas9 control cells although growth rates were highly similar. Preliminary results show successful expression of single nucleoside transporters for detailed characterisation.
Conclusion: We successfully created a L. mexicana super knockout cell line that is null for NT1.1, NT1.2 and NT2, and is suitable for use in further studies to identify any additional ENTs utilised by the parasite. The identification of these transporters will not only improve our understanding of how ENTs work but also potentially allow for therapeutic targeting. 
