Abstract
The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The BT1/PTR1 null mutant is hypersensitive to MTX, but in a step-by-step fashion, we could induce resistance to MTX in these cells. Several resistance mechanisms were found to co-exist including a reduced folate and MTX accumulation, demonstrating that cells with no measurable biopterin uptake but also greatly reduced folate uptake are viable, despite their auxotrophy for each of these substrates. The resistant cells have also amplified the gene coding for the MTX target dihydrofolate reductase. Finally, we found a marked reduction in MTX polyglutamylation in resistant cells. These studies further highlight the formidable ability of Leishmania cells to bypass the blockage of key metabolic pathways.
Highlights
The protozoan parasite Leishmania is responsible for a number of diseases with a wide range of clinical symptoms [1]
Generation of a BT1 and PTR1 Null Mutant of L. tarentolae—L. tarentolae cells becoming resistant to MTX by reducing folate/MTX uptake compensate by overexpressing the biopterin transporter BT1 gene [4]
L. tarentolae cells disrupted in either PTR1 or BT1 were previously reported to be more sensitive to MTX, whereas cells transfected with PTR1 and BT1 were found to be resistant to MTX [4, 20]
Summary
The protozoan parasite Leishmania is responsible for a number of diseases with a wide range of clinical symptoms [1]. Leishmania differs from its mammalian host in that it cannot synthesize pterins from GTP and differs from several microorganisms in that it cannot synthesize folates de novo It lacks enzymes necessary for the conjugation of the three building blocks (pterin, para aminobenzoic acid, and glutamate), leading to folates (reviewed in Ref. 3). Leishmania cells often resist the antifolate drug methotrexate (MTX) by reducing the accumulation of the drug (16 –18) This can be achieved by gene deletion of some of the folate transporter FT genes [7]. The BT1 gene has been inactivated in a number of species [4, 5, 15], and cells can grow in culture flasks, they are less virulent in animal models [19], demonstrating an important role for pterin transport in parasite biology. Selection of the BT1/PTR1 cells for MTX resistance led to unexpected results that will be presented here
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
