Cloning of Leishmania nucleoside transporter genes by rescue of a transport-deficient mutant

Abstract

All parasitic protozoa studied to date are incapable of purine biosynthesis and must therefore salvage purine nucleobases or nucleosides from their hosts. This salvage process is initiated by purine transporters on the parasite cell surface. We have used a mutant line (TUBA5) of Leishmania donovani that is deficient in adenosine/pyrimidine nucleoside transport activity (LdNT1) to clone genes encoding these nucleoside transporters by functional rescue. Two such genes, LdNT1.1 and LdNT1.2, have been sequenced and shown to encode deduced polypeptides with significant sequence identity to the human facilitative nucleoside transporter hENT1. Hydrophobicity analysis of the LdNT1.1 and LdNT1.2 proteins predicted 11 transmembrane domains. Transfection of the adenosine/pyrimidine nucleoside transport-deficient TUBA5 parasites with vectors containing the LdNT1.1 and LdNT1.2 genes confers sensitivity to the cytotoxic adenosine analog tubercidin and concurrently restores the ability of this mutant line to take up [3H]adenosine and [3H]uridine. Moreover, expression of the LdNT1.2 ORF in Xenopus oocytes significantly increases their ability to take up [3H]adenosine, confirming that this single protein is sufficient to mediate nucleoside transport. These results establish genetically and biochemically that both LdNT1 genes encode functional adenosine/pyrimidine nucleoside transporters.

Figure 1

Restriction map of the T1E1 cosmid. The map at the top includes the entire ≈40-kb T1E1 insert, whereas the expanded map below contains only the ≈15-kb EcoRI restriction fragment. Restriction fragments marked by arrows and designated 1, 2, A, B, and C are described in the text. □ designated NT1.1 and NT1.2 indicate the two LdNT1 ORFs. The direction of translation of these ORFs is from left to right. The symbol “+” indicates that the relevant restriction fragment conferred sensitivity to 10 μM tubercidin when subcloned into the pSNAR shuttle vector and transfected into TUBA5 cells. The symbol “−” indicates that this restriction fragment did not confer sensitivity to tubercidin when transfected into TUBA5 cells. Symbols for restriction sites are as follows: R, EcoRI; H, HindIII, X, XbaI; N, NotI; B, BglII; Xh, XhoI. The HindIII, XbaI, BglII, and XhoI sites were mapped only within restriction fragments 1 and 2.

Figure 2

Deduced amino acid sequence of LdNT1.1 (Upper) compared with the human equilibrative nucleoside transporter (18) hENT1 (Bottom) using the pileup program (14) with a gap weight of 4 and a gap length weight of 3. Amino acids that are identical between the two sequences are shown in white over a black background. The solid lines over the LdNT1 sequence and under the hENT1 sequence designate the predicted (15) transmembrane domains of each protein. The asterisks designate amino acids that are different in LdNT1.2. The amino acids P43, M107, T160, A489, T490, Y491 in LdNT1.1 are S43, I107, A160, E489, R490, H491 in LdNT1.2.

Figure 3

Southern blots of genomic DNA and Northern blots of RNA from DI700 and TUBA5 cell lines. Genomic DNA (5 μg) from the DI700 (A) and TUBA5 (B) lines was digested with the indicated restriction enzymes: R, EcoRI; H, HindIII; X, XbaI; S, SacI; P, PstI; Xh, XhoI; Sa, SacII; B, BglII. (C) Polyadenylylated RNA (4 μg) from the TUBA5 (lanes 1, 3, and 6) and DI700 (lane 2, 4, and 7) cell lines and 50 ng of LdNT1.2 cRNA (lane 5) were separated on agarose-formaldehyde gels and transferred to a nylon membrane for hybridization. For each figure, the numbers at the left indicate the position of molecular mass markers with sizes given in kb pairs for A and B and kb for C. Blots were probed with the LdNT1.2 ORF (lanes 1 and 2), a 750-bp SphI/XbaI fragment from the 3′-untranslated region of LdNT1.2 (lanes 3–5), and a 1-kb NdeI/XbaI fragment from the 3′-untranslated region of LdNT1.1 (lanes 6 and 7).

Figure 4

Functional expression of LdNT1 genes in TUBA5 cells and in Xenopus oocytes. TUBA5 cells transfected with the LdNT1.2 ORF in the pSNAR vector (solid symbols) or with the pSNAR vector alone (open symbols) were assayed for uptake of 200 nM [³H]adenosine (A) or 1 μM [³H]uridine (B) (mean ± SD, n = 2). For each time point, samples were assayed in duplicate, and the results were plotted as the mean and SD (error bars). (C) Xenopus oocytes were microinjected with either water or ≈25 ng of LdNT1 cRNA, incubated at 15°C for 6 days, and assayed (16) for uptake of 50 μM [³H]adenosine over 1 h (mean ± SD, n = 4).

Figure 5

[³H]adenosine (A and C) and [³H]uridine (B and D) substrate saturation curves for TUBA5 cells transfected with constructs containing LdNT1.1 (fragment 1 in Fig. 1) (A and B) and LdNT1.2 (either fragment 2 in Fig. 1 or the LdNT1.2 ORF) (C and D) in the pSNAR vector.