Plasmodium falciparum purine nucleoside phosphorylase is critical for viability of malaria parasites

Abstract

Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (Deltapfpnp). Lysates of the Deltapfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the Deltapfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and Deltapfpnp parasites. The Deltapfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins.

FIGURE 1.

Genetic manipulation strategy and molecular characterization of Δpfpnp clones. A, schematic of pBSΔmini/pfpnp plasmid integration into the endogenous pnp locus by single crossover homologous recombination. The resultant recombinant locus contains two non-functional pnp remnants, each lacking sequences for key residues involved in the catalytic activity of the PfPNP. PCR primers used for molecular characterization are indicated by arrows. Fragments from restriction enzyme digestion are indicated on the schematic. E, EcoRI; H, HindIII. B, representative PCR detection of recombinant pnp clones and WT 3D7. Lane 1, wt pnp, p1/p2; lanes 2 and 3, intact plasmid, p3/4 and p3/5; lane 4, recombination event upstream, p1/p4; lane 5, recombination event downstream, p3/p6; lane 6, hrpII integration, p7/p4; lane 7, cam integration, p8/p9. C, Southern hybridization of gDNA from untransfected 3D7 and Δpnp 1 digested with EcoRI or HindIII and probed with pnp.

FIGURE 2.

Characterization of PfPNP expression in Δpfpnp lines. A, reverse transcriptase-PCR results show the absence of transcripts for pnp in all three Δpfpnp lines, whereas the transcript for ada remains intact. B, Western blot of wild-type 3D7 and Δpfpnp clones were probed with antibodies against PfPNP and PfADA. A band at the expected size (∼27 kDa) of PfPNP was detected in the wild type and absent from the Δpfpnp clones, whereas PfADA was observed at the expected size (∼42 kDa) in all parasite samples.

FIGURE 3.

Purine requirements for Δpfpnp lines. Using [³H]ethanolamine as an indicator of growth, the Δpfpnp lines show a greater need for exogenous hypoxanthine (A), inosine (B), and adenosine (C). Error bars represent the mean ± S.D., and two-tailed unpaired t tests were carried out to determine the statistical significance for each disrupted line as compared with the wild-type control line (hrpII integrant). ^*, p =<0.0001; ^**, p =<0.001. The need for exogenous purines is confirmed using the lactate dehydrogenase assay (D), where the intensity of blue color reflects parasite lactate dehydrogenase activity, which is an indication of parasite growth.

FIGURE 4.

Immucillin dose-response curves. The Δpfpnp lines are more sensitive to ImmH (A), a transition state inhibitor that binds tighter to hPNP, and less sensitive to MT-ImmH (B), an inhibitor that binds 100-fold more tightly to PfPNP. Data shown in panels A and B are normalized to untreated controls. IC₅₀ values ± S.E. from multiple independent experiments were calculated and are represented in Table 3. Overall growth of Δpfpnp as represented by total ethanolamine incorporation is lower than wild-type controls, as can be seen when inhibition curves are represented as total incorporated ethanolamine counts (C; subtracting uninfected erythrocyte controls; see Fig. 3 also). The disassociation constants and structures for the immucillins are presented in panel D (5, 7). All assays were performed without hypoxanthine supplementation of the culture media.

FIGURE 5.

The interactions of host and parasite purine pathways in P. falciparum-infected erythrocytes. Purine bases and nucleosides are taken up by erythrocyte equilibrative transporters. Enzymes present in the erythrocyte cytosol include methylthioadenosine phosphorylase (MTAP), adenine phosphoribosyl transferase (APRT), adenosine kinase (AK), ADA, PNP, and HGPRT. The AK and ADA reactions are irreversible but PNP and HG(X)PRT reactions are reversible. At low concentrations of adenosine the AK reaction is favored over the ADA reaction in erythrocytes. P. falciparum encodes dual-specificity ADA and PNP enzymes that salvage purines and also recycle methylthioadenosine, a product of polyamine synthesis. Parasite HGPRT (HGXPRT) also utilizes xanthine. Both purine bases and nucleosides are taken up by parasite-encoded nucleoside transporters. PfNT1 is the major transporter responsible for the low affinity uptake of adenosine, inosine, and hypoxanthine. The higher affinity of PfPNP for inosine and PfHGXPRT for hypoxanthine compared with human counterparts results in metabolic trapping of purines transported by PfNT1. Other transporters may also be present on the plasma membrane but the identities and propertiesof these have not been determined. Specific activities of host and parasite enzymes (in μmol g^-1 min^-1) are as reported by Reyes et al. (42).