Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

Abstract

Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome.

Keywords: gene expression; intraerythrocytic development; malaria; peptide conjugated morpholino oligomer; vivo morpholino oligomer.

Fig. 1.

Inhibition of translation and splicing using VMOs (A–C) Schematic representation of the binding sites of luciferase-VMO. (A), PfPMT-VMO (B), and PfCRT-VMO (C) on their respective sites on the mRNA or pre-mRNA. Arrows indicate the sites and orientation of the primers used for qPCR. cDNA was made from VMO-treated 3D7 parasites. (D) Luciferase-expressing parasites were treated with 1 or 2 µM Ctrl-VMO and luciferase-VMO; 72 h (one cycle) and 96 h (two cycles) later parasites lysates were used for luciferase assay. Luciferase activity is plotted after normalization to Ctrl-VMO. (E and G) qPCR studies with primers 2F and 2R to amplify PfPMT or PfCRT steady-state transcripts. (F and H) qPCR analyses carried out using primers 1F and 1R, which amplify PfPMT or PfCRT unspliced transcripts. The results represent three independent experiments, and the error bars indicate SE of mean. The level of significance in the graph is indicated with an asterisk (*P < 0.01). (I) cDNA from PBS (lane 1), PfPMT-VMO (lane 2), and PfCRT-VMO (lane 3) treated 3D7 parasites along with genomic DNA (lane 4) were amplified using PfPMT 3F and 3R, and the PCR products were separated on a 1% agarose gel.

Fig. S1.

Wild-type (3D7) parasite growth examined by flow cytometry. Luciferase-expressing parasites were treated with 1 and 2 µM Ctrl-VMO and luciferase-VMO, (A) 72 h (one cycle) and (B) 96 h (two cycles) later parasite growth was examined by flow cytometry. (C) 3D7 parasite growth following PfPMT-VMO (D) and PfCRT-VMO treatment was examined by flow cytometry. The gating used on flow data is indicated in the flow plot. R indicates ring stage whereas T indicates trophozoite-stage infected RBC. The experiment was repeated three times. The result represents data from a representative experiment; error bars indicate the SD of the average from three biological replicates.

Fig. 2.

PfPMT-VMO and PfCRT-VMO conjugates inhibit parasite growth. (A) 3D7 parasites were treated with 1.75 µM of control-VMO, PfPMT-VMO, or PfCRT-VMO. Four representative images of Giemsa-stained smears after two cycles posttreatment are shown. (B–E) Luciferase-expressing parasites were treated with 1.25 or 1.75 µM of Ctrl-VMO, PfPMT-VMO, or PfCRT-VMO, and luciferase activity was determined after one or two cycles of intraerythrocytic development. Growth as percentage of luciferase activity of PfPMT-VMO– (B and C) or PfCRT-VMO– (D and E) treated parasites normalized to Ctrl-VMO is shown. The experiment was carried out three times. The result represents data from a representative experiment; error bars indicate the SD of the average from three biological replicates.

Fig. 3.

PfCRT-VMO enhances sensitivity of Dd2 parasites to chloroquine. Dd2 parasites were treated with 1, 2, or 3 µM of Ctrl-VMO or PfCRT-VMO. Parasite inhibition was assessed by flow cytometry. Parasite growth in the presence of PfCRT-VMO was normalized to Ctrl-VMO as shown after one cycle (A) and two cycles (B). The effect of PfCRT-VMO on Dd2 sensitivity to chloroquine (CQ) was examined by treating Dd2 parasites with 2 µM Ctrl-VMO or PfCRT-VMO in the absence or presence of 50 nM CQ (C). Parasite growth was examined by flow cytometry. The percentage inhibition was obtained by calculating the difference between Ctrl-VMO and PfCRT-VMO treatments in the absence or presence of CQ as a percentage of Ctrl-VMO treatment. (D) A representative flow plot comparing different treatments is shown. The presence of PfCRT-VMO increases parasite sensitivity to CQ, whereas a similar effect is not seen with Ctrl-VMO. The experiment was performed three times. The result represents data from a single experiment with the error bars indicative of the SD of the average from three biological replicates. The level of significance in the graph is indicated with an asterisk (*P < 0.01). R: ring-stage parasites; T: trophozoite-stage parasites.

Fig. 4.

PfDXR-PPMO down-regulates PfDXR gene expression and alters growth of both 3D7 and artemisinin slow-clearance parasites. (A) Schematic representation of the PfDXR transcript and the PfDXR-PPMO–binding site. (B) Cleavage of PfDXR mRNA by E. coli RNase P in vitro. (Lane 1) DXR mRNA; (lane 2) DXR mRNA+ E. coli RNAse P; (lane 3) DXR mRNA+ E. coli RNAse P + DXR 130EGS; (lane 4) DXR mRNA+ E. coli RNAse P + DXR 145EGS; (lane 5) DXR mRNA+ HeLaRNAse P; (lane 6) DXR mRNA+ HeLaRNAse P + DXR 130EGS; (lane 7) DXR mRNA+ HeLaRNAse P + DXR 145EGS. (C) cDNA was made from RNA isolated from PfDXR-PPMO–treated 3D7 parasites. qPCR carried out using PfDXR-specific primers shows a dose-dependent reduction in PfDXR transcript. (D and E) 3D7 parasites were treated with PfDXR-PPMO, SaGyr-PPMO (negative control), and dihydroartemisinin. (E) Representative images of infected red blood cells one cycle posttreatment. (F) Inhibition of artemisinin slow-clearance parasites (ART-SL) by PfDXR-PPMO and dihydroartemisinin (positive control). The experiment was repeated twice, and the error bars indicate SD of the average of experimental values. Significant difference is indicated with an asterisk (**P < 0.001, ***P < 0.0001).

Fig. 5.

PfDXR-PPMO–mediated inhibition of P. falciparum is complemented by IPP supplementation. 3D7 parasites were treated with PfDXR-PPMO, SaGyr-PPMO (negative control), or fosmidomycin (fos). (A) Parasite growth was examined by flow cytometry after one and two cycles. (B) The effect of fosmidomyin on parasite growth was determined by flow cytometry. (C) 3D7 parasites were treated with fosmidomycin (2 µM), Ctrl-PPMO (SaGyr-PPMO), or PfDXR-PPMO (12.5 µM) in the absence or presence of 200 µM IPP, and parasite growth was examined by flow cytometry. (D) 3D7 parasites were treated with fosmidomycin (1 µM), PfDXR-PPMO + fos (1 µM), or SaGyr-PPMO + fos (1 µM) in the presence or absence of IPP. Percentage growth was calculated compared with untreated controls. The experiment was repeated twice, and the error bars indicate SD of the average of experimental values. The level of significance in the graph is indicated with an asterisk (*P < 0.01, ** P < 0.001).

Fig. S2.

Effect of SaGyr and PfDXR-PPMO in the growth of 3D7 parasites. (A) A representative flow plot comparing the effect of SaGyr-PPMO and PfDXR-PPMO on parasite growth at 15 µM. Dihydroethidium was used for nuclear staining. The gating used is shown for infected RBCs (iRBCs). (B) A representative flow for combination treatment of PfDXR-PPMO or SaGyr-PPMO with fosmidomycin is shown. Dihydroethidium was used for nuclear staining. The experiment was repeated twice.