Use of peptide nucleic acids to manipulate gene expression in the malaria parasite Plasmodium falciparum.

One of the major concerns in treating malaria by conventional small drug molecules is the rapid emergence of drug resistance. Specific silencing of essential genes by antisense oliogomers has been proposed as an alternative approach that may result in antimalarial activity which is not associated with drug resistance. In addition, such an approach could be an important biological tool for studying many genes' function by reverse genetics. Here we present a novel methodology of using peptide nucleic acids (PNAs) as a useful tool for gene silencing in Plasmodium falciparum. PNAs, designed as specific antisense molecules, were conjugated to a cell penetrating peptide (CPP); namely, octa-D-lysine via the C-terminus, to allow facile delivery through cell membranes. PNAs added to P. falciparum cultures were found exclusively in infected erythrocytes and were eventually localized in nuclei of the parasites at all stages of intra erythrocytic development. We show that these PNAs specifically down regulated both a stably expressed transgene as well as an endogenous essential gene, which significantly reduced parasites' viability. This study paves the way for a simple approach to silence a variety of P. falciparum genes as means of deciphering their function and potentially to develop highly specific and potent antimalarial agents

Specific inhibition of gene expression using PNAs in transgenic <i>P. falciparum</i> parasites.

<p>Dose dependent inhibition of expression of <i>luciferase</i> reporter gene in NF54-luc (GB). These parasites constitutively express <i>luciferase</i> from a chromosomal locus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086802#pone.0086802-Salazar1" target="_blank">[20]</a>. Increasing concentrations of 0.5, 0.7, 0.9, 1.2, and 1.5 µM Luc-PNA were added to the culture media as well as similar concentrations of the scrambled PNA sequence (Scr-Luc-PNA). Parasites were incubated with the PNA molecules for 48h (left panel), then washed and cultured for an additional 48h (96h post incubation, right panel) in the absence of PNA in the media. Specific dose dependent inhibition of <i>luciferase</i> expression was found to be more robust after 96h <b>(A)</b>, reaching up to 70% inhibition <b>(B).</b> LRU, luminescence rate units. ScrPNA, Scr-Luc-PNA. <b>(C).</b> PNA treatment down regulate luciferase expression but does influence the levels of steady state mRNA. Left panel: steady state mRNA levels of luciferase in GB parasites treated with 1.2 µM LucPNA vs untreated parasites (GB). RCN, Relative copy number. Right panel: inhibition of luciferase expression following incubation with 1.2 µM LucPNA for 48h. All experiments were done in triplicate and the average is presented with SE.</p

PNA sequences and their corresponding Maldi-TOF MS assignments.

<p>PNA sequences and their corresponding Maldi-TOF MS assignments.</p

Specific down regulation of endogenous gene in <i>P. falciparum</i> using PNA molecules.

<p><b>(A)</b>, Specific dose dependent down-regulation of PfSec13 expression in parasites incubated with 1.2, 2.4, 4.8 and 9.6 µM Sec13-PNA compared with scrambled PNA sequence (Scr-Sec13-PNA) observed by western blot analysis using antibodies against endogenously tagged PfSec13 (α-HA) and aldolase. (–), no treatment. ScrPNA, Scr-Sec13-PNA. <b>(B)</b>, Down-regulation of an essential gene using specific Sec13-PNA molecules reduces parasites' viability. NF54-<i>luc</i> parasites constitutively expressing the <i>luciferase</i> reporter gene were incubated with 1.2, 2.4, 4.8 and 9.6 µM of either Sec13-PNA or Scr-Sec13-PNA. 72h post incubation the parasitemia was counted by direct microscopy (left panel) as well as inhibition of <i>luciferase</i> expression (right panel). The parasitemia decreased in a dose dependent manner only in parasite treated with Sec13-PNA. The gradual increase in inhibition of parasites' viability by Sec13-PNA was calculated as the percentage of <i>luciferase</i> reads compared with untreated NF54-<i>luc</i> parasites <b>(C).</b> The inhibition in growth is correlated with decrease in luciferase expression in parasite treated with Sec13-PNA. R² = 0.99, P<0.05. Dashed line represents 95% confidence interval slope <b>(D)</b>. Over time inhibition of parasites viability using PNA. Parasites were incubated with 4.8 µM Sec13-PNA or Scr-Sec13-PNA and luciferase expression was measured 24h, 48h, 72h, and 96h post incubation. The specific reduction in parasites viability is presented as percentage of inhibition of <i>lucifearse</i> expression compared with untreated NF54-<i>luc</i> parasites as above. All experiments were done in triplicate and the average is presented with SE.</p

PNA is targeted to the nuclei of intra-erythrocytic plasmodium parasites

<p>. <b>(A)</b>, Schematics of the PNAs molecules. <b>(B)</b>, <i>In vivo</i> imaging of Luc-PNA in infected RBCs 96h after addition of 0.6 µM Luc-PNA into unsynchronized parasites' culture. The PNAs were included in the culture media for the initial 24h, after which the culture media was replaced daily without PNAs. The Luc-PNA is seen in nuclei of parasites in various stages of the IDC. Arrows in the wide field point to the iRBC presented in high magnification. Images were taken using exposure time of at least 700 ms. (C), Quantification of PNA molecules observed in parasites 24h (N = 473) and 48h (N = 449) after incubation into the culture media. The percentage of parasites in which the PNAs could be observed is presented on the right columns. Images were taken using exposure time of 40 ms.</p