A Histone Methyltransferase Inhibitor Can Reverse Epigenetically Acquired Drug Resistance in the Malaria Parasite Plasmodium falciparum

Abstract

Malaria parasites invade and replicate within red blood cells (RBCs), extensively modifying their structure and gaining access to the extracellular environment by placing the plasmodial surface anion channel (PSAC) into the RBC membrane. Expression of members of the cytoadherence linked antigen gene 3 (clag3) family is required for PSAC activity, a process that is regulated epigenetically. PSAC is a well-established route of uptake for large, hydrophilic antimalarial compounds, and parasites can acquire resistance by silencing clag3 gene expression, thereby reducing drug uptake. We found that exposure to sub-IC₅₀ concentrations of the histone methyltransferase inhibitor chaetocin caused substantial changes in both clag3 gene expression and RBC permeability, and reversed acquired resistance to the antimalarial compound blasticidin S that is transported through PSACs. Chaetocin treatment also altered progression of parasites through their replicative cycle, presumably by changing their ability to modify chromatin appropriately to enable DNA replication. These results indicate that targeting histone modifiers could represent a novel tool for reversing epigenetically acquired drug resistance in P. falciparum.

Keywords: PSAC; Plasmodium falciparum; clag; drug resistance; epigenetics.

FIG 1

Steady-state mRNA levels expressed from clag3.1 and clag3.2 in cultured parasites. RNA was extracted from synchronized cultures of late-stage parasites from the P. falciparum FCB isolate. Levels of mRNA were determined using qRT-PCR from cDNA prepared from wild-type parasites (FCB-WT) or parasites that had been selected for resistance to blasticidin (FCB-BR). The resistant parasites were either cultured continuously in the presence of blasticidin (+blast), in the absence of blasticidin for 2 weeks (−blast 2 wks) or in the absence of blasticidin and in the presence of chaetocin for 2 weeks (−blast +chae 2 wks). Expression levels were normalized to the seryl tRNA synthetase housekeeping gene and are displayed as relative copy number ± SEM. Statistically significant differences were determined using t tests with P values as displayed above the compared values.

FIG 2

Chaetocin treatment can reverse resistance to blasticidin. Blasticidin-resistant FCB parasites display reduced CLAG expression and are less sensitive to treatment with blasticidin. (A) Growth rates of resistant parasites grown in the presence of 450, 600, or 750 nM chaetocin, 2.5 μg/ml blasticidin, or both compounds over time. Sensitivity to blasticidin is observed after approximately 6 days of chaetocin treatment (red arrow, right panel). (B) Sybr-green based drug-sensitivity curves to determine sensitivity to blasticidin. The y axis displays Sybr green fluorescence as a percentage of that observed in parasites grown in the absence of blasticidin while the x axis displays the log of molar blasticidin concentration. Blasticidin-resistant parasites grown in the absence of chaetocin are shown in blue while parasites grown in the presence of 450, 600, or 750 nM chaetocin for 13 days are shown in red, green, or purple, respectively. (C) Blasticidin sensitivity curves for wild-type FCB parasites grown in the absence of chaetocin (blue) or in the presence of 450, 600, or 750 nM chaetocin (red, green, or purple, respectively). Values in (B) and (C) represent means ± SEM for triplicate wells on each plate (technical replicates) and in three independent plates (biological replicates). For statistical comparisons, mean EC₅₀ values for 0 nM chaetocin were compared to those treated with chaetocin by t tests (P < 0.05).

FIG 3

Uptake of PUR-1 in wild-type (top), blasticidin-resistant (middle), or blasticidin-resistant parasites grown in the presence of chaetocin (bottom). PUR-1 uptake assays were performed as described by Kelly et al. (28).

FIG 4

Progression through schizogony in the presence of absence of chaetocin. Cultured parasites were grown in the absence (untreated) or presence (chaetocin treated) of the histone methyltransferase inhibitor chaetocin. Cultures were tightly synchronized and monitored by flow cytometry as they progressed through the replicative cycle. Proportions of rings, trophozoites, and schizonts were determined by both RNA content (thiazole orange fluorescence) and DNA content (Hoechst 33342 fluorescence) using a gating strategy described in the Materials and Methods section. The approximate time after initial red cell invasion is shown above each set of charts.