A genome-wide chromatin-associated nuclear peroxiredoxin from the malaria parasite Plasmodium falciparum

Abstract

Malaria parasites are subjected to high levels of oxidative stress during their development inside erythrocytes and the ability of the parasite to defend itself against this assault is critical to its survival. Therefore, Plasmodium possesses an effective antioxidant defense system that could potentially be used as a target for the development of inhibitor-based therapy. We have identified an unusual peroxiredoxin protein that localizes to the nucleus of Plasmodium falciparum and have renamed it PfnPrx (PF10_0268, earlier called MCP1). Our work reveals that PfnPrx has a broad specificity of substrate being able to utilize thioredoxin and glutaredoxin as reductants and having the ability to reduce simple and complex peroxides. Intriguingly, chromatin immunoprecipitation followed by deep sequencing reveals that the enzyme associates with chromatin in a genome-wide manner with a slight enrichment in coding regions. Our results represent the first description of a dedicated chromatin-associated peroxiredoxin and potentially represent an ingenious way by which the parasite can survive the highly oxidative environment within its human host.

FIGURE 1.

PfnPrx is expressed throughout the erythrocytic stage. A, domain organization of PfnPrx PF10_0268. The alignment shows the conserved AhpC-TSA family domain with the peroxidatic cysteine. Comparison was realized with Praline software. The Genbank^TM accession numbers of the aligned sequences are as follows: PfnPrx, AAC46600; Saccharomyces cerevisiae DOT5, P40553; PfTPx1, XP_001348542; PfTPx2, XP_001350554; Pf1-Cys_Prx, AAG14353; and PfAOP, AY306209. The asterisks represent conserved residues, and the arrow highlights the peroxidatic cysteine. B, time course of PfnPrx expression during the blood stage using an affinity-purified rabbit anti-serum raised against the first 100 residues of PfnPrx.

FIGURE 2.

PfnPrx is a nuclear protein. A, immunofluorescence using the mouse monoclonal anti-PfnPrx antibody demonstrates localization of PfnPrx in the nucleus throughout the erythrocytic stage. Troph, trophozoite. B, PfnPrx does not localize to the tight junction of merozoites in the process of invasion. Immunofluorescence with mouse monoclonal anti-nPrx and rabbit anti-RON4 antibody shows that PfnPrx is restricted to the nucleus in parasites invading a red blood cell. C, immunoelectron microscopy using the rabbit anti-PfnPrx antibody on schizont stage parasites reveals that PfnPrx is in the electron dense nuclear periphery. The image shows a single merozoite located within a schizont.

FIGURE 3.

Endogenously tagging nPrx confirms its status as a nuclear protein. A, schematic of the strategy employed to generate the 3D7 nPrx-3′ GFP line. hDHFR, human dihydrofolate reductase. B, Southern blots confirming integration of the targeting construct at the endogenous nPrx locus. C, Western blots using the mouse monoclonal anti-nPrx or an anti-GFP antibody confirms that the 3D7nPrx-3′ GFP parasite line expresses the nPrx-GFP chimera. D, epifluorescence microscopy on the 3D7nPrx-3′ GFP line demonstrates that the tagged nPrx localizes to the nucleus in all blood stages. X-over; crossover.

FIGURE 4.

PfnPrx is a peroxiredoxin with unusual characteristics. A, purification of the N-terminal domain of PfnPrx. B, glutamine synthetase (GS) protection assays revealing that PfnPrx is an antioxidant protein and that this activity was dependent on its conserved peroxidatic cysteine. C–E, peroxidase assays showing that glutaredoxin (C) is a potent reductant of PfnPrx and that PfnPrx efficiently reduces hydrogen peroxide (D) and cumene hydroperoxide (E). 2 μm PfnPrx was used for all assays, 20 μm hydrogen peroxide was used for the variable Pf glutaredoxin 1 assay, and 10 μm PfGrx1 was used for the variable peroxide assays. Results are representative of at least three independent experiments.

FIGURE 5.

PfnPrx shows genome wide chromatin association. A and B, broad distribution of PfnPrx (A), its enrichment in coding and depletion at centromeric region (B) demonstrated by coverage plots and ratio track of PfnPrx-GFP ChIP-seq data obtained from schizont stage parasites. C, average gene profile of PfnPrx occupancy. D, quantitative PCR data confirming enrichment of PfnPrx in coding region and its depletion at centromeres at three different stages of intraerythrocytic development. E, scatter plot analysis of correlation between PfnPrx enrichment in coding body of genes and steady state mRNA levels (RNA-seq data of schizont stage parasites from Bartfai et al. (33)).

FIGURE 6.

PfnPrx-GFP associates with a broad range of proteins. The immunoprecipitated proteins were grouped in categories and plotted according the total number of peptides identified. HSPs, heat shock proteins; RNA/DNA, proteins involved in RNA/DNA metabolism and/or binding. The list of proteins with accession numbers is available in the supplemental Table 1. No GFP peptides were recovered because a P. falciparum-specific database was used for peptide identification.