A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression

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Abstract

The process of merozoite release in Plasmodium falciparum involves rupture of the parasitophorous vacuole membrane and erythrocyte plasma membrane. Through the use of protease inhibitors that halt the merozoite release, a number of parasite proteases, especially serine, aspartic, and cysteine proteases, have been implicated in the schizont rupture. To understand the precise role of cysteine proteases in the merozoite release, in the present study, we treated P. falciparum cultures with siRNAs corresponding to falcipain-1, falcipain-2, and falcipain-3, the three papain-family proteases of the parasite. Treatment of malaria parasites with either of the falcipain siRNAs considerably reduced parasite growth. Morphological examination of the siRNA treated parasite cultures revealed that most of the parasites in falcipain-2 siRNA treated cultures were arrested at schizont stage. Analysis of a transgenic P. falciparum line expressing chimeric-GFP upon treatment with falcipain-2 siRNA revealed block in the rupture of erythrocyte membrane at the time of merozoite egression. These results suggest that falcipain-2 is an important parasitic protease that participates in hemoglobin degradation and in the merozoite release.

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Materials and methods

SiRNA design and synthesis. Three siRNAs—fp-1, fp-2, fp-3, and AP-N were designed for falcipain-1, falcipain-2, falcipain-3, and aminopeptidase-N genes, respectively (Fig. 1A), according to the criteria described by Schwarz et al. [11] and Khvorova et al. [12]. All the siRNAs were obtained from Dharmocon research (Colorado) in annealed and lyophilized form. These siRNAs were suspended in RNase–DNase free water at a concentration of 5 μg/μl.

Parasite stains, culture, and siRNA treatment.

Falcipain siRNA treatment alters parasite growth

We have previously shown that silencing of falcipain-1 and -2 using corresponding dsRNAs brings about substantial inhibitory effects on the parasite growth [16]. To determine whether similar effects can also be produced by siRNAs corresponding to three falcipain genes on P. falciparum culture, synchronized parasites at the late ring stage were treated with siRNAs corresponding to each of the three cysteine protease genes separately at 10 and 100 μg/ml, concentrations. Unrelated siRNA

Discussion

The merozoite release from RBCs has been proposed to be a two-step process that involves rupture of PVM and the rupture of RBC membrane [9], [10]. Even though events of rupture have not been fully characterized, current evidence suggests that malaria proteases in particular cysteine or serine proteases are involved in the rupture of parasitized RBCs [4], [5], [6]. RNA interference/PTGS is fast emerging as an important tool to carry out functional genomic studies in different organisms including

Acknowledgments

We are grateful to Alan Cowman for providing 3D7+His parasites, Chetan Chitnis for anti-PfEMP-1 antibodies and D. Jacobus for the drug WR99210. We thank Reshma Khorde for the technical help. The Confocal Microscope Facility at ICGEB, New Delhi, is funded through an International Senior Research Fellowship of the Wellcome Trust (UK) to Shahid Jameel. P.V.N.D., M.J.H., and A.K. were supported by CSIR Research Fellowships. We also thank Department of Biotechnology, India, for the financial support

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    These authors contributed equally to this work.

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