Biochemical and Biophysical Research Communications
A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression
Section snippets
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.