A nuclear targeting system in Plasmodium falciparum

Kanjana Wittayacom,Chairat Uthaipibull,Theerarat Kochakarn,Ruchanok Tinikul,Pucharee Songprakhon,Thanat Chookajorn,Krittikorn Kumpornsin

doi:10.1186/1475-2875-9-126

Abstract

BackgroundThe distinct differences in gene control mechanisms acting in the nucleus between Plasmodium falciparum and the human host could lead to new potential drug targets for anti-malarial development. New molecular toolkits are required for dissecting molecular machineries in the P. falciparum nucleus. One valuable tool commonly used in model organisms is protein targeting to specific sub-cellular locations. Targeting proteins to specified locations allows labeling of organelles for microscopy, or testing of how the protein of interest modulates organelle function. In recent years, this approach has been developed for various malaria organelles, such as the mitochondrion and the apicoplast. A tool for targeting a protein of choice to the P. falciparum nucleus using an exogenous nuclear localization sequence is reported here.MethodsTo develop a nuclear targeting system, a putative nuclear localization sequence was fused with green fluorescent protein (GFP). The nuclear localization sequence from the yeast transcription factor Gal4 was chosen because of its well-defined nuclear localization signal. A series of truncated Gal4 constructs was also created to narrow down the nuclear localization sequence necessary for P. falciparum nuclear import. Transfected parasites were analysed by fluorescent and laser-scanning confocal microscopy.ResultsThe nuclear localization sequence of Gal4 is functional in P. falciparum. It effectively transported GFP into the nucleus, and the first 74 amino acid residues were sufficient for nuclear localization.ConclusionsThe Gal4 fusion technique enables specific transport of a protein of choice into the P. falciparum nucleus, and thus provides a tool for labeling nuclei without using DNA-staining dyes. The finding also indicates similarities between the nuclear transport mechanisms of yeast and P. falciparum. Since the nuclear transport system has been thoroughly studied in yeast, this could give clues to research on the same mechanism in P. falciparum.

Highlights

The distinct differences in gene control mechanisms acting in the nucleus between Plasmodium falciparum and the human host could lead to new potential drug targets for anti-malarial development
Its nuclear localization sequence can function in other model organisms [22], making it a promising candidate for developing a nuclear targeting system in P. falciparum
Residues 1-147 of Gal4 protein (Gal4p) were fused to the N-terminus of the green fluorescent protein (GFP) protein marker and expressed under the control of 5' and 3' flanking sequences of P. falciparum heat shock protein 86 (Pfhsp86 5') and Plasmodium berghei dihydrofolate reductase-thymidylate synthase (Pbdhfr-ts 3'), respectively

Summary

Introduction

The distinct differences in gene control mechanisms acting in the nucleus between Plasmodium falciparum and the human host could lead to new potential drug targets for anti-malarial development. Proteins are transported into the apicoplast using a bipartite signal sequence to facilitate apicoplast targeting and membrane translocation [13,14] These findings open new opportunities for developing molecular toolkits for targeting a protein of choice to various malaria organelles. An example of this organelle targeting approach is the use of Pfhsp N-terminal sequence to send proteins to the mitochondrion [15]. The nuclear localization signal of Gal4p was shown to be compatible with P. falciparum and can effectively transport a protein of choice into the nucleus. The Gal4-nuclear targeting system was shown to be compatible with different P. falciparum strains

Methods

Results

Conclusion