Centromere Plasmid: A New Genetic Tool for the Study of Plasmodium falciparum

Shiroh Iwanaga,Tomomi Kato,Masao Yuda,Izumi Kaneko,Georges Snounou

doi:10.1371/journal.pone.0033326

Abstract

The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite.

Highlights

Plasmodium falciparum causes the most severe form of malaria in humans, and infection with this parasite is responsible for considerable morbidity and mortality worldwide
We developed a centromere plasmid, pFCEN, for P. falciparum by incorporating a shortened centromere, pfcen5-1.5, into the plasmid DNA
These results clearly demonstrate that pfcen5-1.5 functioned as a centromere in P. falciparum

Summary

Introduction

Plasmodium falciparum causes the most severe form of malaria in humans, and infection with this parasite is responsible for considerable morbidity and mortality worldwide. The ability to introduce transgenes is a fundamental technique in the genetic manipulation in P. falciparum; currently, transgenes are introduced by integration into the parasite genome by single crossover recombination [4]. This approach is widely used but still involves technical limitations because single crossover events rarely occur; it usually takes several months (e.g., 2,6 months) to generate stable transgenic parasites. Episomal plasmids unevenly segregate into daughter parasites during cell division, and the parasites readily lose the plasmid unless selective pressure is maintained These technical problems hinder the progress of the molecular analysis of P. falciparum; a new genetic tool is required

Methods

Results

Conclusion