Transfection of the human malaria parasite Plasmodium falciparum

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Abstract

In the past few years, methods have been developed which allow the introduction of exogenous DNA into the human malaria parasite Plasmodium falciparum. This important technical advance known as parasite transfection, provides powerful new tools to study the function of Plasmodium proteins and their roles in biology and disease. Already it has allowed the analysis of promoter function and has been successfully applied to establish the role of particular molecules and/or mutations in the biology of this parasite. This review summarises the current state of the technology and how it has been applied to dissect the function of the P. falciparum genome.

Introduction

Human malaria is caused by four species of parasite belonging to the genus Plasmodium. Plasmodium falciparum is the most important of these and is responsible for the death of between 1 and 2 million people per year. Many DNA sequences from P. falciparum have been described and as the P. falciparum genome project gathers momentum, a vast database of sequence information is becoming readily available. Ultimately, functional assessment of sequences of interest will be needed to assign biological roles to particular sequences.

Transfection is the transfer of exogenously derived nucleic acid sequences into an organism. Controlled genetic modification of P. falciparum using transfection technology has provided an important tool to aid in the functional study of genes. Successful DNA transfer was originally achieved by Wu et al.[1]. Plasmids were able to be maintained transiently as episomes allowing gene expression and promoter dissection experiments. Shortly thereafter, parasite lines were produced that had integrated plasmids into their haploid genome2, 3. Single recombination events have been reported to occur by homologous and non-homologous recombination in stable transfectants. Successful stable transfection of P. falciparum relies on vectors carrying sequences able to mediate gene expression and confer a selectable phenotype such as drug resistance. Plasmids must also possess the ability to replicate within the parasite.

This review focusses on P. falciparum transfection vector design and transfection techniques in use and published to date. Transfection technology in P. falciparum is still very much in its infancy and is limited in its applications. A great deal of information is still required to boost efforts in this field of molecular parasitology.

Section snippets

Promoters and terminators

Within Plasmodium species, homologous and heterologous promoters are able to confer transgene expression1, 3, 4, 5. Table 1 lists promoter and terminator sequences utilised in P. falciparum transfection vectors to date. The promoter regions vary in their patterns of stage-specific expression. The 5′ upstream regions of P. falciparum heat shock protein 86 (HSP86) and histidine-rich protein 3 (HRP3) are active during the majority of the intra-erythrocytic life-cycle (rings to schizonts)1, 6

Vectors

Plasmids used to transfect P. falciparum take two forms; those having a single expression cassette1, 2, 3, 11and plasmids possessing two expression cassettes (Fig. 1)[5]. Plasmids carrying two expression cassettes use one as a drug selection cassette and the other for transgene expression. Single cassette systems have a drug-resistance or reporter gene flanked by 5′ and 3′ control regions (Fig. 1A). These plasmids can be used for either transient or stable transformation. Expression of a gene

Properties of plasmid DNA in transfection

Propagation of stable transfectants is slow in the presence of selection drug. Pyknotic parasite forms are present in cultures indicating plasmid instability[2]. Stable transfection plasmids that have not integrated into the parasite genome need to replicate episomally within the parasite in order to be maintained through generations. DpnI resistance in episomes extracted from stable transfectants confirms that plasmids are able to replicate within the parasite2, 3, 11. An origin of replication

Transfection technique

Electroporation is the only method available for introducing foreign DNA into Plasmodium. Electroporation of P. falciparum (10% ring-infected erythrocyte) was initially developed using standard bacterial settings of 2.5 kV, 25 μF, 200 Ω in 0.4 cm gap cuvettes[1]. Recently, these conditions have been modified to 0.31 kV, 960 μF, 0.2 cm cuvettes in order to improve the transfer of supercoiled DNA. These studies reported a four- to 10-fold improvement in reporter gene signal[11]. While we do not

Functional studies

In P. falciparum, dihydrofolate reductase (DHFR) is present as the first moiety of the bifunctional enzyme DHFR-TS and catalyses the reduction of dihydrofolate to tetrahydrofolate. Functional analysis testing DHFR-TS inhibition by antimalarials has been carried out utilising transfection[11]. Supplementation of P. falciparum DHFR activity was achieved by transfecting parasites sensitive to the antifolates methotrexate and WR99210 with human DHFR. Plasmodium falciparum DHFR is sensitive to these

Future directions

At present there are two selectable markers available for use in P. falciparum transfection: a mutated form of the TgDHFR-TS[22], and human DHFR11, 23. Both genes are able to confer resistance to pyrimethamine24, 25. Human DHFR also confers resistance to the antifolate dihydrotriazine WR99210 and methotrexate. Human DHFR has the potential to be used as a complement to the already well established TgDHFR-TS system since WR99210 retains full potency on lines already resistant to pyrimethamine[26]

Acknowledgements

This work is supported by the National Health and Medical Research Council of Australia. Jacqueline Waterkeyn is supported by a National Health and Research Council Peter Doherty Post-doctoral Fellowship.

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