A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum

Genomic information is rapidly accumulating for the human malaria pathogen, Plasmodium falciparum. Our ability to perform genetic manipulations to understand Plasmodium gene function is limited. Dihydrofolate reductase is the only selectable marker presently available for transfection of P. falciparum. Additional markers are needed for complementation and for expression of mutated forms of essential genes. We tested parasite sensitivity to different drugs for which selectable markers are available. Two of these drugs that were very effective as antiplasmodial inhibitors in culture, blasticidin and geneticin (G418), were selected for further study. The genes BSD, encoding blasticidin S deaminase of Aspergillus terreus, and NEO, encoding neomycin phosphotransferase II from transposon Tn 5, were expressed under the histidine-rich protein III (HRPIII) gene promoter and tested for their ability to confer resistance to blasticidin or G418, respectively. After transfection, blasticidin and G418-resistant parasites tested positive for plasmid replication and BSD or NEO expression. Cross-resistance assays indicate that these markers are independent. The plasmid copy number and the enzymatic activity depended directly on the concentration of the drug used for selection. These markers set the stage for new methods of functional analysis of the P. falciparum genome. Malaria is responsible yearly for about 2 million deaths— mostly children under the age of 5 (1). Appearance of widespread resistance to currently used drugs such as chloroquine, fansidar, and mefloquine has stimulated efforts to identify new antimalarial drug and vaccine targets. The Plasmodium falciparum genome is '30 megabases in size, has a base composition of 82% A1T, and contains 14 chromosomes. Genome sequencing is underway, and genomic as well as expressedsequence-tag databases have been generated (2). Recently the 0.95-megabase chromosome II of this protozoan has been fully sequenced and is predicted to encode 209 proteins (3). Database analyses indicated that 43% of these proteins have no detectable homologs. On this chromosome, 18 ORFs called rifins have been found, and no function has been assigned to them yet. The paucity of genetic tools to study the parasite’s most important pathways, such as mechanisms of drug resistance, parasite invasion, differentiation, and cell cycle, leaves us with a growing database of genes but difficulty in determining their actual functions. Genetic manipulation of the malaria parasite has taken major strides forward. Early transfection success with Plasmodium species causing rodent malaria (4, 5) was followed by a breakthrough in transient transfection of P. falciparum (6). Subsequently, electroporation-based transfection of ring-stage P. falciparum has been used for stable expression and disruption of malaria genes (7–11). When these early intraerythrocytic parasites are electroporated in the presence of plasmid, DNA seems to be able to cross the erythrocytic membrane, the double membrane surrounding the parasite, and the nuclear membrane to gain entry into the nucleus. There, episomal plasmid can be maintained indefinitely under selective pressure. At a low frequency, homologous integration occurs, and events can be selected following drug withdrawal and subsequent reintroduction (7, 8). Integration has been shown to occur by single-crossover homologous-recombination events that can be exploited for allelic exchange or gene disruption (9, 11). Transfection of the malaria parasite has relied on a single marker, the human or Toxoplasma gondii dihydrofolate reductase genes that confer resistance to methotrexate or pyrimethamine (6, 12). This paucity of selectable markers limits our ability to carry out disruption of essential genes, complementation of knockouts, or execution of plasmid-shift experiments to select specific mutants in important biological pathways. In an attempt to prepare tools that can facilitate systematic analysis of the malaria parasite genome, we sought additional genetic markers for P. falciparum transfection. We tested different drugs for their ability to inhibit the growth of the parasite in culture. Based on these results, two drugs were selected, blasticidin S and G418. We sought to construct vectors that would confer stable, episomal resistance to these agents. The present work describes the use of BSD (encoding blasticidin S deaminase of Aspergillus terreus) and NEO (encoding neomycin phosphotransferase II from transposon Tn 5) genes as positive selectable markers for P. falciparum transfection. These markers will facilitate functional analysis of the malaria genome and understanding of the biology of the parasite, which are crucial for developing chemotherapies and vaccines. EXPERIMENTAL PROCEDURES Strains. The clones 3D7 (The Netherlands), HB3 (Honduras), Dd2 (Indochina), and W2 (Indochina) were used in this study. These parasites were kindly provided by Tom Wellems (National Institutes of Health, Bethesda, MD; HB3 and Dd2) and Pradip Rathod (Catholic University, Washington, DC; 3D7 and W2). Cell Culture and Materials. All enzyme reactions and DNA preparations were performed as described by Maniatis et al. (13). Parasites were cultured by the method of Trager and Jensen (14) by using a gas mixture of 3% O2, 3% CO2, and 94% N2. RPMI medium 1640 was supplemented with 30 mgyliter hypoxanthine (Sigma), 25 mM Hepes (Sigma), 0.225% NaHCO3 (Sigma), 0.5% Albumax I (Life Technologies, Grand Island, NY), and 10 mgyml gentamycin (Life Technologies). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. §To whom reprint requests should be addressed. e-mail: goldberg@ borcim.wustl.edu.