We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.

Malaria infection is initiated by Plasmodium sporozoites infecting the liver. Preventing sporozoite infection would block the obligatory first step of the infection and perhaps reduce disease severity. In addition, such an approach would decrease Plasmodium vivax hypnozoite formation and therefore disease relapses. Here we describe the activity of a trisubstituted pyrrole, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine, in inhibiting motility, invasion, and consequently infection by P. berghei sporozoites. In tissue culture, the compound was effective within the first 3 h of sporozoite addition to HepG2 cells. In vivo, intraperitoneal administration of the compound significantly inhibited liver-stage parasitemia in P. yoelii sporozoite-infected mice and prevented the appearance of blood-stage parasites. P. berghei sporozoites lacking the parasite cGMP-dependent protein kinase, the primary target of the compound in erythrocyte-stage parasites, remained infectious to HepG2 cells and sensitive to the drug. These results suggest that the drug has an additional target(s) in sporozoites. We propose that drugs that inhibit sporozoite infection offer a feasible approach to malaria prophylaxis.The malaria parasite, Plasmodium spp., infects 500 million people a year, killing over 1 million globally. Plasmodium parasites are introduced into humans via mosquito bites, in the form of sporozoites that infect the liver to form liver-stage parasites (LS). LS are eventually released into the bloodstream, where they infect erythrocytes and initiate the symptomatic phase of malaria. Although the liver stage of infection by Plasmodium falciparum and P. vivax, the two major human parasites, is asymptomatic, P. vivax infections often result in the formation of dormant LS called "hypnozoites." When reactivated, hypnozoites cause disease relapses up to 1 year after the initial infection. Therefore, achieving global eradication of malaria will require prevention or treatment of P. vivax hypnozoites in addition to treatment of erythrocyte stages of both species.The spread of drug-resistant parasites, limited vector control measures, and lack of an effective vaccine make novel drug discovery vital. Drugs that prevent sporozoite infection of the liver would block an obligatory step in the parasite's life cycle. They would also prevent the formation of hypnozoites by P. vivax and the pathology caused by the erythrocyte stages of all human Plasmodium species. Although the liver stages are asymptomatic, reducing the parasite burden in the liver is likely to significantly diminish blood-stage infection and reduce disease severity in a subpopulation of patients (1). Furthermore, the small number of parasites and the limited number of replications during the liver phase of malaria infection compared to those in the erythrocytic phase reduces the potential for the emergence of drug-resistant parasites (13). However, of the currently licensed drugs, only primaquine has been formally demonstrated to act against sporozoit...

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