RESUMEN
Conditional mutagenesis is a powerful tool for genetic analysis in Plasmodium berghei. It allows the study of proteins that function both during the parasite's pre-erythocytic and erythrocytic development. Currently available parasite lines used for conditional mutagenesis were constructed in the NK65 strain, and express a DNA recombinase under the control of pre-erythrocytic stage-specific promoters. However, the integration of the recombinase in these lines is unstable leading to inconsistent excision of the target gene. We describe improved lines of P. berghei with stably integrated DNA recombinase that allow efficient, stage-specific excision of target genes in the widely used ANKA strain.
Asunto(s)
Genética Microbiana/métodos , Biología Molecular/métodos , Mutagénesis , Plasmodium berghei/genética , Eliminación de Gen , Recombinación GenéticaRESUMEN
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.
Asunto(s)
Ingeniería Genética/métodos , Mutagénesis Sitio-Dirigida/métodos , Plasmodium berghei/genética , Animales , Anopheles/parasitología , Técnicas de Inactivación de Genes , Ratones , Ratas , Ratas Wistar , Recombinación Genética , Esporozoítos/fisiologíaRESUMEN
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.