ABSTRACT
Functional analysis of Plasmodium genes by classical reverse genetics is currently limited to mutants that are viable during erythrocytic schizogony, the pathogenic phase of the malaria parasite where transfection is performed. Here, we describe a conceptually simple experimental approach to study the function of genes essential to the asexual blood stages in a subsequent life cycle stage by a promoter-swap approach. As a proof of concept we targeted the unconventional class XIV myosin MyoA, which is known to be required for Toxoplasma gondii tachyzoite locomotion and host cell invasion. By placing the corresponding Plasmodium berghei gene, PbMyoA, under the control of the apical membrane antigen 1 (AMA1) promoter, expression in blood stages is maintained but switched off during transmission to the insect vector, i.e. ookinetes. In those mutant ookinetes gliding motility is entirely abolished resulting in a complete block of life cycle progression in Anopheles mosquitoes. Similar approaches should permit the analysis of gene function in the mosquito forms that are shared with the erythrocytic stages of the malaria parasite.
Subject(s)
Antigens, Protozoan/metabolism , Locomotion , Membrane Proteins/metabolism , Myosins/metabolism , Protozoan Proteins/metabolism , Toxoplasma/pathogenicity , Animals , Anopheles/parasitology , Antigens, Protozoan/genetics , Female , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation , Genes, Protozoan , Genetic Complementation Test , Membrane Proteins/genetics , Mice , Microinjections , Myosins/genetics , Oocysts/metabolism , Plasmodium berghei/genetics , Promoter Regions, Genetic , Protozoan Proteins/genetics , Sporozoites/metabolism , Toxoplasma/genetics , Toxoplasma/metabolism , Toxoplasmosis/parasitology , TransfectionABSTRACT
We studied the transmission-blocking effect of isonicotinic acid hydrazide (INH), a widely used anti-tuberculosis drug, against Plasmodium gallinaceum and Plasmodium berghei. INH-treatment of infected animals did not inhibit parasite development in the blood of the vertebrate host, but did inhibit exflagellation, ookinete formation, and oocyst development in the mosquito. Oocyst development was inhibited in a dose-dependent manner. The ED(50) in the P. gallinaceum/chicken/Aedes aegypti model and P. berghei/mouse/Anopheles stephensi model was 72 and 109 mg/kg, respectively. In marked contrast, in vitro exflagellation and ookinete development were not directly affected by physiological concentrations of INH. We suggest that INH exerts its inhibitory effects on the mosquito stages of the malaria parasite by an indirect, and at present undefined mechanism. Further elucidation of the mechanism how INH inhibits parasite development specifically on mosquito stages may allow us to identify new targets for malaria control strategy.