High efficacy in vitro selection procedure for generating transgenic parasites of Plasmodium berghei using an antibiotic toxic to rodent hosts.

The malaria parasite Plasmodium berghei is one of the main rodent malaria models. A shortcoming of this model parasite is its low flexibility in genetic manipulation. As this parasite cannot be continuously propagated in cell cultures, in vivo drug selection procedures are necessary to isolate genetic mutants. Drugs harmful to rodents therefore cannot be used for drug selection, which restricts the range of genetic manipulation. In this study, we addressed this problem by establishing a novel in vitro culture drug selection method, which we used in combination with other established methods to successfully isolate genetically manipulated parasites. The target mutants were enriched to the desired level within two weeks. We show that our system can also be used for sequential genetic manipulation of parasites carrying the traditionally used selection markers, demonstrate the procedure's versatility, and show its use in isolating specific genetically manipulated parasites. This novel in vitro selection method increases the number of available selection markers, allowing more extensive genetic manipulation in malaria parasite research.

Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei.

Phenotypic observation of thioredoxin peroxidase-1 (TPx-1) gene-disrupted Plasmodium berghei (TPx-1 KO) in the liver-stage was performed with an in vitro infection system in order to investigate defective liver-stage development in a mouse infection model. Indirect immunofluorescence microscopy assay with anti-circumsporozoite protein antibody revealed that in the liver schizont stage, TPx-1 KO parasite cells were significantly smaller than cells of the wild-type parent strain (WT). Indirect immunofluorescence microscopy assay with anti-merozoite surface protein-1 antibody, which was used to evaluate late schizont-stage development, indicated that TPx-1 KO schizont development was similar to WT strain development towards the merozoite-forming stage (mature schizont). However, fewer merozoites were produced in the mature TPx-1 KO schizont than in the mature WT schizont. Taken together, the results suggest that TPx-1 may be involved in merozoite formation during liver schizont development.

Plasmodium knowlesi thioredoxin peroxidase 1 binds to nucleic acids and has RNA chaperone activity.

Malaria parasites are under oxidative attack throughout their life cycle in human body and mosquito vector. Therefore, Plasmodium antioxidant defenses are crucial for its survival and being considered as interesting target for antimalarial drug design. Plasmodium knowlesi has emerged recently from its simian host to human in Southeast Asia and has been recognized as the fifth Plasmodium species that can cause human malaria. In this study, we cloned and characterized thioredoxin peroxidase 1 from P. knowlesi (PkTPx-1). PkTPx-1 gene was cloned, and recombinant protein was produced by heterologous overexpression in Escherichia coli. The recombinant protein was used for evaluation of enzymatic activity and polyclonal antibody production. Using the recombinant PkTPx-1 protein, its antioxidant activity was confirmed in a mixed-function oxidation assay where PkTPx-1 prevented nicking of DNA by hydroxyl radicals. PkTPx-1 was able to bind to double-strand DNA and RNA and had RNA chaperone activity in a nucleic acid melting assay indicating new function of PkTPx-1 other than antioxidant activity. Using specific polyclonal antibodies, it was indicated that PkTPx-1 is expressed in the cytoplasm of the parasite. Altogether, these results suggest that PkTPx-1 not only protects the parasite from the adverse effects of reactive oxygen species but also has RNA chaperone activity.

Development of Monoclonal Antibodies That Target 1-Cys Peroxiredoxin and Differentiate Plasmodium falciparum from P. vivax and P. knowlesi.

Prompt and accurate diagnosis of malarial patients is a crucial factor in controlling the morbidity and mortality of the disease. Effective treatment decisions require a correct diagnosis among mixed-species malarial patients. Differential diagnosis is particularly important in cases of Plasmodium vivax, a species that shares endemicity with P. falciparum in most endemic areas. Moreover, it is difficult to identify P. knowlesi on the basis of morphology alone, and rapid diagnostic tests are still not available for this malaria species. Therefore, the development of diagnostic tests applicable to the field is urgently needed. 1-Cys peroxiredoxin (1-Cys-Prx) in P. falciparum is abundantly expressed in the mature asexual stages, making it a promising candidate as a diagnostic antigen. In this study, we produced five monoclonal antibodies (mAbs) against P. falciparum 1-Cys-Prx (Pf1-Cys-Prx) by immunizing BALB/c mice with recombinant Pf1-Cys-Prx and subsequent hybridoma production. Cross reactivity of established mAbs with the orthologous molecule of Pf1-Cys-Prx in P. vivax (Pv1-Cys-Prx) and P. knowlesi (Pk1-Cys-Prx) was examined. Western blot analyses showed that three mAbs reacted with Pv1-Cys-Prx and Pk1-Cys-Prx but two mAbs did not. These results indicate that the two mAbs were effective in differentiating P. falciparum from P. vivax and P. knowlesi and could be used in differential diagnosis as well as comparative molecular studies of human Plasmodium species.

Expression profiles of peroxiredoxins in liver stage of the rodent malaria parasite Plasmodium berghei.

mRNA and protein expression profiles for three peroxiredoxins (TPx-1, TPx-2 and 1-Cys Prx) of liver stage Plasmodium berghei were examined through quantitative reverse transcription-PCR (RT-PCR) and indirect immunofluorescence microscopy assay (IFA). RT-PCR experiments revealed that mRNA expression for the TPx-1 was detected shortly after the sporozoite infection and kept expressed until the schizont stage. In contrast, the mRNA expression for 1-Cys Prx had begun increasing when the parasite developed into the schizont stage. Using the IFA, TPx-1 and 1-Cys Prx were detected in the cytosol. This finding suggested the developmental stage-specific expression of the cytosolic enzymes in the liver stage parasite. On the other hand, the mRNA expression for TPx-2 had begun increasing at the trophozoite stage and peaked at the schizont stage. In the IFA, TPx-2 was found localized in the mitochondria. The increase of TPx-2 might be explained by the exponential development of the parasite during the schizont stage requiring ATP production which may induce reactive oxygen species (ROS) in the mitochondria.

Mitochondrial peroxidase TPx-2 is not essential in the blood and insect stages of Plasmodium berghei.

BACKGROUND: Malaria parasites actively proliferate in the body of their vertebrate and insect hosts, and are subjected to the toxic effects of reactive oxygen species. The antioxidant defenses of malaria parasites are considered to play essential roles in their survival and are thus considered promising targets for intervention. We sought to identify the cellular function of thioredoxin peroxidase-2 (TPx-2), which is expressed in the mitochondria, by disrupting the TPx-2 gene (pbtpx-2) of the rodent malaria parasite Plasmodium berghei. FINDINGS: In three independent experiments, two disruptant populations (TPx-2 KO) and three wild-type parasite populations with pyrimethamine resistance (dhfr-ts/mt at the DHFR-TS locus) and intact pbtpx-2 (TPx-2 WT) were obtained and cloned. Null expression of TPx-2 in the KO population was confirmed by RT-PCR and Western blot analyses. The TPx-2 KO parasite developed normally in mouse erythrocytes and multiplied at a rate similar to that of the TPx-2 WT parasite during the experimental period. The peak period of gametocytemia was delayed by 1 day in the TPx-2 KO compared with that of the TPx-2 WT and the parent parasite, however, the highest gametocyte number was comparable. The number of midgut oocysts in the TPx-2 KO at 14 days post feeding was comparable to that of the TPx-2 WT. CONCLUSIONS: The present finding suggests that mitochondrial Prx TPx-2 is not essential for asexual and the insect stage development of the malaria parasite.