Kanako Komaki-Yasuda

Disruption of the Plasmodium falciparum 2-Cys peroxiredoxin gene renders parasites hypersensitive to reactive oxygen and nitrogen species.

In parasitism, Plasmodium falciparum is exposed to toxic reactive oxygen species and reactive nitrogen species (RNS). Peroxiredoxins (Prx) are ubiquitously distributed antioxidant enzymes. In bacteria and yeast, Prx have also been implicated in detoxifying RNS. Here, we used a gene targeting strategy to investigate the physiological role of 2‐Cys Prx of P. falciparum, PfTPx‐1, in living parasite cells. The PfTPx‐1‐null parasite line was more sensitive to paraquat (a superoxide donor) and sodium nitroprusside (a nitric oxide donor), than wildtype. These findings suggest that PfTPx‐1 protects the parasite cells from oxidative and nitrosative stresses.

Roles of 1-Cys peroxiredoxin in haem detoxification in the human malaria parasite Plasmodium falciparum

In the present study, we investigated whether Plasmodium falciparum 1‐Cys peroxiredoxin (Prx) (Pf1‐Cys‐Prx), a cytosolic protein expressed at high levels during the haem‐digesting stage, can act as an antioxidant to cope with the oxidative burden of haem (ferriprotoporphyrin IX; FP). Recombinant Pf1‐Cys‐Prx protein (rPf1‐Cys‐Prx) competed with glutathione (GSH) for FP and inhibited FP degradation by GSH. When rPf1‐Cys‐Prx was added to GSH‐mediated FP degradation, the amount of iron released was reduced to 23% of the reaction without the protein (P < 0.01). The rPf1‐Cys‐Prx bound to FP–agarose at pH 7.4, which is the pH of the parasite cytosol. The rPf1‐Cys‐Prx could completely protect glutamine synthetase from inactivation by the dithiothreitol–Fe3+‐dependent mixed‐function oxidation system, and it also protected enolase from inactivation by coincubation with FP/GSH. Incubation of white ghosts of human red blood cells and FP with rPf1‐Cys‐Prx reduced formation of membrane associations with FP to 75% of the incubation without the protein (P < 0.01). The findings of the present study suggest that Pf1‐Cys‐Prx protects the parasite against oxidative stresses by binding to FP, slowing the rate of GSH‐mediated FP degradation and consequent iron generation, protecting proteins from iron‐derived reactive oxygen species, and interfering with formation of membrane‐associated FP.

Expression profiles of peroxiredoxin proteins of the rodent malaria parasite Plasmodium yoelii

Patterns of expression of the 2-Cys and 1-Cys peroxiredoxin (Prx) proteins of the rodent malaria parasite Plasmodium yoelii during its life cycle were observed by immunofluorescent antibody staining and confocal laser scanning microscopy. 2-Cys Prx was expressed in the parasite cytoplasm throughout the life cycle, and the thioredoxin (Trx)-peroxidase activity of 2-Cys Prx revealed with the recombinant protein suggested that the Prx is constitutively expressed and, thus, likely plays a housekeeping role in the parasites intracellular redox control. In contrast, 1-Cys Prx showed stage-specific expression in blood-stage parasites. The limited expression of 1-Cys Prx in the trophozoite cytoplasm suggests that 1-Cys Prx may be involved in haemoglobin metabolism by the parasite, which generates a prooxidative haem iron and increases intracellular oxidative stress. The antioxidant activity of 1-Cys Prx was tested for its ability to protect yeast enolase against inactivation of the mixed-function oxidation system. Differential expression of the two Prx proteins during the erythrocytic and insect stages suggests the importance of these proteins in protecting parasites against oxidative stress, which is generated by the parasites metabolism and also from the environment.

Disruption of the Plasmodium berghei 2-Cys peroxiredoxin TPx-1 gene hinders the sporozoite development in the vector mosquito.

To investigate the physiologic role of cytosolic 2-Cys peroxiredoxin of Plasmodium berghei (PbTPx-1), we infected the vector mosquito Anopheles stephensi with a parasite carrying a targeted knockout of pbtpx-1 (Prx-KO). The number of Prx-KO midgut oocysts at 14-15 days post-feeding (pf) was comparable to that of the parent strain (WT); however, the numbers of sporozoites that formed in midgut oocysts and accumulated in the salivary gland of Prx-KO-infected mosquitoes by 21 days pf were decreased to 10-20% and 3-10%, respectively, of those values in WT-infected mosquitoes. A higher frequency of DNA strand breaks was detected in Prx-KO oocysts than in WT oocysts. Sporozoites carrying the targeted disruption had reduced infectivity in mice; however, the knockout did not affect the ability of the sporozoite to reach the liver parenchyma and initiate exo-erythrocytic form (EEF) development. TPx-1 may be involved in development during exponentially multiplying stages, such as sporozoites and EEF.

2-Cys peroxiredoxin of Plasmodium falciparum is involved in resistance to heat stress of the parasite

In the cytoplasm of Plasmodium falciparum, two peroxiredoxins: PfTPx-1 and Pf1-Cys-Prx, are expressed at different time-points of the parasite cell cycle during the intraerythrocytic stage. In the present study, to gain insight into the functions of Prxs in the cytoplasm of P. falciparum, we investigated the heat stress sensitivity of the previously established PfTPx-1 KO line and found that PfTPx-1 disruption renders the parasite hypersensitive to heat stress. In addition, we established Pf1-Cys-Prx knockout (KO) parasite lines. The phenotypes of Pf1-Cys-Prx KO lines were different to those of the PfTPx-1 KO line and did not show hypersensitivity to reactive oxygen species, reactive nitrogen species, chloroquine or heat stress. These results suggest that the function of Pf1-Cys-Prx in the parasite cytoplasm is independent from that of PfTPx-1. The hyperthermal protective function of the PfTPx-1 is obviously important for the parasite physiology in the human patient body, in which it must survive repeated incidences of fever.

Target proteins of the cytosolic thioredoxin in Plasmodium falciparum

The target proteins of a cytosolic Trx (PfTrx-1) in Plasmodium falciparum with Trx-affinity chromatography were examined. Based on the Trx protein reduction pathway, we generated a cysteine mutant of PfTrx-1, which captures the target protein as a mixed disulfide intermediate. A number of proteins were captured with PfTrx-1(C33S) immobilized on resin and were eluted by DTT treatment. The PfTrx-1(C33S) immobilized resin-captured proteins were trypsin-digested and analyzed on a liquid chromatography-mass spectrometry system. Analysis of the sequence data against databases assigned 20 proteins, four of which had been found previously in P. falciparum, with the remaining 16 being new targets. The potential Trx-target proteins included those in pathways such as the redox cycle, protein biosynthesis, energy metabolism and signal transduction. We captured 4 enzymes in the glycolysis pathway (hexokinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate mutase and L-lactate dehydrogenase (LDH)) as Trx-targets, and we found that PfTrx-1 enhanced the activity of PfGAPDH and PfLDH.

Evaluation of the NOW Malaria Immunochromatographic Test for Quantitative Diagnosis of Falciparum and Vivax Malaria Parasite Density

The NOW® Malaria Test, an immunochromatographic test (ICT), was evaluated to determine its ability to quantitatively detect malaria parasites using 100 blood samples from Thailand, including 50 Plasmodium falciparum (Pf) infections and 50 P. vivax (Pv) infections. Intensities of the thickness of the visible bands of the positive ICT were compared with the parasite densities. In cases of Pf infection, the intensities of both HRP-2 bands (T1 bands: Pf specific bands) and aldolase bands (T2 bands: pan-Plasmodium bands) correlated with the parasite densities. The intensities of T2 bands in Pf positive samples showed better correlation with the parasite densities than the T1 bands. In the cases of Pv infection, the intensities of T2 bands were also well correlated with parasite density. These results suggest that the ICT is useful not only for rapid detection of malaria parasites but also for estimating parasite density.

Identification of a Novel and Unique Transcription Factor in the Intraerythrocytic Stage of Plasmodium falciparum

The mechanisms of stage-specific gene regulation in the malaria parasite Plasmodium falciparum are largely unclear, with only a small number of specific regulatory transcription factors (AP2 family) having been identified. In particular, the transcription factors that function in the intraerythrocytic stage remain to be elucidated. Previously, as a model case for stage-specific transcription in the P. falciparum intraerythrocytic stage, we analyzed the transcriptional regulation of pf1-cys-prx, a trophozoite/schizont-specific gene, and suggested that some nuclear factors bind specifically to the cis-element of pf1-cys-prx and enhance transcription. In the present study, we purified nuclear factors from parasite nuclear extract by 5 steps of chromatography, and identified a factor termed PREBP. PREBP is not included in the AP2 family, and is a novel protein with four K-homology (KH) domains. The KH domain is known to be found in RNA-binding or single-stranded DNA-binding proteins. PREBP is well conserved in Plasmodium species and partially conserved in phylum Apicomplexa. To evaluate the effects of PREBP overexpression, we used a transient overexpression and luciferase assay combined approach. Overexpression of PREBP markedly enhanced luciferase expression under the control of the pf1-cys-prx cis-element. These results provide the first evidence of a novel transcription factor that activates the gene expression in the malaria parasite intraerythrocytic stage. These findings enhance our understanding of the evolution of specific transcription machinery in Plasmodium and other eukaryotes.

A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi

A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient’s blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a “fast PCR enzyme”. In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the “fast PCR enzyme”, with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses.

No Plasmodium falciparum Chloroquine Resistance Transporter and Artemisinin Resistance Mutations, Haiti

We obtained 78 human blood samples from areas in Haiti with high transmission of malaria and found no drug resistance–associated mutations in Plasmodium falciparum chloroquine resistance transporter and Kelch 13 genes. We recommend maintaining chloroquine as the first-line drug for malaria in Haiti. Artemisinin-based therapy can be used as alternative therapy.