Dynamic association of the H3K64 trimethylation mark with genes encoding exported proteins in Plasmodium falciparum.

Epigenetic modifications have emerged as critical regulators of virulence genes and stage-specific gene expression in Plasmodium falciparum. However, the specific roles of histone core epigenetic modifications in regulating the stage-specific gene expression are not well understood. In this study, we report an unconventional trimethylation at lysine 64 on histone 3 (H3K64me3) and characterize its functional relevance in P. falciparum. We show that PfSET4 and PfSET5 proteins of P. falciparum methylate H3K64 and that they prefer the nucleosome as a substrate over free histone 3 proteins. Structural analysis of PfSET5 revealed that it interacts with the nucleosome as a dimer. The H3K64me3 mark is dynamic, being enriched in the ring and trophozoite stages and drastically reduced in the schizont stages. Stage-specific global chromatin immunoprecipitation -sequencing analysis of the H3K64me3 mark revealed the selective enrichment of this methyl mark on the genes of exported family proteins in the ring and trophozoite stages and a significant reduction of the same in the schizont stages. Collectively, our data identify a novel epigenetic mark that is associated with the subset of genes encoding for exported proteins, which may regulate their expression in different stages of P. falciparum.

The High Blood Pressure-Malaria Protection Hypothesis.

RATIONALE: A recently proposed hypothesis states that malaria may contribute to hypertension in endemic areas,1 but the role of angiotensin II (Ang II), a major regulator of blood pressure, was not considered. Elevated levels of Ang II may confer protection against malaria morbidity and mortality, providing an alternative explanation for hypertension in malaria endemic areas. OBJECTIVE: To discuss a possible alternative cause for hypertension in populations who have been under the selective pressure of malaria. METHODS AND RESULTS: We reviewed published scientific literature for studies that could establish a link between Ang II and malaria. Both genetic and functional studies suggested that high levels of Ang II may confer protection against cerebral malaria by strengthening the integrity of the endothelial brain barrier. We also describe strong experimental evidence supporting our hypothesis through genetic, functional, and interventional studies. CONCLUSIONS: A causal association between high levels of Ang II and protection from malaria pathogenesis can provide a likely explanation for the increased prevalence in hypertension observed in populations of African and South Asian origin. Furthermore, this potential causative connection might also direct unique approaches for the effective treatment of cerebral malaria.

Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-(14)C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

Molecular genotyping in a malaria treatment trial in Uganda - unexpected high rate of new infections within 2 weeks after treatment.

Polymerase chain reaction (PCR) genotyping of malaria parasites in drug efficacy trials helps differentiate reinfections from recrudescences. A combination therapy trial of one (n = 115) or three (n = 117) days artesunate (1AS, 3AS 4 mg/kg/day) plus sulphadoxine-pyrimethamine (SP) vs. SP alone (n = 153) was conducted in Mbarara, a mesoendemic area of western Uganda. All paired recurrent Plasmodium falciparum parasitaemias on days 7, 14, 21 and 28 post-treatment were genotyped by PCR amplification and analysis of glutamate-rich protein (glurp) and merozoite surface proteins (msp) 1 and 2 genes to distinguish recrudescent from new infections. A total of 156 (1AS = 61, 3AS = 35, SP alone = 60) of 199 paired recurrent samples were successfully analysed and were resolved as 79 recrudescences (1AS = 32, 3AS = 8, SP = 39) and 77 as new infections (1AS = 29, 3AS = 27, SP = 21). The ratios of proportions of new to recrudescent infections were 0.2, 0.9, 1.4 and 1.9 on days 7, 14, 21 and 28, respectively (P < 0.001, chi(2) test for linear trend). Unexpected high new infection rates were observed early in follow-up on days 7 [5/26 (19.2%)] and 14 [24/51 (47.1%)]. These results impact significantly on resistance monitoring and point to the value of genotyping all recurrent infections in antimalarial trials.

Low efficacy of the combination artesunate plus amodiaquine for uncomplicated falciparum malaria among children under 5 years in Kailahun, Sierra Leone.

OBJECTIVE: In 2004, Sierra Leone adopted artesunate plus amodiaquine as first-line antimalarial treatment. We evaluated the efficacy of this combination in Kailahun, where a previous study had shown 70.2% efficacy of amodiaquine in monotherapy. METHODS: Method and outcome classification of the study complied with WHO guidelines. Children 6-59 months with uncomplicated malaria were followed-up for 28 days. PCR genotyping was used to distinguish recrudescence from reinfection. Reinfections were reclassified as cured. RESULTS: Of 172 children who were referred to the study clinic, 126 satisfied inclusion criteria and were enrolled. No early treatment failures were reported. The day 14, efficacy was 98.2% (95% CI: 93.8-99.8). Of 65 recurrent parasitaemias analysed by PCR, 17 were recrudescences. The PCR-adjusted day 28 efficacy was 84.5% (95% CI: 76.4-90.7). All true failures occurred in the last 8 days of follow-up. Of 110 children who completed the 28-day follow-up, 54 (49.1%) experienced a novel infection. CONCLUSION: The efficacy of this combination was disappointing. The high reinfection rate suggested little prophylactic effect. In Kailahun a more efficacious combination might be necessary in the future. The efficacy of AS + AQ needs to be monitored in Kailahun and in the other regions of Sierra Leone.

Chemosensitizing action of cepharanthine against drug-resistant human malaria, Plasmodium falciparum.

We have established a system of in vitro and in vivo assays to prioritize plant extracts that can serve as a source of drug candidates for the treatment of malaria, an infectious disease that affects nearly 40% of the world's population. In the present study, we have investigated the biological potential of one such plant-derived drug lead, cepharanthine. In vitro growth inhibition studies indicated this compound possessed good antiplasmodial activity without mediating a cytotoxic response. Based on this selectivity, evaluations were performed with an in vivo mouse model. Moderate activity was observed, inhibiting parasite growth by 46% at a dose of 100 mg/kg body weight (BW). We further assessed the ability of cepharanthine to serve as a drug in combination with a standard antimalarial regimen. Like chloroquine, cepharanthine inhibited the trophozoite stage of parasite growth. Isobolographic analyses revealed synergism with chloroquine, but only with the drug-resistant malaria clone, and single-dose drug-interaction studies demonstrated that cepharanthine lowered the half-maximal inhibitory concentration of chloroquine from 148.5 to 37.8 nM. In summary, since activity in the mouse model was only moderate, cepharanthine may be of greater value as a modulator of resistance, capable of prolonging the clinical utility of chloroquine.

Efficacy and effectiveness of the combination of sulfadoxine/pyrimethamine and a 3-day course of artesunate for the treatment of uncomplicated falciparum malaria in a refugee settlement in Zambia.

In the Maheba Refugee Settlement, in the clinics supported by Medecins Sans Frontieres, all children aged up to 5 years with a confirmed diagnosis of uncomplicated falciparum malaria are treated with the combination of sulfadoxine/pyrimethamine (SP) and artesunate (AS). We compared the treatment's efficacy and effectiveness. Patients were randomized in order to receive the treatment supervised (efficacy) or unsupervised (effectiveness). Therapeutic response was determined after 28 days of follow up. The difference between recrudescence and re-infection was ascertained by polymerase chain reaction (PCR). We also assessed genetic markers associated to SP resistance (dhfr and dhps). Eighty-five patients received treatment under supervision and 84 received it unsupervised. On day 28, and after PCR adjustment, efficacy was found to be 83.5% (95% CI: 74.1-90.5), and effectiveness 63.4% (95% CI: 52.6-73.3) (P < 0.01). Point mutations on dhfr (108) and dhps (437) were found for 92.0% and 44.2% respectively of the PCR samples analysed. The significant difference in therapeutic response after supervised and unsupervised treatment intake can only be explained by insufficient patient adherence. When implementing new malaria treatment policies, serious investment in ensuring patient adherence is essential to ascertain the effectiveness of the new treatment schedules.

Artesunate-dapsone-proguanil treatment of falciparum malaria: genotypic determinants of therapeutic response.

The combination of chlorproguanil and dapsone is being considered as an alternative antimalarial to sulfadoxine-pyrimethamine in Africa, because of its greater efficacy against resistant parasites, and its shorter half-lives, which exert less selective pressure for the emergence of resistance. A triple artesunate-chlorproguanil-dapsone combination is under development. In a previous study of relatively low-dose chlorproguanil-dapsone in multidrug-resistant falciparum malaria in Thailand failure rates were high. Proguanil is inexpensive, widely available and very similar to chlorproguanil. The safety and efficacy of artesunate-dapsone-proguanil (artesunate 4 mg/kg, dapsone 2.5mg/kg, proguanil 8 mg/kg daily for three days), was studied prospectively in 48 Thai adult patients with acute falciparum malaria followed daily for 28 days. Eleven of these had a recrudescence of their infection. Genotyping of Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) indicated that the Pfdhfr I164L mutation was the main determinant of therapeutic outcome; all 11 failures carried this mutation (failure rate 11/37; 30%) whereas none of the 11 infections with 'wild type' 164 genotypes failed. The addition of artesunate considerably augments the antimalarial activity of the biguanide-dapsone combination, but this is insufficient for infections with parasites carrying the highly antifol-resistant Pfdhfr I164L mutation.

Influence of hemoglobin E trait on the antimalarial effect of artemisinin derivatives.

To determine whether hemoglobin E trait influences the antimalarial effect of artemisinin derivatives, we retrospectively compared 32 case patients with hemoglobin E trait to 32 control patients who did not have hemoglobin E, beta-thalassemia, glucose-6-phosphate dehydrogenase deficiency, or alpha-thalassemia trait on the basis of a mean corpuscular volume > or =78 femtoliters. All patients were admitted to the Hospital for Tropical Diseases in Bangkok, Thailand, with acute falciparum malaria. Control patients were matched to case patients with hemoglobin E trait by treatment with artemisinin derivatives versus other antimalarial drugs, by ethnic group, and by parasite count. Among 38 patients treated with artemisinin derivatives, the presence of hemoglobin E trait was associated with significantly faster parasite clearance (2.9-fold; 95% confidence interval [CI], 1.4-6.3; P=.006). Among 26 patients treated only with other antimalarial drugs, hemoglobin E trait did not significantly enhance parasite clearance (hazards ratio, 1.1; 95% CI, 0.5-2.5; P=. 8). Hemoglobin E trait may potentiate the antimalarial effect of artemisinin derivatives.

Genetic and dietary adaptation to malaria in human populations.

Plasmodial invasion places a severe oxidant stress on parasitized erythrocytes which can result in red cell damage and removal within the reticuloendothelial system or lysis, thus interrupting the parasitic cycle. The basis of a number of genetic adaptations to malaria--including the hemoglobin variants, the thalassemias, and glucose-6-phosphate dehydrogenase deficiency--is an increased sensitivity of the variant erythrocytes to the oxidant stress of plasmodial parasitization. It is suggested that dietary adaptations of traditional cusines in human populations living in areas where malaria is endemic augment this oxidant stress. It appears that there are three components of this adaptive dietary pattern in most tropical populations: the consumption of 'oxidant fuels', moderate to high iron intake, and limitation of dietary antioxidant intake or storage. It is argued that this dietary pattern maximizes iron-mediated free radical production in parasitized erythrocytes and thus provides a form of diet-mediated antimalarial prophylaxis. African pastoral populations that are heavy consumers of milk appear to manifest a different adaptive pattern involving low intakes of para-aminobenzoic acid, vitamin E, and iron. Periodic food restriction may also contribute to this antimalarial effect.