Chloroquine clinical failures in P. falciparum malaria are associated with mutant Pfmdr-1, not Pfcrt in Madagascar

Molecular studies have demonstrated that mutations in the Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) play a major role in chloroquine resistance, while mutations in P. falciparum multidrug resistance gene (Pfmdr-1) act as modulator. In Madagascar, the high rate of chloroquine treatment failure (44%) appears disconnected from the overall level of in vitro CQ susceptibility (prevalence of CQ-resistant parasites <5%) or Pfcrt mutant isolates (<1%), strongly contrasting with sub-Saharan African countries. Previous studies showed a high frequency of Pfmdr-1 mutant parasites (>60% of isolates), but did not explore their association with P. falciparum chloroquine resistance. To document the association of Pfmdr-1 alleles with chloroquine resistance in Madagascar, 249 P. falciparum samples collected from patients enrolled in a chloroquine in vivo efficacy study were genotyped in Pfcrt/Pfmdr-1 genes as well as the estimation of the Pfmdr-1 copy number. Except 2 isolates, all samples displayed a wild-type Pfcrt allele without Pfmdr-1 amplification. Chloroquine treatment failures were significantly associated with Pfmdr-1 86Y mutant codon (OR = 4.6). The cumulative incidence of recurrence of patients carrying the Pfmdr-1 86Y mutation at day 0 (21 days) was shorter than patients carrying Pfmdr-1 86N wild type codon (28 days). In an independent set of 90 selected isolates, in vitro susceptibility to chloroquine was not associated with Pfmdr-1 polymorphisms. Analysis of two microsatellites flanking Pfmdr-1 allele showed that mutations occurred on multiple genetic backgrounds. In Madagascar, Pfmdr-1 polymorphism is associated with late chloroquine clinical failures and unrelated with in vitro susceptibility or Pfcrt genotype. These results highlight the limits of the current in vitro tests routinely used to monitor CQ drug resistance in this unique context. Gaining insight about the mechanisms that regulate polymorphism in Pfmdr1 remains important, particularly regarding the evolution and spread of Pfmdr-1 alleles in P. falciparum populations under changing drug pressure which may have important consequences in terms of antimalarial use management.     

Plasmodium falciparum: selenium-induced cytotoxicity to P. falciparum

The in vitro antimalarial activity of sodium selenite (NaSe) was investigated and the mechanism of its action was studied. NaSe had antimalarial activity against both the chloroquine-susceptible strain FCR-3 and chloroquine-resistant strain K-1 of Plasmodium falciparum. The shrunken cytoplasm of the parasite was observed in a smear 12 h after treatment with NaSe. Co-treatment with copper sulfate (CuSO(4)) in culture did not affect the antimalarial activity of NaSe, but NaSe cytotoxicity against the mammalian cell line Alexander was decreased significantly. The intracellular reduced glutathione level of parasitized red blood cells was decreased significantly by treatment with NaSe, and the decrease was consistent with their mortality. Treatment with NaSe had a strong inhibitory effect on plasmodial development, and NaSe cytotoxicity to human cells was decreased by co-treatment with CuSO(4). These results suggest that co-treatment with NaSe and CuSO(4) may be useful as a new antimalarial therapy.     

Stage-Dependent Effects of Chloroquine on Plasmodium falciparum In Vitro1

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of ³H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.     

Karyotype comparison between P. chabaudi and P. falciparum: analysis of a P. chabaudi cDNA containing sequences highly repetitive in P. falciparum.

The molecular karyotypes of P. chabaudi and P. falciparum have been compared by pulse field gradient electrophoresis. P. chabaudi has 3 extra chromosomes in the 750-2000 Kb range although the overall number appears to be 14 as is the case for P. falciparum. The chromosomal location of the rRNA genes has been determined for P. chabaudi together with that of a 24 Kd antigen gene. The corresponding cDNA 443 may code for a protein unusually rich in tyrosine and contains sequences highly repetitive in P. falciparum.     

Semaphorin-7A Is an Erythrocyte Receptor for P. falciparum Merozoite-Specific TRAP Homolog,MTRAP

The motility and invasion of Plasmodium parasites is believed to require a cytoplasmic actin-myosin motor associated with a cell surface ligand belonging to the TRAP (thrombospondin-related anonymous protein) family. Current models of invasion usually invoke the existence of specific receptors for the TRAP-family ligands on the surface of the host cell; however, the identities of these receptors remain largely unknown. Here, we identify the GPI-linked protein Semaphorin-7A (CD108) as an erythrocyte receptor for the P. falciparum merozoite-specific TRAP homolog (MTRAP) by using a systematic screening approach designed to detect extracellular protein interactions. The specificity of the interaction was demonstrated by showing that binding was saturable and by quantifying the equilibrium and kinetic biophysical binding parameters using surface plasmon resonance. We found that two MTRAP monomers interact via their tandem TSR domains with the Sema domains of a Semaphorin-7A homodimer. Known naturally-occurring polymorphisms in Semaphorin-7A did not quantitatively affect MTRAP binding nor did the presence of glycans on the receptor. Attempts to block the interaction during in vitro erythrocyte invasion assays using recombinant proteins and antibodies showed no significant inhibitory effect, suggesting the inaccessibility of the complex to proteinaceous blocking agents. These findings now provide important experimental evidence to support the model that parasite TRAP-family ligands interact with specific host receptors during cellular invasion.     

Chloroquine binds in the cofactor binding site of Plasmodium falciparum lactate dehydrogenase

Although the molecular mechanism by which chloroquine exerts its effects on the malarial parasite Plasmodium falciparum remains unclear, the drug has previously been found to interact specifically with the glycolytic enzyme lactate dehydrogenase from the parasite. In this study we have determined the crystal structure of the complex between chloroquine and P. falciparum lactate dehydrogenase. The bound chloroquine is clearly seen within the NADH binding pocket of the enzyme, occupying a position similar to that of the adenyl ring of the cofactor. Chloroquine hence competes with NADH for binding to the enzyme, acting as a competitive inhibitor for this critical glycolytic enzyme. Specific interactions between the drug and amino acids unique to the malarial form of the enzyme suggest this binding is selective. Inhibition studies confirm that chloroquine acts as a weak inhibitor of lactate dehydrogenase, with mild selectivity for the parasite enzyme. As chloroquine has been shown to accumulate to millimolar concentrations within the food vacuole in the gut of the parasite, even low levels of inhibition may contribute to the biological efficacy of the drug. The structure of this enzyme-inhibitor complex provides a template from which the quinoline moiety might be modified to develop more efficient inhibitors of the enzyme.     

Ineffective erythropoiesis in acute human P. falciparum malaria

An analysis of erythroblast cell kinetics utilizing quantitative 14C-autoradiography has been performed in five cases of acute Plasmodium falciparum malaria prior to and, in four patients, 3 or 6 days after the onset of antimalarial therapy. Associated with no or only moderate anemia were changes of erythroblast morphology, a considerable shift in the frequency of red and white blood cell precursors in the bone marrow, and a reduced rate of erythroblast proliferation. There was a marked loss of polychromatic erythroblasts, which was smaller but still detectable during the therapeutic phase. The results provide some quantitative data on the extent of "parenchymal damage" of bone marrow and stress the impact of ineffective erythropoiesis and reduced rate of erythropoietic proliferation on the emergence of anemia in Plasmodium falciparum malaria.     

Chloroquine mediates specific proteome oxidative damage across the erythrocytic cycle of resistant Plasmodium falciparum

Resistance of Plasmodium falciparum to chloroquine hinders malaria control in endemic areas. Current hypotheses on the action mechanism of chloroquine evoke its ultimate interference with the parasite's oxidative defence systems. Through carbonyl derivatization by 2,4-dinitrophenylhydrazine and proteomics, we compared oxidatively modified proteins across the parasite's intraerythrocytic stages in untreated and transiently IC(50) chloroquine-treated cultures of the chloroquine-resistant P. falciparum strain Dd2. Functional plasmodial protein groups found to be most oxidatively damaged were among those central to the parasite's physiological processes, including protein folding, proteolysis, energy metabolism, signal transduction, and pathogenesis. While an almost constant number of oxidized proteins was detected across the P. falciparum life cycle, chloroquine treatment led to increases in both the extent of protein oxidation and the number of proteins oxidized as the intraerythrocytic cycle progressed to mature stages. Our data provide new insights into early molecular effects produced by chloroquine in the parasite, as well as into the normal protein-oxidation modifications along the parasite cycle. Oxidized proteins involved in the particular parasite drug-response suggest that chloroquine causes specific oxidative stress, sharing common features with eukaryotic cells. Targeting these processes might provide ways of combating chloroquine-resistance and developing new antimalarial drugs.     

Further improvements of the P. falciparum humanized mouse model

Background

It has been shown previously that it is possible to obtain growth of Plasmodium falciparum in human erythrocytes grafted in mice lacking adaptive immune responses by controlling, to a certain extent, innate defences with liposomes containing clodronate (clo-lip). However, the reproducibility of those models is limited, with only a proportion of animals supporting longstanding parasitemia, due to strong inflammation induced by P. falciparum. Optimisation of the model is much needed for the study of new anti-malarial drugs, drug combinations, and candidate vaccines.

Materials/Methods

We investigated the possibility of improving previous models by employing the intravenous route (IV) for delivery of both human erythrocytes (huRBC) and P. falciparum, instead of the intraperitoneal route (IP), by testing various immunosuppressive drugs that might help to control innate mouse defences, and by exploring the potential benefits of using immunodeficient mice with additional genetic defects, such as those with IL-2Rγ deficiency (NSG mice).

Results

We demonstrate here the role of aging, of inosine and of the IL-2 receptor γ mutation in controlling P. falciparum induced inflammation. IV delivery of huRBC and P. falciparum in clo-lip treated NSG mice led to successful infection in 100% of inoculated mice, rapid rise of parasitemia to high levels (up to 40%), long-lasting parasitemia, and consistent results from mouse-to-mouse. Characteristics were closer to human infection than in previous models, with evidence of synchronisation, partial sequestration, and receptivity to various P. falciparum strains without preliminary adaptation. However, results show that a major IL-12p70 inflammatory response remains prevalent.

Conclusion

The combination of the NSG mouse, clodronate loaded liposomes, and IV delivery of huRBC has produced a reliable and more relevant model that better meets the needs of Malaria research.     

P. falciparum msp1 and msp2 genetic diversity in P. falciparum single and mixed infection with P. malariae among the asymptomatic population in Southern Benin

Plasmodium falciparum and Plasmodium malariae infections are prevalent in malaria-endemic countries. However, very little is known about their interactions especially the effect of P. malariae on P. falciparum genetic diversity. This study aimed to assess P. falciparum genetic diversity in P. falciparum and mixed infection P. falciparum/P. malariae isolates among the asymptomatic populations in Southern Benin. Two hundred and fifty blood samples (125 of P. falciparum and 125 P. falciparum/P. malariae isolates) were analysed by a nested PCR amplification of msp1 and msp2 genes. The R033 allelic family was the most represented for the msp1 gene in mono and mixed infection isolates (99.2% vs 86.4%), while the K1 family had the lowest frequency (38.3% vs 20.4%). However, with the msp2 gene, the two allelic families displayed similar frequencies in P. falciparum isolates while the 3D7 allelic family was more represented in P. falciparum/P. malariae isolates (88.7%). Polyclonal infections were also lower (62.9%) in P. falciparum/P. malariae isolates (p < 0.05). Overall, 96 individual alleles were identified (47 for msp1 and 49 for msp2) in P. falciparum isolates while a total of 50 individual alleles were identified (23 for msp1 and 27 for msp2) in P. falciparum/P. malariae isolates. The Multiplicity of Infection (MOI) was lower in P. falciparum/P. malariae isolates (p < 0.05). This study revealed a lower genetic diversity of P. falciparum in P. falciparum/P. malariae isolates using msp1 and msp2 genes among the asymptomatic population in Southern Benin.     

Accumulation of heavy metals by aquatic mosses. 3: Seasonal changes

The changes taking place at seven stream and river sites over a 14-month period were followed for the following:

1. % cover of Rhynchostegium riparioides;
2. relative abundance of Rhynchostegium as part of the whole plant community and, where present, also Amblystegium riparium and Fontinalis antipyretica;
3. 18 water chemistry variables;
4. concentrations of nine metals in 2-cm tips of Rhynchostegium at all sites and of Amblystegium and Fontinalis at one site each.

A decrease in the amounts of these mosses occurred in winter at most sites, the effect being most pronounced with Amblystegium. Nevertheless the majority of plants which remained had healthy shoots suitable for sampling for metal analysis at all times of year. There were marked temporal changes in the concentrations of metals in the 2-cm tips at each site, but few of these appeared to be related to seasonal events. The most plausible was for Pb in Rhynchostegium at a main river site, where increased accumulation in winter was probably an indirect effect of other chemical differences in the water during a period of high flows. Provided healthy shoots were available, there was no indication of seasonal differences in metal accumulation resulting from features of the mosses themselves.     

Heritability of P. falciparum and P. vivax Malaria in a Karen Population in Thailand

The majority of studies concerning malaria host genetics have focused on individual genes that confer protection against rather than susceptibility to malaria. Establishing the relative impact of genetic versus non-genetic factors on malaria infection and disease is essential to focus effort on key determinant factors. This relative contribution has rarely been evaluated for Plasmodium falciparum and almost never for Plasmodium vivax. We conducted a longitudinal cohort study in a Karen population of 3,484 individuals in a region of mesoendemic malaria, Thailand from 1998 to 2005. The number of P. falciparum and P. vivax clinical cases and the parasite density per person were determined. Statistical analyses were performed to account for the influence of environmental factors and the genetic heritability of the phenotypes was calculated using the pedigree-based variance components model. The genetic contribution to the number of clinical episodes resulting from P. falciparum and P. vivax were 10% and 19% respectively. There was also moderate genetic contribution to the maximum and overall parasite trophozoite density phenotypes for both P. falciparum (16%&16%) and P. vivax (15%&13%). These values, for P. falciparum, were similar to those previously observed in a region of much higher transmission intensity in Senegal, West Africa. Although environmental factors play an important role in acquiring an infection, genetics plays a determinant role in the outcome of an infection with either malaria parasite species prior to the development of immunity.     

Sexual-stage antibody responses to P. falciparum in endemic populations

Gametocytes and sporogonic stages are responsible for the spread of disease and drug resistance in the population. Sexual stage immunity affects the infectiousness of gametocytes to mosquitoes. Specific antibodies including anti-Pfs48/45 and anti-Pfs230 antibodies are found in individuals with limited prior exposure to malaria. Sexual stage antibodies are rapidly acquired after infection and are relatively prevalent in gametocytaemic individuals. Functional transmission reducing activity (TRA) is found after primary infections and in young children and appears to depend on recent rather than cumulative exposure to gametocytes. Exposure to gametocytes decreases with age most likely as a consequence of the acquisition of asexual-stage immunity that controls asexual parasite density and consequently gametocytaemia. This results in lower exposure to the antigenic load of gametocytes in semi-immune individuals. Since sexual stage immunity is probably short-lived in the absence of gametocytes, we hypothesize that sexual stage immunity will wane, resulting in low antibody and TRA prevalences in clinically semi-immune carriers.     

恶性疟原虫多抗原表位基因的融合与表达

本研究以霍乱毒素B亚基(CT-B)基因为载体,构建了含不同抗原表位的恶性疟原虫的融合基因CTB/ATE和CTB/AWTE。前者除含有恶性疟原虫裂殖子表面主要抗原表位杂合多肽基因SPf66外,还含有很强的T辅助细胞表位CST3和Tc细胞表位;后者在此基础上将我国发现的B细胞表位NKNDD基因经8次串联后融合其中、两种形式的融合基因经测序正确后转入大肠杆菌TK1046中,产量分别为10mg/L及5mg/L。表达产物CTB/AWTE经亲和层析纯化的双抗夹心ELISA测定表明,该融合蛋白在保留了与抗CTB抗体结合的同时,与抗NKNDD单抗的结合效价达1∶8000。