A study on pathogenicity and mosquito transmission success in the rodent malaria parasite Plasmodium chabaudi adami

We investigated the parasitology, pathogenicity (virulence) and infectivity to mosquitoes of blood infections in mice, of two strains, DS and DK, of the rodent malaria parasite Plasmodium chabaudi adami. Blood infections of DS were found to be highly pathogenic; the asexual parasites in these infections were fast-growing and showed no evidence of selectivity in their infection of host erythrocytes. In contrast to DS, blood infections of DK were much less pathogenic; the asexual parasites were slower-growing and showed a moderate degree of selectivity to a subset of erythrocytes which were not reticulocytes. In both DS and DK infections, infectivity to mosquitoes was highest before the peak of asexual parasitaemia had occurred; usually this did not coincide with the time when gametocyte numbers in the blood were highest. Infections with the pathogenic DS strain in CBA mice produced fewer gametocytes than did the less pathogenic DK strain. The DS strain infections in both CBA and C57 mice were also significantly much less infective to mosquitoes than the DK strain. Investigations by others on the related rodent malaria parasite subspecies, Plasmodium chabaudi chabaudi, have indicated that the mosquito infectivity of blood infections in mice tended to be higher in the more pathogenic (virulent) and lower in the less pathogenic strains of this parasite subspecies. This is the converse of the finding of the present investigation of blood infections of P. c. adami in mice in which a more pathogenic, or virulent, strain (DS) of these parasites was significantly much less infective to mosquitoes than was a less pathogenic strain (DK).     

Plasmodium chabaudi: rosetting in a rodent malaria model

Rosetting is a property of many malaria parasite species that has been linked to virulence in the major species infecting humans, Plasmodium falciparum. Here, the basic properties of rosettes in the rodent malaria laboratory model, P. chabaudi, were studied with a view to future studies on the role of rosetting in malaria parasite virulence and transmission. Rosetting occurred in 14 out of the 15 P. chabaudi clones studied, varied consistently between clones, and ranged between 9 and 37% at full parasite maturity. Rosetting frequency markedly declined after the mouse reached peak parasitemia, possibly due to host immunity. Consistent with P. falciparum and P. vivax, rosettes in P. chabaudi were disrupted by treatment with trypsin and EDTA. However, P. chabaudi rosettes were insensitive to sulfated glycoconjugates (heparin, heparan sulfate and fucoidan). The molecular basis of rosetting in P. chabaudi is unknown at present, but the results suggest that the molecules involved may differ from those in human-infecting species.     

Alterations of lecithin-cholesterol acyltransferase activity during Plasmodium chabaudi rodent malaria

In non fatal and synchronous P. chabaudi rodent malaria, we observed at the stage of parasitaemia peak, an alteration (50 % decrease) in LCAT activity. This decrease could be related partly to hepatic dysfunction, and mainly to circulating inhibitors released into blood from parasitized red blood cells at each end of a schizogonic cycle. This decrease in LCAT activity, at this step of the infection, accounts for part of the dyslipoproteinemia previously observed (i.e., increase in cholesterol and phospholipids into VLDL-LDL and decrease in the EC series and delayed conversion of Tg-rich lipoproteins into LDL-HDL. At a prepatent step of infection and after the parasitaemia peak, the alterations observed in LCAT activity, (respectively, increase and then decrease), would be related to similar changes in levels of cholesterol of HDL associated to complex changes in triacylglyceride transport and metabolism.     

Understanding and predicting strain-specific patterns of pathogenesis in the rodent malaria Plasmodium chabaudi

Despite considerable success elucidating important immunological and resource-based mechanisms that control the dynamics of infection in some diseases, little is known about how differences in these mechanisms result in strain differences in patterns of pathogenesis. Using a combination of data and theory, we disentangle the role of ecological factors (e.g., resource abundance) in the dynamics of pathogenesis for the malaria species Plasmodium chabaudi in CD4+ T cell-depleted mice. We build a series of nested models to systematically test a number of potential regulatory mechanisms and determine the "best" model using statistical techniques. The best-fit model is further tested using an independent data set from mixed-clone competition experiments. We find that parasites preferentially invade older red blood cells even when they are more fecund in younger reticulocytes and that inoculum size has a strong effect on burst size in reticulocytes. Importantly, the results suggest that strain-specific differences in virulence arise from differences in red blood cell age-specific invasion rates and burst sizes, since these are lower for the less virulent strain, as well as from differences in levels of erythropoesis induced by each strain. Our analyses highlight the importance of model selection and validation for revealing new biological insights.     

Selection for high and low virulence in the malaria parasite Plasmodium chabaudi.

What stops parasites becoming ever more virulent? Conventional wisdom and most parasite-centred models of the evolution of virulence suppose that risk of host (and, hence, parasite) death imposes selection against more virulent strains. Here we selected for high and low virulence within each of two clones of the rodent malaria parasite Plasmodium chabaudi on the basis of between-host differences in a surrogate measure of virulence--loss of live weight post-infection. Despite imposing strong selection for low virulence which mimicked 50-75% host mortality, the low virulence lines increased in virulence as much as the high virulence lines. Thus, artificial selection on between-host differences in virulence was unable to counteract natural selection for increased virulence caused by within-host selection processes. The parasite''s asexual replication rate and number of sexual transmission forms also increased in all lines, consistent with evolutionary models explaining high virulence. An upper bound to virulence, though not the asexual replication rate, was apparent, but this bound was not imposed by host mortality. Thus, we found evidence of the factors assumed to drive evolution of increased virulence, but not those thought to counter this selection.     

Virulence, drug sensitivity and transmission success in the rodent malaria, Plasmodium chabaudi

Here, we test the hypothesis that virulent malaria parasites are less susceptible to drug treatment than less virulent parasites. If true, drug treatment might promote the evolution of more virulent parasites (defined here as those doing more harm to hosts). Drug-resistance mechanisms that protect parasites through interactions with drug molecules at the sub-cellular level are well known. However, parasite phenotypes associated with virulence might also help parasites survive in the presence of drugs. For example, rapidly replicating parasites might be better able to recover in the host if drug treatment fails to eliminate parasites. We quantified the effects of drug treatment on the in-host survival and between-host transmission of rodent malaria (Plasmodium chabaudi) parasites which differed in virulence and had never been previously exposed to drugs. In all our treatment regimens and in single- and mixed-genotype infections, virulent parasites were less sensitive to pyrimethamine and artemisinin, the two antimalarial drugs we tested. Virulent parasites also achieved disproportionately greater transmission when exposed to pyrimethamine. Overall, our data suggest that drug treatment can select for more virulent parasites. Drugs targeting transmission stages (such as artemisinin) may minimize the evolutionary advantage of virulence in drug-treated infections.     

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 parasite's 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 parasite's metabolism and also from the environment.     

Detection of a system of microsomal monooxygenases in the rodent malaria parasite Plasmodium berghei

A microsomal fraction from the cells of the malaria parasite of rodent Plasmodium berghei was obtained. The spectral properties of microsomal preparations suggest that P. berghei microsomes contain cytochromes b5 and P-420. Electrophoretic separation of microsomal proteins revealed the presence of proteins whose molecular mass corresponds to NADPH-cytochrome c reductase, cytochrome P-450 and epoxide hydratase. The activities of NADPH-cytochrome c reductase and benzpyrene hydroxylase were determined. The spectral parameters, electrophoretic data and enzymatic activities of microsomal proteins indicate that P. berghei cells contain a cytochrome P-450 monooxygenase system. The interrelationship between the activity of the microsomal monooxygenase system and the resistance of P. berghei cells to the antimalaria preparation chloroquine is discussed.     

Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei.

Dihydroorotate dehydrogenase (DHODase) has been purified 400-fold from the rodent malaria parasite Plasmodium berghei to apparent homogeneity by Triton X-100 solubilization followed by anion-exchange, Cibacron Blue F3GA-agarose affinity, and gel filtration chromatography. The purified enzyme has a molecular mass of 52 +/- 2 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of 55 +/- 6 kDa by gel filtration chromatography, and it has a pI of 8.2. It is active in monomeric form, contains 2.022 mol of iron and 1.602 acid-labile sulfurs per mole of enzyme, and does not contain a flavin cofactor. The purified DHODase exhibits optimal activity at pH 8.0 in the presence of the ubiquinone coenzyme CoQ6, CoQ7, CoQ9, or CoQ10. The Km values for L-DHO and CoQ6 are 7.9 +/- 2.5 microM and 21.6 +/- 5.5 microM, respectively. The kcat values for both substrates are 11.44 min-1 and 11.70 min-1, respectively. The reaction product orotate and an orotate analogue, 5-fluoroorotate, are competitive inhibitors of the enzyme-catalyzed reaction with Ki values of 30.5 microM and 34.9 microM, respectively. The requirement of the long-chain ubiquinones for activity supports the hypothesis of the linkage of pyrimidine biosynthesis to the electron transport system and oxygen utilization in malaria by DHODase via ubiquinones [Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].     

The aspartic proteinase from the rodent parasite Plasmodium berghei as a potential model for plasmepsins from the human malaria parasite, Plasmodium falciparum

The gene encoding an aspartic proteinase precursor (proplasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general aspartic proteinase inhibitor, isovaleryl pepstatin and by Ro40-4388, a lead compound for the inhibition of plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proplasmepsin gene in P. berghei. Two plasmepsins have previously been reported in P. falciparum. Here, we describe two further proplasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of plasmepsin inhibitors is discussed.     

Effects of artesunate on parasite recrudescence and dormancy in the rodent malaria model Plasmodium vinckei

Artemisinin (ART) is the recommended first line therapy for treating uncomplicated and drug-resistant Plasmodium falciparum, the most pathogenic form of malaria. However, treatment failure following ART monotherapy is not uncommon and resistance to this rapidly acting drug has been reported in the Thai-Cambodian border. Recent in vitro studies have shown that following treatment with dihydroartemisinin (DHA), the development of ring-stage parasites is arrested for up to 20 days. These arrested (i.e. dormant) rings could be responsible for the recrudescence of infection that is observed following ART monotherapy. To develop a better understanding of the stage-specific effects of ART and determine if dormancy occurs in vivo, the ART derivative artesunate (AS) was used to treat mice infected with the synchronous rodent malaria parasites P. vinckei petteri (non-lethal) and P. v. vinckei (lethal). Results show that in both the non-lethal and lethal strains, ring-stage parasites are the least susceptible to treatment with AS and that the day of treatment has more of an impact on recrudescence than the total dose administered. Additionally, 24 hrs post-treatment with AS, dormant forms similar in morphology to those seen in vitro were observed. Finally, rate of recrudescence studies suggest that there is a positive correlation between the number of dormant parasites present and when recrudescence occurs in the vertebrate host. Collectively, these data suggest that dormancy occurs in vivo and contributes to recrudescence that is observed following AS treatment. It is possible that this may represent a novel mechanism of parasite survival following treatment with AS.     

Inhibitory effect of rhodamine 123 on the growth of the rodent malaria parasite, Plasmodium yoelii

The effect of the cationic permeant fluorescent dye, rhodamine 123 (R123), on the in vivo growth of Plasmodium yoelii was examined. Plasmodium yoelii-infected mouse erythrocytes were incubated in vitro with R123 and injected intravenously into mice. Examination of daily parasitemias showed that R123 delayed parasite growth whereas rhodamine 110, a neutral compound, and fluorescein, a negatively charged fluorescent dye, did not. Infected erythrocytes treated with R123 were not cleared from the circulation even 7 h after injection. Quantitation of cell-associated R123 by spectrophotometry revealed that infected cells with increased levels of R123 considerably prolonged the 2% prepatent period, the time required for the parasite to develop a 2% parasitemia. Degenerating parasites within and outside the host erythrocytes were observed on day 1 of infection in the mice. Thus it follows that R123, which accumulated in infected erythrocytes, inhibits the growth of P. yoelii; moreover, when R123-labeled infected erythrocytes were treated with 1-10 microM carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton ionophore, to release R123 from the cells, the inhibitory effect on the growth rate of P. yoelii was partially reversed.     

Critical roles of the mitochondrial complex II in oocyst formation of rodent malaria parasite Plasmodium berghei

It is generally accepted that the mitochondria play central roles in energy production of most eukaryotes. In contrast, it has been thought that Plasmodium spp., the causative agent of malaria, rely mainly on cytosolic glycolysis but not mitochondrial oxidative phosphorylation for energy production during blood stages. However, Plasmodium spp. possesses all genes necessary for the tricarboxylic acid (TCA) cycle and most of the genes for electron transport chain (ETC) enzymes. Therefore, it remains elusive whether oxidative phosphorylation is essential for the parasite survival. To elucidate the role of TCA metabolism and ETC in malaria parasites, we deleted the gene for flavoprotein (Fp) subunit, Pbsdha, one of four components of complex II, a catalytic subunit for succinate dehydrogenase activity. The Pbsdha(-) parasite grew normally at blood stages in mouse. In contrast, ookinete formation of Pbsdha(-) parasites in the mosquito stage was severely impaired. Finally, Pbsdha(-) ookinetes failed in oocyst formation, leading to complete malaria transmission blockade. These results suggest that malaria parasite may switch the energy metabolism from glycolysis to oxidative phosphorylation to adapt to the insect vector where glucose is not readily available for ATP production.     

Effect of anti-mosquito antibodies on the infectivity of the rodent malaria parasite Plasmodium berghei to Anopheles farauti

The effect of mouse anti-mosquito antibodies, present in the bloodmeal, on the infectivity of Plasmodium berghei Vincke to Anopheles farauti Laveran was investigated. Significantly fewer oocysts developed in mosquitoes feeding on mice immunized with sugar-fed mosquito midgut antigens than in mosquitoes feeding on control mice. Mosquitoes feeding on mice immunized with the midgut antigens derived from sugar-fed mosquitoes also showed reduced mortality and had lower infection rates than those fed on unimmunized mice. Blood-fed midgut antigen was less effective in producing these effects than sugar-fed midgut antigen.     

Wolbachia strain wAlbB enhances infection by the rodent malaria parasite Plasmodium berghei in Anopheles gambiae mosquitoes

Wolbachia, a common bacterial endosymbiont of insects, has been shown to protect its hosts against a wide range of pathogens. However, not all strains exert a protective effect on their host. Here we assess the effects of two divergent Wolbachia strains, wAlbB from Aedes albopictus and wMelPop from Drosophila melanogaster, on the vector competence of Anopheles gambiae challenged with Plasmodium berghei. We show that the wAlbB strain significantly increases P. berghei oocyst levels in the mosquito midgut while wMelPop modestly suppresses oocyst levels. The wAlbB strain is avirulent to mosquitoes while wMelPop is moderately virulent to mosquitoes pre-blood meal and highly virulent after mosquitoes have fed on mice. These various effects on P. berghei levels suggest that Wolbachia strains differ in their interactions with the host and/or pathogen, and these differences could be used to dissect the molecular mechanisms that cause interference of pathogen development in mosquitoes.     

Proteomics of the human malaria parasite Plasmodium falciparum

The lethal species of malaria parasite, Plasmodium falciparum, continues to exact a huge toll of mortality and morbidity, particularly in sub-Saharan Africa. Completion of the genome sequence of this organism and advances in proteomics and mass spectrometry have opened up unprecedented opportunities for understanding the complex biology of this parasite and how it responds to drug challenge and other interventions. This review describes recent progress that has been made in applying proteomics technology to this important pathogen and provides a look forward to likely future developments.     

High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei

This protocol describes a method of genetic transformation for the rodent malaria parasite Plasmodium berghei with a high transfection efficiency of 10(-3)-10(-4). It provides methods for: (i) in vitro cultivation and purification of the schizont stage;(ii) transfection of DNA constructs containing drug-selectable markers into schizonts using the nonviral Nucleofector technology; and (iii) injection of transfected parasites into mice and subsequent selection of mutants by drug treatment in vivo. Drug selection is described for two (antimalarial) drugs, pyrimethamine and WR92210. The drug-selectable markers currently in use are the pyrimethamine-resistant dihydrofolate reductase (dhfr) gene of Plasmodium or Toxoplasma gondii and the DHFR gene of humans that confer resistance to pyrimethamine and WR92210, respectively. This protocol enables the generation of transformed parasites within 10-15 d. Genetic modification of P. berghei is widely used to investigate gene function in Plasmodium, and this protocol for high-efficiency transformation will enable the application of large-scale functional genomics approaches.     

The contribution of Plasmodium chabaudi to our understanding of malaria

Malaria kills close to a million people every year, mostly children under the age of five. In the drive towards the development of an effective vaccine and new chemotherapeutic targets for malaria, field-based studies on human malaria infection and laboratory-based studies using animal models of malaria offer complementary opportunities to further our understanding of the mechanisms behind malaria infection and pathology. We outline here the parallels between the Plasmodium chabaudi mouse model of malaria and human malaria. We will highlight the contribution of P. chabaudi to our understanding of malaria in particular, how the immune response in malaria infection is initiated and regulated, its role in pathology, and how immunological memory is maintained. We will also discuss areas where new tools have opened up potential areas of exploration using this invaluable model system.