Glycosylphosphatidyl-inositols in murine malaria: Plasmodium yoelii yoelii

Glycosylphosphatidyl-inositols (GPIs) are vital major glycoconjugates in intraerythrocytic stages of Plasmodium. Here, we report on the biosynthesis and the characterization of GPIs synthesized by the murine malarial parasite P. yoelii yoelii YM. Parasitized erythrocytes were labeled in vivo and in vitro with either radioactive nucleotide sugar precursors, ethanolamine or glucosamine. The pathway leading to the formation of GPI precursors was found to resemble that described for P. falciparum; however, in P. yoelii, the formation of an additional hydrophilic precursor containing an acid-labile modification was detected. The data suggest that this modification is linked to the fourth mannose attached to the trimannosyl backbone in an alpha1-2 linkage. The modification was susceptible to hydrofluoric acid (HF), but not to nitrous acid (HNO(2)). Data obtained from size-exclusion chromatography on Bio-Gel P4, and Mono Q analysis of the fragments generated by HNO(2) deamination suggest that the modification is due to the presence of an additional ethanolamine linked to the fourth mannose via a phosphodiester bond.     

Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle

Plasmodium yoelii is a rodent parasite commonly used as a model to study malaria infection. It is the preferred model parasite for liver-stage immunological studies and is also widely used to study hepatocyte, erythrocyte and mosquito infection. We have generated a P. yoelii yoelii 17XNL line that is stably transfected with the green fluorescent protein (gfp) gene. This parasite line constitutively expresses high levels of GFP during the complete parasite life cycle including liver, blood and mosquito stages. These fluorescent parasites can be used in combination with fluorescence activated cell sorting or live microscopy for a wide range of experimental applications.     

Endoprotease in Plasmodium yoelii nigeriensis

1. P. yoelli nigeriensis has an acid endoprotease (cathepsin D) and an endoarylamidase. 2. The acid endoprotease is specific towards haemogloblin. It is found in 2 molecular forms, of molecular weight 100,000 and 50,000. It is inhibited by hematin and pepsatin. 3. In mouse normal red blood cells we find an acid protease having physico-chemical properties similar to those of the enzyme present in P. yoelii nigeriensis extracts, except as regards the pHi. 4. In parasite extracts there exists an enzyme active on the synthesis substrate N-acetyl alanine 4 nitro anilide. The main properties of this enzyme have been determined. 5. This enzyme must be also involved in the mechanism of haemoglobin degradation.     

Oxidative phosphorylation and rotenone-insensitive malate- and NADH-quinone oxidoreductases in Plasmodium yoelii yoelii mitochondria in situ

Respiration, membrane potential, and oxidative phosphorylation of mitochondria of Plasmodium yoelii yoelii trophozoites were assayed in situ after permeabilization with digitonin. ADP induced an oligomycin-sensitive transition from resting to phosphorylating respiration in the presence of oxidizable substrates. A functional respiratory chain was demonstrated. In addition, the ability of the parasite to oxidize exogenous NADH, as well as the insensitivity of respiration to rotenone and its sensitivity to flavone, suggested the presence of an alternative NADH-quinone (NADH-Q) oxidoreductase. Rotenone-insensitive respiration and membrane potential generation in the presence of malate suggested the presence of a malate-quinone oxidoreductase. These results are in agreement with the presence of genes in P. yoelii encoding for proteins with homology to NADH-Q oxidoreductases of bacteria, plant, fungi, and protozoa and malate-quinone oxidoreductases of bacteria. The complete inhibition of respiration by antimycin A and cyanide excluded the presence of an alternative oxidase as described in other parasites. An uncoupling effect of fatty acids was partly reversed by bovine serum albumin and GTP but was unaffected by carboxyatractyloside. These results provide the first biochemical evidence of the presence of an alternative NADH-Q oxidoreductase and a malate-quinone oxidoreductase and confirm the operation of oxidative phosphorylation in malaria parasites.     

Scanning electron microscopic observations of the oocyst, sporoblast and sporozoite of Plasmodium yoelii yoelii

Scanning electron microscopy was used to study the surface characteristics of the oocyst, sporoblast and sporozoite of Plasmodium yoelii yoelii. Observations were made of the sporogonic stages of 6-12 day infections of the malaria parasite in Anopheles stephensi. Oocyst and sporoblast development were not synchronous. The surface of the undifferentiated (early stage) oocyst appeared smooth, whereas that of differentiated (late stage) oocysts were rough or wrinkled. The wall of the differentiated oocysts showed numerous micropores at higher magnification (x15,000-20,000) the biological significance of which is not known. Small, bud-like satellite bodies were seen attached to some oocysts. Various forms of different stages of the sporoblast were described. Sporozoite budding took place on the surface of the sporoblast body. The sporozoite was elongate, curved and with a blunt anterior end.     

约氏疟原虫配子体形成的形态学特征

应用六氰酸铁钾和锇酸双染色法的透射电镜技术,对红内期疟原虫配子体形成过程中的形态生物学特征进行识别。结果表明,配子体是一渐进发育的单核、惰性(团块样、虫体不活跃)虫体,其核旁有一个由扁平囊和泡状小体极性排列组成的细胞器——高尔基体(Golgi complex),虫体被膜下有嗜锇小体。其雌性配子体的结构特点是核质较致密、内质网丰富和被膜下嗜锇小体多;而雄性配子体却是核质较疏松、线粒体发达、内织网和嗜锇小体少。结论是红内期疟原虫配子体形成与裂体增殖过程中形态结构的明显差别,雌雄配子体在结构与发育上明显的差异,都具有特定的生物学与生态学意义。     

Ovary specific immune response during Plasmodium yoelii yoelii infection in malaria vector Anopheles stephensi (Diptera:Insecta)

Innate immune related polypeptides expression during three gonotrophic cycles in the ovaries of major disease vector mosquito A. stephensi has been analyzed following infection by malaria parasite, P. yoelii yoelii. Seventeen polypeptides were induced in the ovaries of various stages due to parasitic infection. Most of proteins were induced systemically during early stages of infection suggesting the possibility of immune related signalling process. The reduction in the quantity of protein contents in infected mosquitoes has been ascribed to the repression of seven polypeptides and in turn correlated with the fecundity reduction. The mechanism of these responses and their significance for malaria transmission and fecundity reduction are discussed.     

Artemisinin-Ginkgo biloba extract combination therapy for Plasmodium yoelii

Malaria remains a widespread life-threatening infectious disease, leading to an estimated 219 million cases and around 435,000 deaths. After an unprecedented success, the antimalarial progress is at a standstill. Therefore, new methods are urgently needed to decrease drug resistant and enhance antimalarial efficacy. According to the alteration of erythrocyte biomechanical properties and the immune evasion mechanism of parasites, drugs, which can improve blood circulation, can be chosen to combine with antimalarial drugs for malaria treatment. Ginkgo biloba extract (GBE), one of drug for vascular disease, was used to combine with artemisinin for Plasmodium yoelii therapy. Artemisinin-GBE combination therapy (AGCT) demonstrated remarkable antimalarial efficacy by decreasing infection rate, improving blood microcirculation and modulating immune system. Besides, the expression of invasion related genes, such as AMA1, MSP1 and Py01365, can be suppressed by AGCT, hindering invasion process of merozoites. This new antimalarial strategy, combining antimalarial drugs with drugs that improve blood circulation, may enhance the antimalarial efficacy and ameliorate restoration ability, proving a potential method for finding ideal compatible drugs to improve malaria therapy     

Purification and characterization of Plasmodium yoelii adenosine deaminase

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K_m values of 41 μM and 34 μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K_i value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.     

Plasmodium yoelii sporozoites infect CD36-deficient mice

Sinnis, P. and Febbraio, M. 2001. Plasmodium yoelii sporozoites infect CD36-deficient mice. Experimental Parasitology100, 12-16.     

Genetic control of immunity to Plasmodium yoelii sporozoites

Using a rodent malaria system, we have shown that protective immunity to the preerythrocytic stages of malaria is genetically controlled by MHC and non-MHC genes. Ten congenic strains of mice were immunized with irradiated sporozoites of Plasmodium yoelii. When challenged with viable sporozoites, only two strains had a high proportion of animals that did not develop blood stage infections. Immunity did not correlate with antisporozoite antibody levels. Two protective mechanisms exist determined by non-H-2 genes, and each mechanism is further controlled by H-2-linked Ir genes. On the BALB background only H-2d mice are protected, and protection is abolished by depleting CD8+ T cells. In contrast, on the B10 background only H-2q mice are strongly protected, and protection is not affected by CD8+ T cell depletion. If similar complex genetic regulation of immunity occurs in the human malarias, it will be a major hurdle for vaccine development.     

In vitro culture of Plasmodium yoelii blood stages

Lewis-Hughes P. H. and Howell M. J. 1984. In vitro culture of Plasmodium yoelii blood stages. International Journal for Parasitology14: 447–451. Plasmodium yoelii infected reticulocytes were cultured for 72 h at either 37 or 20°C in MEM (Eagle's modification) medium containing, in addition, glucose, para-aminobenzoic acid and 5% foetal calf serum, buffered at pH 7.3 with sodium bicarbonate/ HEPES and maintained under 10% CO₂ in air. Red blood cell numbers were more stable at 20°C than at 37°C. Culture at both temperatures resulted in an increase in parasitaemia of the reticulocyte population over the initial 36 h at 37°C and for at least 72 h at 20°C. The effects of different temperatures appeared to be related to the continued presence of target cells. Parasites were not detected after 72 h culture at 37°C, but persisted for up to 120 h at 20°C. Increasing parasitaemia at both temperatures was associated with changes in the numbers of some parasite development types. Early falls in schizont numbers were associated with an increase in the numbers of ring forms. Trophozoite numbers tended to remain constant throughout the culture period. Viability of parasites cultured for 36 h was confirmed by their infectivity to CBA mice. In addition, parasites progressively incorporated H³-leucine into TCA-precipitable material over the initial 36 h of culture.     

Plasmodium yoelii: induction of attenuated mutants by irradiation

When erythrocytic forms of Plasmodium yoelii nigeriensis, which is invariably fatal in mice, were exposed to X rays, the dose to reduce surviving parasites to one millionth was 100 gray (10 Krad). A suspension of 5 X 10(6) per ml of parasitized erythrocyte was irradiated at 100 gray, and 0.2 ml aliquots were inoculated into 22 mice. Eleven mice showed patent parasitemia, and in these the growth curves were less steep than that found in nonirradiated parasites. The infections of 8 mice of the 11 were self-resolving, and the attenuated feature of the parasites maintained following a limited number of blood passages. The parasites were slowly growing even in nude mice and cause self-resolving infections in intact mice. BALB/c mice immunized with the attenuated parasites were protected against subsequent challenge infections with the original virulent erythrocytic and sporogonic forms. These findings indicate that attenuated mutants of malaria parasites can be readily induced by this method.     

Susceptibility of species A, B, and C of Anopheles culicifacies complex to Plasmodium yoelii yoelii and Plasmodium vinckei petteri infections

The comparative susceptibilities of colonized species A, B, and C of Anopheles culicifacies complex and Anopheles stephensi were determined for 2 rodent malaria parasites Plasmodium vinckei petteri and Plasmodium yoelii yoelii. All the 3 members of the complex were found to support complete sporogony with varying success. Controls, A. stephensi, become readily infected, with >70% developing oocysts. Of the test groups, species A had the highest percentage of mosquitoes with oocysts (>25%) and sporozoites (>15%). Anopheles culicifacies species B were least susceptible; less than 10% had oocysts and sporozoites in the salivary glands. The results demonstrate that A. culicifacies species A is most susceptible and species B is least susceptible to infections with both the parasites.     

Plasmodium yoelii can ablate vaccine-induced long-term protection in mice

Malaria is a serious cause of morbidity and mortality for people living in endemic areas, but unlike many other infections, individuals exposed to the parasite do not rapidly become resistant to subsequent infections. High titers of Ab against the 19-kDa C-terminal fragment of the merozoite surface protein-1 can mediate complete protection in model systems; however, previous studies had not determined whether this vaccine generated long-term protection. In this study, we report that functional memory cells generated by merozoite surface protein-1, per se, do not offer any protection. This is because the parasite induces deletion of vaccine-specific memory B cells as well as long-lived plasma cells including those specific for bystander immune responses. Our study demonstrates a novel mechanism by which Plasmodium ablates immunological memory of vaccines, which would leave the host immuno-compromised.     

Nucleoside permeation in mouse erythrocytes infected with Plasmodium yoelii

In normal mouse erythrocytes, nucleoside permeation was almost completely blocked in the presence of binding site-saturating concentrations of nitrobenzylthioinosine, whereas permeation in erythrocytes infected with the malarial parasite, Plasmodium yoelii, was substantial under these conditions, suggesting the presence of a permeation mechanism of low sensitivity to nitrobenzylthioinosine in the infected cells. Binding sites for nitrobenzylthioinosine were more numerous on infected erythrocytes than on uninfected cells. When mice infected with P. yoelii were treated with combinations of tubercidin and nitrobenzylthioinosine 5'-monophosphate, progression of parasitemia was delayed and survival times were increased.     

The effects of host diet on Plasmodium yoelii nigeriensis

A comparative study carried out on infected mice to investigate the effect of host diet on Plasmodium yoelii nigeriensis showed that concentrations of blood protein, hemoglobin, and erythrocytes began to decrease in infected mice on day 2 after inoculation and reached the lowest levels on day 8. The greatest decrease was among mice fed on protein-rich mouse cubes, whereas the least decrease was among mice fed on cassava meal. Inflammation of the spleens and livers of infected mice also was noticed. Leucocyte numbers and parasitemia in infected mice reached their peaks on day 8. Again, the greatest prevalence of these abnormalities was apparent among mice whose diet contained the highest amount of protein in comparison to the other diets used in this study. The abnormalities decreased proportionately among the other groups of infected mice, corresponding to the protein content of their various diets. Mice fed on cassava meal, with the lowest content of protein, had the fewest abnormalities. Elevated body temperature, characteristic of severe malaria, and extensive liver damage were highest among mice with the greatest amount of protein in their diet. In view of these observations, it was surmised that during malaria, host diets high in protein heighten the severity of this disease.     

Plasmodium yoelii: axenic development of the parasite mosquito stages

Study of the parasite mosquito stages of Plasmodium and its use in the production of sporozoite vaccines against malaria has been hampered by the technical difficulties of in vitro development. Here, we show the complete axenic development of the parasite mosquito stages of Plasmodium yoelii. While we demonstrate that matrigel is not required for parasite development, soluble factors produced and secreted by Drosophila melanogaster S2 cells appear to be crucial for the ookinete to oocyst transition. Parasites cultured axenically are both morphologically and biologically similar to mosquito-derived ookinetes, oocysts, and sporozoites. Axenically derived sporozoites were capable of producing an infection in mice as determined by RT-PCR; however, the parasitemia was significantly much less than that produced by mosquito-derived sporozoites. Our cell free system for development of the mosquito stages of P. yoelii provides a simplified approach to generate sporozoites that may be for biological assays and genetic manipulations.     

The plasma membrane and mitochondrial membrane potentials of Plasmodium yoelii

1. The plasma membrane potential and the mitochondrial membrane potential of P. yoellii was examined by fluorescence microscopy using rhodamine 123 and by transmembrane distribution of tetraphenylphosphonium. 2. The mitochondrion of P. yoelii, free of gametocyte stage, maintained a high negative inside membrane potential. 3. Deprivation of glucose in incubation medium largely abolished the plasma membrane potential but not the mitochondrial membrane potential. 4. Studies with metabolic inhibitors showed that the mitochondrial membrane potential constituted a marginal portion as compared with the plasma membrane potential in intact infected erythrocytes.