The malaria parasite Plasmodium falciparum infects 5-10% of the world's population and kills two million people annually. Fatalities are thought to result in part from pathological reactions initiated by a malarial toxin. Glycosylphosphatidylinositol (GPI) originating from the parasite has the properties predicted of a toxin; however, a requirement for toxins in general and GPI in particular in malarial pathogenesis and fatality remains unproven. As anti-toxic vaccines can be highly effective public health tools, we sought to determine whether anti-GPI vaccination could prevent pathology and fatalities in the Plasmodium berghei/rodent model of severe malaria. The P. falciparum GPI glycan of the sequence NH(2)-CH(2)-CH(2)-PO(4)-(Man alpha 1-2)6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH(2)alpha 1-6myo-inositol-1,2-cyclic-phosphate was chemically synthesized, conjugated to carriers, and used to immunize mice. Recipients were substantially protected against malarial acidosis, pulmonary oedema, cerebral syndrome and fatality. Anti-GPI antibodies neutralized pro-inflammatory activity by P. falciparum in vitro. Thus, we show that GPI is a significant pro-inflammatory endotoxin of parasitic origin, and that several disease parameters in malarious mice are toxin-dependent. GPI may contribute to pathogenesis and fatalities in humans. Synthetic GPI is therefore a prototype carbohydrate anti-toxic vaccine against malaria.

Severe malarial anemia (SMA) is the most frequent life-threatening complication of malaria and may contribute to the majority of malarial deaths worldwide. To explore the mechanisms of pathogenesis, we developed a novel murine model of SMA in which parasitemias peaked around 1.0% of circulating red blood cells (RBCs) and yet hemoglobin levels fell to 47% to 56% of baseline. The severity of anemia was independent of the level of peak or cumulative parasitemia, but was linked kinetically to the duration of patent infection. In vivo biotinylation analysis of the circulating blood compartment revealed that anemia arose from accelerated RBC turnover. Labeled RBCs were reduced to 1% of circulating cells by 8 days after labeling, indicating that the entire blood compartment had been turned over in approximately one week. The survival rate of freshly transfused RBCs was also markedly reduced in SMA animals, but was not altered when RBCs from SMA donors were transferred into naive recipients, suggesting few functional modifications to target RBCs. Anemia was significantly alleviated by depletion of either phagocytic cells or CD4+ T lymphocytes. This study demonstrates that immunologic mechanisms may contribute to SMA by promoting the accelerated turnover of uninfected RBCs.

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BackgroundGenome sequencing of Leishmania species that give rise to a range of disease phenotypes in the host has revealed highly conserved gene content and synteny across the genus. Only a small number of genes are differentially distributed between the three species sequenced to date, L. major, L. infantum and L. braziliensis. It is not yet known how many of these genes are expressed in the disease-promoting intracellular amastigotes of these species or whether genes conserved between the species are differentially expressed in the host.Methods/Principal FindingsWe have used customised oligonucleotide microarrays to confirm that all of the differentially distributed genes identified by genome comparisons are expressed in intracellular amastigotes, with only a few of these subject to regulation at the RNA level. In the first large-scale study of gene expression in L. braziliensis, we show that only ∼9% of the genes analysed are regulated in their RNA expression during the L. braziliensis life cycle, a figure consistent with that observed in other Leishmania species. Comparing amastigote gene expression profiles between species confirms the proposal that Leishmania transcriptomes undergo little regulation but also identifies conserved genes that are regulated differently between species in the host. We have also investigated whether host immune competence influences parasite gene expression, by comparing RNA expression profiles in L. major amastigotes derived from either wild-type (BALB/c) or immunologically compromised (Rag2−/− γc −/−) mice. While parasite dissemination from the site of infection is enhanced in the Rag2−/− γc −/− genetic background, parasite RNA expression profiles are unperturbed.Conclusion/SignificanceThese findings support the hypothesis that Leishmania amastigotes are pre-adapted for intracellular survival and undergo little dynamic modulation of gene expression at the RNA level. Species-specific parasite factors contributing to virulence and pathogenicity in the host may be limited to the products of a small number of differentially distributed genes or the differential regulation of conserved genes, either of which are subject to translational and/or post-translational controls.

Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5′-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites.

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The natural killer complex (NKC) is a genetic region of highly linked genes encoding several receptors involved in the control of NK cell function. The NKC is highly polymorphic, and allelic variability of various NKC loci has been demonstrated in inbred mice. Making use of BALB.B6-Cmv1 r congenic mice, in which the NKC from disease-susceptible C57BL/6 mice has been introduced into the disease-resistant BALB/c background, we show here that during murine malaria infection, the NKC regulates a range of pathophysiological syndromes such as cerebral malaria, pulmonary edema, and severe anemia, which contribute to morbidity and mortality in human malaria. Parasitemia levels were not affected by the NKC genotype, indicating that control of malarial fatalities by the NKC cells does not operate through effects on parasite growth rate. Parasitespecific antibody responses and the proinflammatory gene transcription profile, as well as the TH1/TH2 balance, also appeared to be influenced by NKC genotype, providing evidence that this region, known to control innate immune responses via NK and/or NK T-cell activation, can also significantly regulate acquired immunity to infection. To date, NKC-encoded innate system receptors have been shown mainly to regulate viral infections. Our data provide evidence for critical NKC involvement in the broad immunological responses to a protozoan parasite.

Leishmaniasis is a neglected disease resulting in a global morbidity of 2,090 thousand Disability-Adjusted Life Years and a mortality rate of approximately 60,000 per year. Among the three clinical forms of leishmaniasis (cutaneous, mucosal, and visceral), visceral leishmaniasis (VL) accounts for the majority of mortality, as if left untreated VL is almost always fatal. Caused by infection with Leishmania donovani or L. infantum, VL represents a serious public health problem in endemic regions and is rapidly emerging as an opportunistic infection in HIV patients. To date, no vaccine exists for VL or any other form of leishmaniasis. In endemic areas, the majority of those infected do not develop clinical symptoms and past infection leads to robust immunity against reinfection. Thus the development of vaccine for Leishmania is a realistic public health goal, and this paper summarizes advances in vaccination strategies against VL.