Complex genetic control of susceptibility to malaria: positional cloning of the Char9 locus

Mouse strains AcB55 and AcB61 are resistant to malaria by virtue of a mutation in erythrocyte pyruvate kinase (PklrI90N). Linkage analysis in [AcB55 × A/J] F2 mice detected a second locus (Char9; logarithm of odds = 4.74) that regulates the blood-stage replication of Plasmodium chabaudi AS independently of Pklr. We characterized the 77 genes of the Char9 locus for tissue-specific expression, strain-specific alterations in gene expression, and polymorphic variants that are possibly associated with differential susceptibility. We identified Vnn1/Vnn3 as the likely candidates responsible for Char9. Vnn3/Vnn1 map within a conserved haplotype block and show expression levels that are strictly cis-regulated by this haplotype. The absence of Vnn messenger RNA expression and lack of pantetheinase protein activity in tissues are associated with susceptibility to malaria and are linked to a complex rearrangement in the Vnn3 promoter region. The A/J strain also carries a unique nonsense mutation that leads to a truncated protein. Vanin genes code for a pantetheinase involved in the production of cysteamine, a key regulator of host responses to inflammatory stimuli. Administration of cystamine in vivo partially corrects susceptibility to malaria in A/J mice, as measured by reduced blood parasitemia and decreased mortality. These studies suggest that pantetheinase is critical for the host response to malaria

Proteomic Analysis of Excretory-Secretory Products of Heligmosomoides polygyrus Assessed with Next-Generation Sequencing Transcriptomic Information

The murine parasite Heligmosomoides polygyrus is a convenient experimental model to study immune responses and pathology associated with gastrointestinal nematode infections. The excretory-secretory products (ESP) produced by this parasite have potent immunomodulatory activity, but the protein(s) responsible has not been defined. Identification of the protein composition of ESP derived from H. polygyrus and other relevant nematode species has been hampered by the lack of genomic sequence information required for proteomic analysis based on database searches. To overcome this, a transcriptome next generation sequencing (RNA-seq) de novo assembly containing 33,641 transcripts was generated, annotated, and used to interrogate mass spectrometry (MS) data derived from 1D-SDS PAGE and LC-MS/MS analysis of ESP. Using the database generated from the 6 open reading frames deduced from the RNA-seq assembly and conventional identification programs, 209 proteins were identified in ESP including homologues of vitellogenins, retinol- and fatty acid-binding proteins, globins, and the allergen V5/Tpx-1-related family of proteins. Several potential immunomodulators, such as macrophage migration inhibitory factor, cysteine protease inhibitors, galectins, C-type lectins, peroxiredoxin, and glutathione S-transferase, were also identified. Comparative analysis of protein annotations based on the RNA-seq assembly and proteomics revealed processes and proteins that may contribute to the functional specialization of ESP, including proteins involved in signalling pathways and in nutrient transport and/or uptake. Together, these findings provide important information that will help to illuminate molecular, biochemical, and in particular immunomodulatory aspects of host-H. polygyrus biology. In addition, the methods and analyses presented here are applicable to study biochemical and molecular aspects of the host-parasite relationship in species for which sequence information is not available

Bisphosphoglycerate mutase deficiency protects against cerebral malaria and severe malaria-induced anemia

The replication cycle and pathogenesis of the Plasmodium malarial parasite involves rapid expansion in red blood cells (RBCs), and variants of certain RBC-specific proteins protect against malaria in humans. In RBCs, bisphosphoglycerate mutase (BPGM) acts as a key allosteric regulator of hemoglobin/oxyhemoglobin. We demonstrate here that a loss-of-function mutation in the murine Bpgm (BpgmL166P) gene confers protection against both Plasmodium-induced cerebral malaria and blood-stage malaria. The malaria protection seen in BpgmL166P mutant mice is associated with reduced blood parasitemia levels, milder clinical symptoms, and increased survival. The protective effect of BpgmL166P involves a dual mechanism that enhances the host’s stress erythroid response to Plasmodium-driven RBC loss and simultaneously alters the intracellular milieu of the RBCs, including increased oxyhemoglobin and reduced energy metabolism, reducing Plasmodium maturation, and replication. Overall, our study highlights the importance of BPGM as a regulator of hemoglobin/oxyhemoglobin in malaria pathogenesis and suggests a new potential malaria therapeutic target

STAT6-mediated suppression of erythropoiesis in an experimental model of malarial anemia

The findings of this study indicate a STAT6-dependent mechanism involving interleukin-4 in mediating erythropoietic suppression during blood-stage malaria. See related perspective article on page 157

The mouse Char10 locus regulates severity of pyruvate kinase deficiency and susceptibility to malaria

Pyruvate kinase (PKLR) deficiency protects mice and humans against blood-stage malaria.Although mouse strain AcB62 carries a malaria-protective PklrI90N genetic mutation, it is phenotypicallysusceptible to blood stage malaria induced by infection with Plasmodium chabaudiAS, suggesting a genetic modifier of the PklrI90N protective effect. Linkage analysis in aF2 cross between AcB62 (PklrI90N) and another PK deficient strain CBA/Pk (PklrG338D) mapsthis modifier (designated Char10) to chromosome 9 (LOD = 10.8, 95% Bayesian CI = 50.7-75Mb). To study the mechanistic basis of the Char10 effect, we generated an incipient congenicline (Char10C) that harbors the Char10 chromosome 9 segment from AcB62 fixed onthe genetic background of CBA/Pk. The Char10 effect is shown to be highly penetrant as theChar10C line recapitulates the AcB62 phenotype, displaying high parasitemia following P.chabaudi infection, compared to CBA/Pk. Char10C mice also display a reduction in anemiaphenotypes associated with the PklrG338D mutation including decreased splenomegaly,decreased circulating reticulocytes, increased density of mature erythrocytes, increasedhematocrit, as well as decreased iron overload in kidney and liver and decreased serum iron.Erythroid lineage analyses indicate that the number of total TER119+ cells as well as thenumbers of the different CD71+/CD44+ erythroblast sub-populations were all found to belower in Char10C spleen compared to CBA/Pk. [...