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

Discovery and characterization of PKLR gene variants from malaria endemic regions

Malaria has subjected a strong selective pressure on the human genome. Several well-known erythrocyte protein variants, including hemoglobin S (causing sickle cell anemia) and functional variants in glucose-6-phosphate dehydrogenase (leading to deficiency in this enzyme) have been associated with protection against severe malarial disease and are distributed in regions which are historically and currently endemic for malaria. First identified in mice, then confirmed in vitro in human erythrocytes, pyruvate kinase deficiency appears to protect against Plasmodium infection. We hypothesize that functional variants within the pyruvate kinase erythrocyte gene (PKLR) will be identified within populations from malarial endemic regions, and that these variants will be associated with reduced severity of malarial phenotypes. The exons and intron/exon junctions of the PKLR gene were sequenced for a subset of participants within three longitudinal studies from areas of high and intermediate endemicity (Senegalese villages, Dielmo and Ndiop, and cluster of villages along the Thai-Myanmar border). Three non-synonymous mutations were discovered in the sequenced cohort: the L272V mutation within the Ndiop community, as well as the V269F and R41Q mutations from the Thai community. Genotyping of the R41Q mutation (not previously reported in the literature) within the Thai study population revealed a minor allele frequency of approximately 4.5%. While a biochemical assay of this mutation failed to indicate any effect on enzymatic activity, women heterozygous for R41Q exhibited a significant decrease in the number of P. falciparum attacks in comparison to non-variant homozygotes.La malaria a soumis le génome humain à une forte pression de sélection. En effets, plusieurs variantes de protéine érythrocytaires bien connues, tel que l’hémoglobine S (causant l’anémie falciforme) et des variantes fonctionnelles causant une déficience en glucose-6-phosphate déshydrogénase, ont été associés à une protection contre des formes sévères de malaria. Ces variantes protectrices sont retrouvées dans des régions qui sont historiquement et présentement endémiques pour la malaria. Initialement identifié chez la souris et ensuite confirmé in vitro dans des érythrocytes humains, le déficit en pyruvate kinase confère une protection contre les infections par le Plasmodium. Nous formulons donc l’hypothèse que des variantes fonctionnels du gène du pyruvate kinase des érythrocytes (PKLR) pourront être identifiés dans des populations provenant de ces régions endémiques et que ceux-ci seront associés à une réduction de la sévérité des symptômes de la malaria. Les exons et les jonctions exons/introns du gène PKLR ont été séquencés chez un sous-groupe de participants de trois études longitudinales effectuées dans des régions d’endémicité haute à intermédiaire; plus particulièrement dans les villages Sénégalais Dielmo et Ndiop, de même que dans un groupe de villages situés le long de la frontière entre la Thaïlande et le Myanmar. Dans la cohorte analysée, trois mutations non-synonymes furent identifiées : la mutation L272V dans la communauté Ndiop, de même que les mutations V269F et R41Q dans la communauté Thaïlandaise. Le génotypage de la mutation R41Q (non rapportée précédemment dans la littérature) dans la population Thaïlandaise nous indique une fréquence allélique mineure approximant 4,5%. Une évaluation biochimique de la mutation ne démontre pas d’effet sur l’activité enzymatique de la protéine. Néanmoins, les femmes hétérozygotes pour la mutation R41Q présentent une réduction significative des attaques de P. falciparum en comparaison avec les homozygotes non-variantes

Vaccinia Virus E3 Suppresses Expression of Diverse Cytokines through Inhibition of the PKR, NF-κB, and IRF3 Pathways▿

The vaccinia virus double-stranded RNA binding protein E3 has been demonstrated to inhibit the expression of cytokines, including beta interferon (IFN-β) and tumor necrosis factor alpha (TNF-α). However, few details regarding the molecular mechanisms of this inhibition have been described. Using real-time PCR arrays, we found that E3 suppressed the induction of a diverse array of cytokines representing members of the IFN, interleukin (IL), TNF, and transforming growth factor cytokine families. We discovered that the factor(s) responsible for the induction of IL-6, TNF-α, and inhibin beta A (INHBA) was associated with the early and late phases of virus infection. In contrast, the factor(s) which regulates IFN-β induction was associated with the late phase of replication. We have found that expression of these cytokines can be induced by transfection of cells with RNA isolated from vaccinia virus-infected cells. Moreover, we provide evidence that E3 antagonizes both PKR-dependent and PKR-independent pathways to regulate cytokine expression. PKR-dependent activation of p38 and NF-κB was required for vaccinia virus-induced INHBA expression, whereas induction of TNF-α required only PKR-dependent NF-κB activation. In contrast, induction of IL-6 and IFN-β was largely PKR independent. IL-6 induction is regulated by NF-κB, while IFN-β induction is mediated by IFN-β promoter stimulator 1 and IFN regulatory factor 3/NF-κB. Collectively, these results indicate that E3 suppresses distinct but interlinked host signaling pathways to inhibit the expression of a diverse array of cytokines

Mouse ENU Mutagenesis to Understand Immunity to Infection: Methods, Selected Examples, and Perspectives

International audienc

Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR) Variants in a Thai Population

Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival) of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria

Selective neuronal degeneration in MATR3 S85C knock-in mouse model of early-stage ALS

MATR3 is an ALS-linked gene. Here the authors describe a mutant MATR3 knockin mouse, which mimics some aspects of early-stage ALS

PKLR gene variants discovered in both the Senegalese and Thai community.

<p><b>(a)</b> Location of coding and non-coding variants within the <i>PKLR</i> gene. Encoded on chromosome 1q22 (155259084–155270792 base pairs), the erythrocyte isoform, transcribed from a tissue specific promoter has 11 exons (blocks). <b>(b)</b> Haplotypes and haplotype frequencies for sequenced population. Determined using the Haploview software, haplotypes were defined by the SNPs with a MAF ≥ 10% that defined the Senegalese and Thai populations. Seven of the SNPs were shared within the three study populations, while rs4620533 was specific for the Senegalese populations and rs3762272 was found only within the Thai population. Haplotype frequency distribution were calculated for the complete data set (ALL), or subdivided on country (Thailand, THA; Senegal, SEN) or village (Dielmo, DLM; Ndiop, NDP).</p

Effect of R41Q on PKLR protein stability.

<p><b>(a)</b> Transiently transfected HEK293T cells expressing either the wild-type (WT) or the R41Q mutant PKLR proteins were metabolically labelled with [³⁵S]-methionine followed by chase in label-free media for the indicated time period (in hours). Cell lysates were prepared and PKLR was immunoprecipitated, followed by gel electrophoresis. <b>(b)</b> The radiolabelled PKLR species were quantitated using ImageJ software, and averages from 2–3 independent experiments were calculated as a fraction of the maximum labelling observed at 0 hr.</p

Effect of human and mouse PKLR variants on malaria phenotypes.

<p><b>(a)</b> Association of the R41Q mutation and <i>Plasmodium falciparum</i> attacks in the genotyped Thai population. Residual <i>Plasmodium falciparum</i> attacks for heterozygous (R41Q) and homozygous wild-type (R41R) individuals. Both parametric and non-parametric tests indicate statistically significant decrease in attacks for heterozygous females only (ANOVA p = 0.0260 Kruskal-Wallis (KW) p = 0.0053). <b>(b)</b> Residual <i>Plasmodium vivax</i> attacks for heterozygous (R41Q) and homozygous wild-type (R41R) individuals. A statistically significant increase in attacks was noted in heterozygous females though only for the parametric test (ANOVA p = 0.0007), not the non-parametric test (KW p = 0.1789). <b>(c)</b> Time course of <i>P</i>. <i>chabaudi chabaudi</i> AS infection in PK-sufficient (CBA/CaHn-Btk^xid/J, circles), PK-deficient (CBA/Pk^slc, triangles) and F1 generation mice (squares). <b>(d)</b> Peak parasitemia (8dpi). Results are separated based on gender. Statistical significance was determined via unpaired, two-tailed t-tests, calculated by GraphPad (Prism) where “*” represents p<0.05. Statistically significant differences between PK-sufficient and F1 heterozygotes was noted for both genders (females: p = 0.0264, males: p = 0.0378).</p