Rocaglates as dual-targeting agents for experimental cerebral malaria

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.We thank Susan Gauthier, Genevieve Perreault, and Patrick Senechal for technical assistance. This work was supported by a research grant (to P.G.) from the Canadian Institutes of Health Research (CIHR) (Foundation Grant). J.P. and P.G. are supported by a James McGill Professorship salary award. D.L. is supported by fellowships from the Fonds de recherche sante Quebec, the CIHR Neuroinflammation training program. J.P. is supported by CIHR Research Grant FDN-148366. M.S. is supported by a CIHR Foundation grant. J.A.P. is supported by NIH Grant R35 GM118173. Work at the Boston University Center for Molecular Discovery is supported by Grant R24 GM111625. K.C.K. was supported by a CIHR Foundation Grant and the Canada Research Chair program. (Canadian Institutes of Health Research (CIHR); James McGill Professorship salary award; Fonds de recherche sante Quebec; CIHR Neuroinflammation training program; FDN-148366 - CIHR Research Grant; CIHR Foundation grant; R35 GM118173 - NIH; Canada Research Chair program; R24 GM111625

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

Leishmania promastigotes: building a safe niche within macrophages

Upon their internalization by macrophages, Leishmania promastigotes inhibit phagolysosome biogenesis. The main factor responsible for this inhibition is the promastigote surface glycolipid lipophosphoglycan (LPG). This glycolipid has a profound impact on the phagosome, causing periphagosomal accumulation of F-actin and disruption of phagosomal lipid microdomains. Functionally, this LPG-mediated inhibition of phagosome maturation is characterized by an impaired assembly of the NADPH oxidase and the exclusion of the vesicular proton-ATPase from phagosomes. In this chapter, we review the current knowledge concerning the nature of the intra-macrophage compartment in which Leishmania donovani promastigotes establish infection. We also describe how LPG enables this parasite to remodel the parasitophorous vacuole

Leishmania dices away cholesterol for survival.

International audienceHost lipid alterations are centrally involved in Leishmania donovani infection, and infected patients exhibit hypocholesterolemia. In this issue of Cell Host & Microbe, Ghosh et al. (2013) show that the metalloprotease GP63 released by L. donovani in the liver cleaves DICER1, inhibiting miR-122 maturation, which regulates cholesterol metabolism. These events decrease serum cholesterol and promote parasite growth

Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63.

The protozoan Leishmania parasitizes macrophages and evades the microbicidal consequences of phagocytosis through the inhibition of phagolysosome biogenesis. In this study, we investigated the impact of this parasite on LC3-associated phagocytosis, a non-canonical autophagic process that enhances phagosome maturation and functions. We show that whereas internalization of L. major promastigotes by macrophages promoted LC3 lipidation, recruitment of LC3 to phagosomes was inhibited through the action of the parasite surface metalloprotease GP63. Reactive oxygen species generated by the NOX2 NADPH oxidase are necessary for LC3-associated phagocytosis. We found that L. major promastigotes prevented, in a GP63-dependent manner, the recruitment of NOX2 to phagosomes through a mechanism that does not involve NOX2 cleavage. Moreover, we found that the SNARE protein VAMP8, which regulates phagosomal assembly of the NADPH oxidase NOX2, was down-modulated by GP63. In the absence of VAMP8, recruitment of LC3 to phagosomes containing GP63-deficient parasites was inhibited, indicating that VAMP8 is involved in the phagosomal recruitment of LC3. These findings reveal a role for VAMP8 in LC3-associated phagocytosis and highlight a novel mechanism exploited by L. major promastigotes to interfere with the host antimicrobial machinery

Absence of VAMP8 does not affect intracellular survival of <i>L</i>. <i>major</i> in macrophages.

<p>(A) Quantification of opsonized <i>L</i>. <i>major</i> GLC94 promastigotes internalization and replication in WT or <i>Vamp8</i>^-/- C57BL/6 x 129 BMM, assessed by Giemsa staining at 1, 24, 48, 72, and 96 h after infection. Data are presented as the mean ± SEM of values from three independent experiments. *p<0.05. (B) Images of Giemsa-stained WT or <i>Vamp8</i>^-/- BMM infected with opsonized <i>L</i>. <i>major</i> GLC94 for 2, 24, and 72h.</p

Genotype–Phenotype Correlations in Iranian Myotonic Dystrophy Type I Patients

Objectives: Myotonic Dystrophy type I (DM1) is a dominantly inherited disorder with a multisystemic pattern affecting skeletal muscle, heart, eye, endocrine and central nervous system. DM1 is associated with the expansion and instability of CTG repeat in the 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene located on chromosome 19q13.3. The aim of this study was to determine clinical and genetic characteristic of DM1 in Iranian patients. Genotype-phenotype correlation was also assessed in a small group of studied patients. Methods: Twenty six DM1 patients belonging to seventeen families were analyzed. Clinical assessment was based on the muscular disability rating scale (MDRS) and a sum of symptoms score (SSS). Molecular analysis (PCR and Southern blot) was used to clarify uncertain clinical diagnosis and in order to confirm clinical findings. Results: There was an inverse and significant correlation between age of onset&nbsp; and expanded allele&nbsp; length (P=0.026, tau-b=-0.360) based on Kendall's tau-b correlation coefficient, while there was no significant correlation between age of onset and severity of the clinical symptoms (P<0.05). Also no significant correlation was observed between the two severity scales of the disease (MDRS and SSS) and expanded allele length (P<0.05). Expanded allele length was correlated with hypogonadism (P=0.007) and cognitive impairment (P=0.034). Discussion: There was no correlation between cataract and endocrine dysfunction with the expansion size in DM1 patients. Generally it seems there is discordant correlation between clinical symptoms and expanded allele length

<i>L</i>. <i>major</i> GP63 cleaves VAMP8 and prevents its recruitment to phagosomes.

<p>(A) VAMP8 cleavage in C57BL/6 x 129 BMM infected for 2 h with opsonized WT, Δ<i>gp63</i> or Δ<i>gp63</i>+<i>gp63 L</i>. <i>major</i> NIH S promastigotes was assessed by immunoblot analysis. VAMP8 band intensities were measured by spot densitometry, normalized to the β-actin loading control and compared to the uninfected control (Ctl) cells. (B) Confocal microscopy images of C57BL/6 x 129 BMM fed zymosan particles or infected with opsonized WT, Δ<i>gp63</i>, or Δ<i>gp63</i>+<i>gp63 L</i>. <i>major</i> NIH S promastigotes for 1 h. VAMP8 is in green; nuclei are in blue. Asterisks indicate phagosomes containing zymosan particles, white arrows indicate parasite nuclei, and red-filled arrowheads point to VAMP8 recruitment. Scale bar, 5 μM. (C) Quantification of VAMP8-positive phagosomes at 1 and 2 h after infection. Data are presented as the mean ± SEM of values from triplicate samples of an experiment representative of more than three others. *p<0.05, **p<0.01.</p

<i>L</i>. <i>major</i> GP63 prevents LC3 recruitment to phagosomes.

<p>(A, C) Confocal microscopy images of BALB/c BMM infected for 1 h with opsonized WT, Δ<i>gp63</i>, or Δ<i>gp63</i>+<i>gp63 L</i>. <i>major</i> NIH S promastigotes. LC3 (A) and Beclin (C) are in green; nuclei are in blue. White arrows indicate parasite nuclei; red-filled arrowheads point to LC3 recruitment (A). Scale bar, 5 μM. (B) Quantification of LC3-positive phagosomes at 1 h after infection. Data are presented as the mean ± standard error of the mean (SEM) of values from three independent experiments. **p<0.01.</p