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Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2?) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum.

ABSTRACT: In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2? factor, an eukaryotic initiation factor phosphorylated by eIF2? kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2? phosphorylation pattern as a molecular mechanism for its action.

SUBMITTER: Alves E

PROVIDER: S-EPMC4768138 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2α) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum.

Alves Eduardo E Maluf Fernando V FV Bueno Vânia B VB Guido Rafael V C RV Oliva Glaucius G Singh Maneesh M Scarpelli Pedro P Costa Fahyme F Sartorello Robson R Catalani Luiz H LH Brady Declan D Tewari Rita R Garcia Celia R S CR

Scientific reports 20160226

In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite developm ...[more]

PMID: 26915471

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Project description:BackgroundPlasmodium enolase is a target for the growth neutralizing antibodies. Interestingly, the three invasive stages i.e. sporozoites, merozoites, and ookinetes express this protein on their cell surface. Polyclonal anti-Plasmodium falciparum enolase (Pfeno) antibodies disrupt traversal of ookinete through mosquito mid-gut wall as well as have inhibitory effect on parasite growth at erythrocytic stage. In a recent study, it was observed that immunization with a unique epitope of parasite enolase (EWGWS) could confer partial protection against mouse malaria. Further validation is needed for the protective potential of this unique epitope in otherwise highly conserved enolase.MethodsIn order to investigate the efficacy of growth inhibitory potential of the epitope of P falciparum enolase, a monoclonal antibody specific to EWGWS is generated. In vitro parasite growth inhibition assays and passive immunization of Plasmodium yoelii (or Plasmodium berghei) infected mice were used to assess the parasite growth neutralizing activity of the antibody.ResultsScreening a panel of monoclonal antibodies raised against recombinant Pfeno that were specific to EWGWS resulted in isolation of H12E1. This antibody recognized only EWGWS epitope containing enolases. H12E1 strongly inhibited parasite growth in culture. This inhibition was strain transcending. Passive infusion of this antibody in P. yoelii or P. berghei infected mice showed significant reduction in parasitemia as compared to controls (p < 0.001). Surface Plasmon Resonance measurements indicated high affinity binding of H12E1 to P. falciparum enolase (KD ~ 7.6 × 10-9M).ConclusionsA monoclonal antibody directed against EWGWS epitope of Pfeno was shown to inhibit the growth of blood stage malarial parasites. This inhibition was species/strain transcending and is likely to arise due to blockade of enolase on the surface of merozoites, functionally implicating Pfeno in invasion related events. Presence of enolase on the cell surface of merozoites and ookinetes could potentially result in inhibition of host cell invasions at erythrocytic and transmission stages in the parasite life cycle. It is suggested that antibodies against EWGWS epitope have the potential to confer dual stage, species and strain transcending protection against malaria.

Project description:The sphingolipid pool is key regulator of vital cellular functions in Plasmodium falciparum a causative agent for deadly malaria. Erythrocytes, the host for asexual stage of Plasmodium, are major reservoir for Sphingosine-1-phosphate (S1P). Erythrocyte possesses Sphingosine kinase (SphK) that catalyzed its biosynthesis from sphingosine (Sph). Since, Plasmodium lacks SphK homologous protein it can be envisaged that it co-opts sphingolipids from both intraerythrocytic as well as extracellular pools for its growth and development. Herein, by sphingosine-NBD probing, we report that infected erythrocytes imports Sph from extracellular pool, which is converted to S1P and thereby taken by P. falciparum. Next, by targeting of the SphK through specific inhibitor N,N-Dimethylsphingosine DMS, we show a reduction in erythrocyte endogenous S1P pool and SphK-phosphorylation that led to inhibition in growth and development of ring stage P. falciparum. Owing to the role of S1P in erythrocyte glycolysis we analyzed uptake of NBD-Glucose and production of lactate in DMS treated and untreated plasmodium. DMS treatment led to decreased glycolysis in Plasmodium. Interestingly the host free Plasmodium did not show any effect on glycolysis with DMS treatment indicating its host-mediated effect. Further to understand the in-vivo anti-plasmodial effects of exogenous and endogenous erythrocyte S1P level, Sphingosine-1-phosphate lyase (S1PL) inhibitor (THI), S1P and SphK-1 inhibitor (DMS), were used in Plasmodium berghei ANKA (PbA) mice model. DMS treatment led to reduction of endogenous S1P conferred significant decrease in parasite load, whereas the plasma level S1P modulated by (THI) and exogenous S1P have no effect on growth of Plasmodium. This suggested erythrocyte endogenous S1P pool is important for Plasmodium growth whereas the plasma level S1P has no effect. Altogether, this study provides insight on cellular processes regulated by S1P in P. falciparum and highlights the novel mechanistically distinct molecular target i.e. SphK-1.

Dataset Information

Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2?) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum.

Publications

Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2α) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum.

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