Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite, Plasmodium vivax, and characterization of its properties

Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni2+-NTA resin giving a yield of 25-30 mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4 x 10(8) min(-1) M-1, 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors

Plasmodium berghei glycine cleavage system T-protein is non-essential for parasite survival in vertebrate and invertebrate hosts

T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N-5, N-10-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in alpha-lcetoacid dehydrogenase reactions. (C) 2014 Elsevier B.V. All rights reserved

Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections

Abstract Background The native gut microbiota of Anopheles mosquitoes is known to play a key role in the physiological function of its host. Interestingly, this microbiota can also influence the development of Plasmodium in its host mosquitoes. In recent years, much interest has been shown in the employment of gut symbionts derived from vectors in the control of vector-borne disease transmission. In this study, the midgut microbial diversity has been characterized among laboratory-reared adult Anopheles stephensi mosquitoes, from the colony created by rearing progeny of wild-caught mosquitoes (obtained from three different locations in southern India) for multiple generations, using 16S ribosomal RNA (rRNA) gene sequencing approach. Further, the influence of native midgut microbiota of mosquitoes on the development of rodent malaria parasite Plasmodium berghei in its host has been studied. Methods The microbial diversity associated with the midgut of An. stephensi mosquitoes was studied by sequencing V3 region of 16S ribosomal RNA (rRNA) gene. The influence of native midgut microbiota of An. stephensi mosquitoes on the susceptibility of the mosquitoes to rodent malaria parasite P. berghei was studied by comparing the intensity and prevalence of P. berghei infection among the antibiotic treated and untreated cohorts of mosquitoes. Results The analysis of bacterial diversity from the midguts of An. stephensi showed Proteobacteria as the most dominant population among the three laboratory-reared strains of An. stephensi studied. Major genera identified among these mosquito strains were Acinetobacter, Pseudomonas, Prevotella, Corynebacterium, Veillonella, and Bacillus. The mosquito infectivity studies carried out to determine the implication of total midgut microbiota on P. berghei infection showed that mosquitoes whose native microbiota cleared with antibiotics had increased susceptibility to P. berghei infection compared to the antibiotic untreated mosquitoes with its natural native microbiota. Conclusions The use of microbial symbiont to reduce the competence of vectors involved in disease transmission has gained much importance in recent years as an emerging alternative approach towards disease control. In this context, the present study was aimed to identify the midgut microbiota composition of An. stephensi, and its effect on the development of P. berghei. Interestingly, the analysis of midgut microbiota from An. stephensi revealed the presence of genus Veillonella in Anopheles species for the first time. Importantly, the study also revealed the negative influence of total midgut microbiota on the development of P. berghei in three laboratory strains of An. stephensi, emphasizing the importance of understanding the gut microbiota in malaria vectors, and its relationship with parasite development in designing strategies to control malaria transmission

Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

<div><p>Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme <i>de novo</i>, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of <i>Plasmodium berghei</i> (<i>Pb</i>). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out <i>in vitro</i> radiolabeling of heme in <i>Pb</i>-infected mouse reticulocytes and <i>Plasmodium falciparum</i>-infected human RBCs using [4-¹⁴C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the <i>de novo</i> pathway is absolutely essential for parasite development in the mosquito and liver stages. <i>Pb</i>KO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in <i>Pb</i>ALASKO parasites recovered when mosquitoes received an ALA supplement. <i>Pb</i>ALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.</p></div

Growth curves for intraerythrocytic stages of <i>P. berghei</i> WT and KO parasites in mice.

<p>Mice were injected intraperitoneally with 10⁵<i>P. berghei</i> infected-RBCs/reticulocytes and the parasite growth was routinely monitored as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003522#s4" target="_blank">Materials and Methods</a>. Multiple fields were used to quantify the parasite infected cells. The data provided represent the mean ± S.D. obtained from 6 animals.</p

Oocyst and sporozoite formation in <i>P.berghei</i>-infected (WT and KOs) mosquitoes.

<p>(A) Mercurochrome staining of oocysts in the midgut preparations. Arrows indicate oocysts and the magnified images of oocysts are provided in insets. Scale bar: 100 µm. (B) Sporozoites in the salivary glands. Magnified images of sporozoites are provided in insets. Scale bar: 50 µm. (C) Quantification of oocysts. P values for <i>Pb</i>ALASKO and <i>Pb</i>FCKO with respect to WT are <0.02. P value for <i>Pb</i>ALASKO(Mq^+ALA) with respect to <i>Pb</i>ALASKO is <0.01 and <i>Pb</i>FCKO(Mq^+Blood) with respect to <i>Pb</i>FCKO is >0.05. The data represent 90 mosquitoes from 3 different batches. (D) Quantification of sporozoites. P values for <i>Pb</i>ALASKO, <i>Pb</i>FCKO, <i>Pb</i>ALASKO(Mq^+ALA) and <i>Pb</i>FCKO(Mq^+Blood) with respect to WT are <0.01. The data represent 90 mosquitoes from 3 different batches. UI, uninfected; Mq, mosquitoes; <i>Pb</i>ALASKO(Mq^+ALA) and <i>Pb</i>FCKO(Mq^+Blood), <i>P. berghei</i> KO parasites from mosquitoes supplemented with ALA and blood feeding, respectively.</p

Ability of <i>P.berghei</i> sporozoites (WT and KOs) to infect mice with and without ALA supplement to the animals.

<p>Mosquitoes were allowed to feed on mice (30 mosquitoes/mouse) and parasitemia in blood and mortality of the animals were assessed. The data represent 9 mice each from three different batches. Mq, mosquito; Mi, mice; <i>Pb</i>ALASKO(Mq^+ALAMi^+ALA), <i>Pb</i>ALASKO supplemented with ALA in mosquitoes and mice; <i>Pb</i>ALASKO(Mq^+ALAMi^−ALA), <i>Pb</i>ALASKO supplemented with ALA in mosquitoes but not in mice; <i>Pb</i>FCKO(Mq^+Blood), <i>Pb</i>FCKO supplemented with blood feeding in mosquitoes.</p

<i>De novo</i> heme-biosynthetic pathway of <i>P. falciparum</i>.

<p>The enzymes are localized in three different cellular compartments - mitochondrion, apicoplast and cytosol. The transporters involved in the shuttling of intermediates are yet to be identified. Red bars represent the knockouts generated in <i>P. berghei</i> for the first (ALAS) and last (FC) enzymes of this pathway.</p

Model depicting the possible routes of heme transport from hemoglobin and biosynthetic heme in the intraerythrocytic stages of malaria parasite.

<p>H, heme; Hb, hemoglobin; FV, food vacuole; M, mitochondrion; Ap, apicoplast; Gly, glycine; SCoA, succinyl CoA; PBG, porphobilinogen; UROG, uroporphyrinogen III; COPROG, coproporphyrinogen III; PROTOG, protoporphyrinogen IX; PROTO, protoporphyrin IX.</p

Ookinete formation in the midgut of <i>P.berghei</i>-infected (WT and KOs) mosquitoes.

<p>(A) Quantification of ookinetes formed <i>in vitro</i> using gametocyte cultures. The data represent three independent experiments; P>0.05. (B) Ookinetes formed <i>in vitro</i> and stained with Giemsa reagent. Scale bar: 5 µm. (C) Quantification of ookinetes formed <i>in vivo</i>. (D) Ookinetes formed <i>in vivo</i> and stained with Giemsa reagent. Scale bar: 5 µm. The <i>in vivo</i> data are from 30 mosquitoes from 3 different batches; P>0.05.</p