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.

Histone H3K9 acetylation level modulates gene expression and may affect parasite growth in human malaria parasite Plasmodium falciparum

Three-dimensional positioning of the nuclear genome plays an important role in the epigenetic regulation of genes. Although nucleographic domain compartmentalization in the regulation of epigenetic state and gene expression is well established in higher organisms, it remains poorly understood in the pathogenic parasite Plasmodium falciparum. In the present study, we report that two histone tail modifications, H3K9Ac and H3K14Ac, are differentially distributed in the parasite nucleus. We find colocalization of active gene promoters such as Tu1 (tubulin-1 expressed in the asexual stages) with H3K9Ac marks at the nuclear periphery. By contrast, asexual stage inactive gene promoters such as Pfg27 (gametocyte marker) and Pfs28 (ookinete marker) occupy H3K9Ac devoid zones at the nuclear periphery. The histone H3K9 is predominantly acetylated by the PCAF/GCN5 class of lysine acetyltransferases, which is well characterized in the parasite. Interestingly, embelin, a specific inhibitor of PCAF/GCN5 family histone acetyltransferase, selectively decreases total H3K9Ac acetylation levels (but not H3K14Ac levels) around the var gene promoters, leading to the downregulation of var gene expression, suggesting interplay among histone acetylation status, as well as subnuclear compartmentalization of different genes and their activation in the parasites. Finally, we found that embelin inhibited parasitic growth at the low micromolar range, raising the possibility of using histone acetyltransferases as a target for antimalarial therapy.

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.

Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.

Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections

The proteins of Plasmodium, the malaria parasite, are strikingly rich in asparagine. Plasmodium depends primarily on host haemoglobin degradation for amino acids and has a rudimentary pathway for amino acid biosynthesis, but retains a gene encoding asparagine synthetase (AS). Here we show that deletion of AS in Plasmodium berghei (Pb) delays the asexual-and liver-stage development with substantial reduction in the formation of ookinetes, oocysts and sporozoites in mosquitoes. In the absence of asparagine synthesis, extracellular asparagine supports suboptimal survival of PbAS knockout (KO) parasites. Depletion of blood asparagine levels by treating PbASKO-infected mice with asparaginase completely prevents the development of liver stages, exflagellation of male gametocytes and the subsequent formation of sexual stages. In vivo supplementation of asparagine in mice restores the exflagellation of PbASKO parasites. Thus, the parasite life cycle has an absolute requirement for asparagine, which we propose could be targeted to prevent malaria transmission and liver infections.

Mass spectrometric analysis of dimer-disrupting mutations in Plasmodium triosephosphate isomerase

Electrospray ionization mass spectrometry (ESI MS) under nanospray conditions has been used to examine the effects of mutation at two key dimer interface residues, Gln (Q) 64 and Thr (T) 75, in Plasmodium falciparum triosephosphate isomerase. Both residues participate in an intricate network of intra- and intersubunit hydrogen bonds. The gas phase distributions of dimeric and monomeric protein species have been examined for the wild type enzyme (TWT) and three mutants, Q64N, Q64E, and 175S, under a wide range of collision energies (40-160 eV). The results established the order of dimer stability as TWT > T75S > Q64E similar to Q64N. The mutational effects on dimer stability are in good agreement with the previously reported estimates, based on the concentration dependence of enzyme activity. Additional experiments in solution, using inhibition of activity by a synthetic dimer interface peptide, further support the broad agreement between gas phase and solution studies. (C) 2016 Elsevier Inc. All rights reserved.

WHO guidelines for antimicrobial treatment in children admitted to hospital in an area of intense Plasmodium falciparum transmission: prospective study.

OBJECTIVES: To assess the performance of WHO's "Guidelines for care at the first-referral level in developing countries" in an area of intense malaria transmission and identify bacterial infections in children with and without malaria. DESIGN: Prospective study. SETTING: District hospital in Muheza, northeast Tanzania. PARTICIPANTS: Children aged 2 months to 13 years admitted to hospital for febrile illness. MAIN OUTCOME MEASURES: Sensitivity and specificity of WHO guidelines in diagnosing invasive bacterial disease; susceptibility of isolated organisms to recommended antimicrobials. RESULTS: Over one year, 3639 children were enrolled and 184 (5.1%) died; 2195 (60.3%) were blood slide positive for Plasmodium falciparum, 341 (9.4%) had invasive bacterial disease, and 142 (3.9%) were seropositive for HIV. The prevalence of invasive bacterial disease was lower in slide positive children (100/2195, 4.6%) than in slide negative children (241/1444, 16.7%). Non-typhi Salmonella was the most frequently isolated organism (52/100 (52%) of organisms in slide positive children and 108/241 (45%) in slide negative children). Mortality among children with invasive bacterial disease was significantly higher (58/341, 17%) than in children without invasive bacterial disease (126/3298, 3.8%) (P<0.001), and this was true regardless of the presence of P falciparum parasitaemia. The sensitivity and specificity of WHO criteria in identifying invasive bacterial disease in slide positive children were 60.0% (95% confidence interval 58.0% to 62.1%) and 53.5% (51.4% to 55.6%), compared with 70.5% (68.2% to 72.9%) and 48.1% (45.6% to 50.7%) in slide negative children. In children with WHO criteria for invasive bacterial disease, only 99/211(47%) of isolated organisms were susceptible to the first recommended antimicrobial agent. CONCLUSIONS: In an area exposed to high transmission of malaria, current WHO guidelines failed to identify almost a third of children with invasive bacterial disease, and more than half of the organisms isolated were not susceptible to currently recommended antimicrobials. Improved diagnosis and treatment of invasive bacterial disease are needed to reduce childhood mortality.

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in > 2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH2] Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.

Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the x-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the x-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA- M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.

The Plasmodium falciparum Malaria M1 Alanyl Aminopeptidase (PfA-M1): Insights of Catalytic Mechanism and Function from MD Simulations

Malaria caused by several species of Plasmodium is major parasitic disease of humans, causing 1-3 million deaths worldwide annually. The widespread resistance of the human parasite to current drug therapies is of major concern making the identification of new drug targets urgent. While the parasite grows and multiplies inside the host erythrocyte it degrades the host cell hemoglobin and utilizes the released amino acids to synthesize its own proteins. The P. falciparum malarial M1 alanyl-aminopeptidase (PfA-M1) is an enzyme involved in the terminal stages of hemoglobin digestion and the generation of an amino acid pool within the parasite. The enzyme has been validated as a potential drug target since inhibitors of the enzyme block parasite growth in vitro and in vivo. In order to gain further understanding of this enzyme, molecular dynamics simulations using data from a recent crystal structure of PfA-M1 were performed. The results elucidate the pentahedral coordination of the catalytic Zn in these metallo-proteases and provide new insights into the roles of this cation and important active site residues in ligand binding and in the hydrolysis of the peptide bond. Based on the data, we propose a two-step catalytic mechanism, in which the conformation of the active site is altered between the Michaelis complex and the transition state. In addition, the simulations identify global changes in the protein in which conformational transitions in the catalytic domain are transmitted at the opening of the N-terminal 8 angstrom-long channel and at the opening of the 30 angstrom-long C-terminal internal chamber that facilitates entry of peptides to the active site and exit of released amino acids. The possible implications of these global changes with regard to enzyme function are discussed.

The M17 leucine aminopeptidase of the malaria parasite Plasmodium falciparum:importance of active site metal ions in the binding of substrates and inhibitors

The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays a role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal ion at site 1 is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn(2+) > Mn(2+) > Co(2+) > Mg(2+). While it is likely that native PfLAP contains a Zn(2+) in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.

Seasonality of Plasmodium falciparum transmission: a systematic review

Background: Although Plasmodium falciparum transmission frequently exhibits seasonal patterns, the drivers of malaria seasonality are often unclear. Given the massive variation in the landscape upon which transmission acts, intra-annual fluctuations are likely influenced by different factors in different settings. Further, the presence of potentially substantial inter-annual variation can mask seasonal patterns; it may be that a location has "strongly seasonal" transmission and yet no single season ever matches the mean, or synoptic, curve. Accurate accounting of seasonality can inform efficient malaria control and treatment strategies. In spite of the demonstrable importance of accurately capturing the seasonality of malaria, data required to describe these patterns is not universally accessible and as such localized and regional efforts at quantifying malaria seasonality are disjointed and not easily generalized. 

Methods: The purpose of this review was to audit the literature on seasonality of P. falciparum and quantitatively summarize the collective findings. Six search terms were selected to systematically compile a list of papers relevant to the seasonality of P. falciparum transmission, and a questionnaire was developed to catalogue the manuscripts. 

Results and discussion: 152 manuscripts were identified as relating to the seasonality of malaria transmission, deaths due to malaria or the population dynamics of mosquito vectors of malaria. Among these, there were 126 statistical analyses and 31 mechanistic analyses (some manuscripts did both). 

Discussion: Identified relationships between temporal patterns in malaria and climatological drivers of malaria varied greatly across the globe, with different drivers appearing important in different locations. Although commonly studied drivers of malaria such as temperature and rainfall were often found to significantly influence transmission, the lags between a weather event and a resulting change in malaria transmission also varied greatly by location. 

Conclusions: The contradicting results of studies using similar data and modelling approaches from similar locations as well as the confounding nature of climatological covariates underlines the importance of a multi-faceted modelling approach that attempts to capture seasonal patterns at both small and large spatial scales.

Persistent endothelial activation and inflammation after Plasmodium falciparum Infection in Malawian children

Endothelial dysregulation is central to the pathogenesis of acute Plasmodium falciparum infection. It has been assumed that this dysregulation resolves rapidly after treatment, but this return to normality has been neither demonstrated nor quantified. We therefore measured a panel of plasma endothelial markers acutely and in convalescence in Malawian children with uncomplicated or cerebral malaria. Evidence of persistent endothelial activation and inflammation, indicated by increased plasma levels of soluble intracellular adhesion molecule 1, angiopoetin 2, and C-reactive protein, were observed at 1 month follow-up visits. These vascular changes may represent a previously unrecognized contributor to ongoing malaria-associated morbidity and mortality.

Role of plasmodium translationally repressed gene products in malaria transmission

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2015