ResMAP-a saturation mutagenesis platform enabling parallel profiling of target-specific resistance-conferring mutations in Plasmodium.

New and improved drugs are required for the treatment and ultimate eradication of malaria. The efficacy of front-line therapies is now threatened by emerging drug resistance; thus, new tools to support the development of drugs with a lower propensity for resistance are needed. Here, we describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium drug targets. Proof-of-concept studies focused on interrogating the antimalarial drug target, Plasmodium falciparum lysyl tRNA synthetase (PfKRS). Saturation mutagenesis was used to construct a plasmid library encoding all conceivable mutations within a 20-residue span at the base of the PfKRS ATP-binding pocket. The superior transfection efficiency of Plasmodium knowlesi was exploited to generate a high coverage parasite library expressing PfKRS bearing all possible amino acid changes within this region of the enzyme. The selection of the library with PfKRS inhibitors, cladosporin and DDD01510706, successfully identified multiple resistance-conferring substitutions. Genetic validation of a subset of these mutations confirmed their direct role in resistance, with computational modeling used to dissect the structural basis of resistance. The application of ResMAP to inform the development of resistance-resilient antimalarials of the future is discussed. IMPORTANCE: An increase in treatment failures for malaria highlights an urgent need for new tools to understand and minimize the spread of drug resistance. We describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium spp, the causative agent of malaria. Saturation mutagenesis was used to generate a mutation library containing all conceivable mutations for a region of the antimalarial-binding site of a promising drug target, Plasmodium falciparum lysyl tRNA synthetase (PfKRS). Screening of this high-coverage library with characterized PfKRS inhibitors revealed multiple resistance-conferring substitutions including several clinically relevant mutations. Genetic validation of these mutations confirmed resistance of up to 100-fold and computational modeling dissected their role in drug resistance. We discuss potential applications of this data including the potential to design compounds that can bypass the most serious resistance mutations and future resistance surveillance.

Cloning, Expression, Purification, and Characterization of Lactate Dehydrogenase from Plasmodium knowlesi: A Zoonotic Malaria Parasite.

Plasmodium knowlesi is the only Plasmodium that causes zoonotic disease among the Plasmodium that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by Plasmodium spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of P. knowlesi LDH (PkLDH) using a bacterial expression system for studying malaria caused by P. knowlesi. Recombinant pET-21a(+)-PkLDH plasmid was constructed by inserting the PkLDH gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing Escherichia coli Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by P. knowlesi.

Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from Plasmodium knowlesi for the Development of Anti-Malarial Drugs.

Plasmodium lactate dehydrogenase (pLH) is one of the enzymes in glycolysis with potential target for chemotherapy. This study aimed to clone, overexpress and characterize soluble recombinant lactate dehydrogenase from Plasmodium knowlesi in a bacterial system. Synthetic P. knowlesi lactate dehydrogenase (Pk-LDH) gene was cloned into pET21a expression vector, transformed into Escherichia coli strain BL21 (DE3) expression system and then incubated for 18 h, 20 °C with the presence of 0.5 mM isopropyl ß-d-thiogalactoside in Terrific broth supplemented with Magnesium sulfate, followed by protein purifications using Immobilized Metal Ion Affinity Chromatography and size exclusion chromatography (SEC). Enzymatic assay was conducted to determine the activity of the enzyme. SDS-PAGE analysis revealed that protein of 34 kDa size was present in the soluble fraction. In SEC, a single peak corresponding to the size of Pk-LDH protein was observed, indicating that the protein has been successfully purified. From MALDI-TOF analysis findings, a peptide score of 282 was established, which is significant for lactate dehydrogenase from P. knowlesi revealed via MASCOT analysis. Secondary structure analysis of CD spectra indicated 79.4% α helix and 1.37% ß strand structure. Specific activity of recombinant Pk-LDH was found to be 475.6 U/mg, confirming the presence of active protein. Soluble Pk-LDH that is biologically active was produced, which can be used further in other malaria studies.

Diagnostic Characteristics of Lactate Dehydrogenase on a Multiplex Assay for Malaria Detection Including the Zoonotic Parasite Plasmodium knowlesi.

Plasmodium lactate dehydrogenase (pLDH) is a common target in malaria rapid diagnostic tests (RDTs). These commercial antibody capture assays target either Plasmodium falciparum-specific pLDH (PfLDH), P. vivax-specific pLDH (PvLDH), or a conserved epitope in all human malaria pLDH (PanLDH). However, there are no assays specifically targeting P. ovale, P. malariae or zoonotic parasites such as P. knowlesi and P. cynomolgi. A malaria multiplex array, carrying the specific antibody spots for PfLDH, PvLDH, and PanLDH has been previously developed. This study aimed to assess potential cross-reactivity between pLDH from various Plasmodium species and this array. We tested recombinant pLDH proteins, clinical samples for P. vivax, P. falciparum, P. ovale curtisi, and P. malariae; and in vitro cultured P. knowlesi and P. cynomolgi. P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) cross-reacted with the PfLDH and PanLDH spots. Plasmodium Knowlesi-specific pLDH (PkLDH) and P. cynomolgi-specific pLDH (PcLDH) cross-reacted with the PvLDH spot, but only PkLDH was recognized by the PanLDH spot. Plasmodium ovale and P. malariae can be differentiated from P. falciparum by the concentration ratios of PanLDH/PfLDH, which had mean (range) values of 4.56 (4.07-5.16) and 4.56 (3.43-6.54), respectively, whereas P. falciparum had a lower ratio of 1.12 (0.56-2.61). Plasmodium knowlesi had a similar PanLDH/PvLDH ratio value, with P. vivax having a mean value of 2.24 (1.37-2.79). The cross-reactivity pattern of pLDH can be a useful predictor to differentiate certain Plasmodium species. Cross-reactivity of the pLDH bands in RDTs requires further investigation.

High-throughput screening for phosphatidylserine decarboxylase inhibitors using a distyrylbenzene-bis-aldehyde (DSB-3)-based fluorescence assay.

Phosphatidylserine decarboxylases (PSDs) catalyze the decarboxylation of phosphatidylserine to generate phosphatidylethanolamine, a critical step in phospholipid metabolism in both prokaryotes and eukaryotes. Most PSDs are membrane-bound, and classical radioisotope-based assays for determining their activity in vitro are not suitable for high-throughput drug screening. The finding that the PkPSD from Plasmodium knowlesi can be purified in a soluble and active form and the recent development of a fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay to measure PSD activity in vitro have laid the groundwork for screening chemical libraries for PSD inhibitors. Using this assay, here we conducted a high-throughput screen of a structurally diverse 130,858-compound library against PkPSD. Further characterization of the hits identified in this screening yielded five PkPSD inhibitors with IC50 values ranging from 3.1 to 42.3 µm Lead compounds were evaluated against the pathogenic yeast Candida albicans in the absence or presence of exogenous ethanolamine, and YU253467 and YU254403 were identified as inhibiting both native C. albicans PSD mitochondrial activity and C. albicans growth, with an MIC50 of 22.5 and 15 µg/ml without ethanolamine and an MIC50 of 75 and 60 µg/ml with ethanolamine, respectively. Together, these results provide the first proof of principle for the application of DSB-3-based fluorescent readouts in high-throughput screening for PSD inhibitors. The data set the stage for future analyses to identify more selective and potent PSD inhibitors with antimicrobial or antitumor activities.

Expression and functional analysis of the TatD-like DNase of Plasmodium knowlesi.

BACKGROUND: In recent years, human infection by the simian malaria parasite Plasmodium knowlesi has increased in Southeast Asia, leading to growing concerns regarding the cross-species spread of the parasite. Consequently, a deeper understanding of the biology of P. knowlesi is necessary in order to develop tools for control of the emerging disease. TatD-like DNase expressed at the surface of P. falciparum has recently been shown to counteract host innate immunity and is thus a potential malaria vaccine candidate. METHODS: The expression of the TatD DNase of P. knowlesi (PkTatD) was confirmed by both Western-blot and immunofluorescent assay. The DNA catalytic function of the PkTatD was confirmed by digestion of DNA with the recombinant PkTatD protein in the presence of various irons. RESULTS: In the present study, we investigated the expression of the homologous DNase in P. knowlesi. The expression of TatD-like DNase in P. knowslesi (PkTatD) was verified by Western blot and indirect immunofluorescence assays. Like that of the P. falciparum parasite, PkTatD was also found to be located on the surface of erythrocytes infected by the parasites. Biochemical analysis indicated that PkTatD can hydrolyze DNA and this activity is magnesium-dependent. CONCLUSIONS: We identified that PkTatD expressed on the surface of P. knowlesi-infected RBCs is a Mg2+-dependent DNase and exhibits a stronger hydrolytic capacity than TatD from P. falciparum. The data support our previous findings that TatD-like DNase is a unanimously expressed virulence factor of Plasmodium parasites.

Preparation of Asymmetric Liposomes Using a Phosphatidylserine Decarboxylase.

Lipid asymmetries between the outer and inner leaflet of the lipid bilayer exist in nearly all biological membranes. Although living cells spend great effort to adjust and maintain these asymmetries, little is known about the biophysical phenomena within asymmetric membranes and their role in cellular function. One reason for this lack of insight into such a fundamental membrane property is the fact that the majority of model-membrane studies have been performed on symmetric membranes. Our aim is to overcome this problem by employing a targeted, enzymatic reaction to prepare asymmetric liposomes with phosphatidylserine (PS) primarily in the inner leaflet. To achieve this goal, we use a recombinant version of a water soluble PS decarboxylase from Plasmodium knowlesi, which selectively decarboxylates PS in the outer leaflet, converting it to phosphatidylethanolamine. The extent of decarboxylation is quantified using high-performance thin-layer chromatography, and the local concentration of anionic PS in the outer leaflet is monitored in terms of the ζ potential. Starting, for example, with 21 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine sodium salt, the assay leads to liposomes with 21 mol % in the inner and 6 mol % PS in the outer leaflet. This asymmetry persists virtually unchanged for at least 4 days at 20°C and at least 2 days at 40°C. The use of a highly specific enzyme carries the advantage that a minor component such as PS can be adjusted without affecting or being affected by the other lipid species present in the model membrane. The phenomena governing the residual outside PS content are addressed but warrant further study.

Genetic and haplotype analyses targeting cytochrome b gene of Plasmodium knowlesi isolates of Malaysian Borneo and Peninsular Malaysia.

Malaria is a notorious disease which causes major global morbidity and mortality. This study aims to investigate the genetic and haplotype differences of Plasmodium knowlesi (P. knowlesi) isolates in Malaysian Borneo and Peninsular Malaysia based on the molecular analysis of the cytochrome b (cyt b) gene. The cyt b gene of 49 P. knowlesi isolates collected from Sabah, Malaysian Borneo and Peninsular Malaysia was amplified using PCR, cloned into a commercialized vector and sequenced. In addition, 45 cyt b sequences were retrieved from humans and macaques bringing to a total of 94 cyt b gene nucleotide sequences for phylogenetic analysis. Genetic and haplotype analyses of the cyt b were analyzed using MEGA6 and DnaSP ver. 5.10.01. The haplotype genealogical linkage of cyt b was generated using NETWORK ver. 4.6.1.3. Our phylogenetic tree revealed the conservation of the cyt b coding sequences with no distinct cluster across different geographic regions. Nucleotide analysis of cyt b showed that the P. knowlesi isolates underwent purifying selection with population expansion, which was further supported by extensive haplotype sharing between the macaques and humans from Malaysian Borneo and Peninsular Malaysia in the median-joining network analysis. This study expands knowledge on conservation of the zoonotic P. knowlesi cyt b gene between Malaysian Borneo and Peninsular Malaysia.

Expression and characterization of functional domains of FK506-binding protein 35 from Plasmodium knowlesi.

The FK506-binding protein of Plasmodium knowlesi (Pk-FKBP35) is considerably a viable antimalarial drug target, which belongs to the peptidyl-prolyl cis-trans isomerase (PPIase) protein family member. Structurally, this protein consists of an N-terminal FK506-binding domain (FKBD) and a C-terminal tetratricopeptide repeat domain (TPRD). This study aims to decipher functional properties of these domains as a platform for development of novel antimalarial drugs. Accordingly, full-length Pk-FKBP35 as well as its isolated domains, Pk-FKBD and Pk-TPRD were overexpressed, purified, and characterized. The results showed that catalytic PPIase activity was confined to the full-length Pk-FKBP35 and Pk-FKBD, suggesting that the catalytic activity is structurally regulated by the FKBD. Meanwhile, oligomerization analysis revealed that Pk-TPRD is essential for dimerization. Asp55, Arg60, Trp77 and Phe117 in the Pk-FKBD were considerably important for catalysis as underlined by significant reduction of PPIase activity upon mutations at these residues. Further, inhibition activity of Pk-FKBP35 towards calcineurin phosphatase activity revealed that the presence of FKBD is essential for the inhibitory property, while TPRD may be important for efficient binding to calcineurin. We then discussed possible roles of FKBP35 in Plasmodium cells and proposed mechanisms by which the immunosuppressive drug, FK506, interacts with the protein.

A novel fluorescence assay for measuring phosphatidylserine decarboxylase catalysis.

Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and animals. We developed a fluorescence-based assay for selectively monitoring production of PE in reactions using a maltose-binding protein fusion with Plasmodium knowlesi PSD (MBP-His6-Δ34PkPSD) as the enzyme. The PE detection by fluorescence (λex = 403 nm, λem = 508 nm) occurred after the lipid reacted with a water-soluble distyrylbenzene-bis-aldehyde (DSB-3), and provided strong discrimination against the phosphatidylserine substrate. The reaction conditions were optimized for enzyme, substrate, product, and DSB-3 concentrations with the purified enzyme and also tested with crude extracts and membrane fractions from bacteria and yeast. The assay is readily amenable to application in 96- and 384-well microtiter plates and should prove useful for high-throughput screening for inhibitors of PSD enzymes across diverse phyla.

Comparison of the susceptibility of Plasmodium knowlesi and Plasmodium falciparum to antimalarial agents.

BACKGROUND: The simian malaria parasite Plasmodium knowlesi is now a well-recognized pathogen of humans in South-East Asia. Clinical infections appear adequately treated with existing drug regimens, but the evidence base for this practice remains weak. The availability of P. knowlesi cultures adapted to continuous propagation in human erythrocytes enables specific studies of in vitro susceptibility of the species to antimalarial agents, and could provide a surrogate system for testing investigational compounds against Plasmodium vivax and other non-Plasmodium falciparum infections that cannot currently be propagated in vitro. OBJECTIVES: We sought to optimize protocols for in vitro susceptibility testing of P. knowlesi and to contrast outputs with those obtained for P. falciparum under comparable test conditions. METHODS: Growth monitoring of P. knowlesi in vitro was by DNA quantification using a SYBR Green fluorescent assay or by colorimetric detection of the lactate dehydrogenase enzyme. For comparison, P. falciparum was tested under conditions identical to those used for P. knowlesi. RESULTS: The SYBR Green I assay proved the most robust format over one (27 h) or two (54 h) P. knowlesi life cycles. Unexpectedly, P. knowlesi displays significantly greater susceptibility to the dihydrofolate reductase inhibitors pyrimethamine, cycloguanil and trimethoprim than does P. falciparum, but is less susceptible to the selective agents blasticidin and DSM1 used in parasite transfections. Inhibitors of dihydroorotate dehydrogenase also demonstrate lower activity against P. knowlesi. CONCLUSIONS: The fluorescent assay system validated here identified species-specific P. knowlesi drug susceptibility profiles and can be used for testing investigational compounds for activity against non-P. falciparum malaria.

Fast and robust single PCR for Plasmodium sporozoite detection in mosquitoes using the cytochrome oxidase I gene.

BACKGROUND: Molecular tools for detecting malaria-infected mosquitoes with improved practicality, sensitivity and specificity, and high-throughput are required. A common PCR technique used to detect mosquitoes infected with Plasmodium spp. is a nested PCR assay based on the 18s-rRNA gene. However, this technique has several technical limitations, is laborious and time consuming. METHODS: In this study, a PCR-based on the Plasmodium cytochrome oxidase I (COX-I) gene was compared with the 18s-rRNA nested PCR using serial dilutions (330-0.0012 pg) of DNA from Plasmodium vivax, Plasmodium falciparum and Plasmodium knowlesi and with DNA from 48 positive and negative Kenyan mosquitoes (previously detected by using both ELISA and PCR). This assay for Plasmodium spp. DNA detection using the fast COX-I PCR assay was then performed individually on 2122 field collected mosquitoes (from the Solomon Islands) in which DNA was extracted from head and thorax. RESULTS: The fast COX-I PCR assay took 1 h to run and consistently detected as low as to 0.043 pg of parasite DNA (equivalent to two parasites) in a single PCR, while analyses with the 18s-rRNA nested PCR required 4 h to complete with a consistent detection threshold of 1.5 pg of DNA. Both assays produced concordant results when applied to the 48 Kenyan control samples with known Plasmodium spp. infection status. The fast COX-I PCR identified 23/2122 Plasmodium-infected mosquitoes from the Solomon Islands. CONCLUSIONS: This new COX-I PCR adapted for a single PCR reaction is a faster, simpler, cheaper, more sensitive technique amenable to high-throughput analyses for Plasmodium DNA detection in mosquitoes and is comparable to the 18s-rRNA nested PCR. The improved sensitivity seen with the fast COX-I PCR will improve the accuracy of mosquito infection rate determination.

Investigating the antiplasmodial activity of primary sulfonamide compounds identified in open source malaria data.

In the past decade there has been a significant reduction in deaths due to malaria, in part due to the success of the gold standard antimalarial treatment - artemisinin combination therapies (ACTs). However the potential threat of ACT failure and the lack of a broadly effective malaria vaccine are driving efforts to discover new chemical entities (NCEs) to target this disease. The primary sulfonamide (PS) moiety is a component of several clinical drugs, including those for treatment of kidney disease, glaucoma and epilepsy, however this chemotype has not yet been exploited for malaria. In this study 31 PS compounds sourced from the GlaxoSmithKline (GSK) Tres Cantos antimalarial set (TCAMS) were investigated for their ability to selectively inhibit the in vitro growth of Plasmodium falciparum asexual stage malaria parasites. Of these, 14 compounds were found to have submicromolar activity (IC50 0.16-0.89 µM) and a modest selectivity index (SI) for the parasite versus human cells (SI > 12 to >43). As the PS moiety is known to inhibit carbonic anhydrase (CA) enzymes from many organisms, the PS compounds were assessed for recombinant P. falciparum CA (PfCA) mediated inhibition of CO2 hydration. The PfCA inhibition activity did not correlate with antiplasmodial potency. Furthermore, no significant difference in IC50 was observed for P. falciparum versus P. knowlesi (P > 0.05), a Plasmodium species that is not known to contain an annotated PfCA gene. Together these data suggest that the asexual intraerythrocytic stage antiplasmodial activity of the PS compounds examined in this study is likely unrelated to PfCA inhibition.

Transfusion-transmitted severe Plasmodium knowlesi malaria in a splenectomized patient with beta-thalassaemia major in Sabah, Malaysia: a case report.

BACKGROUND: Transfusion-transmitted malaria (TTM) is a well-recognized risk of receiving blood transfusions, and has occurred with Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. The simian parasite Plasmodium knowlesi is also known to be transmissible through inoculation of infected blood, and this species is now the most common cause of malaria in Malaysia with a high rate of severity and fatal cases reported. No confirmed case of accidental transfusion-transmitted P. knowlesi has yet been reported. CASE PRESENTATION: A 23-year old splenectomized patient with beta thalassaemia major presented with fever 11 days after receiving a blood transfusion from a pre-symptomatic donor who presented with knowlesi malaria 12 days following blood donation. The infection resulted in severe disease in the recipient, with a parasite count of 84,000/µL and associated metabolic acidosis and multi-organ failure. She was treated with intravenous artesunate and made a good recovery. Sequencing of a highly diverse 649-base pair fragment of the P. knowlesi bifunctional dihydrofolate reductase-thymidylate synthase gene (pkdhfr) revealed that the recipient and donor shared the same haplotype. CONCLUSIONS: This case demonstrates that acquisition of P. knowlesi from blood transfusion can occur, and that clinical consequences can be severe. Furthermore, this case raises the possibility that thalassaemic patients, particularly those who are splenectomized, may represent a high-risk group for TTM and severe malaria. With rising P. knowlesi incidence, further studies in Sabah are required to determine the risk of TTM in order to guide screening strategies for blood transfusion services.

Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah, Malaysia.

BACKGROUND: Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission. METHODS: The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket. RESULTS: Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates. CONCLUSION: Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.

Development of monoclonal antibodies against Plasmodium falciparum thioredoxin peroxidase 1 and its possible application for malaria diagnosis.

Rapid diagnostic tests (RDTs) have been considered as an ideal alternative for light microscopy to detect malaria parasites especially in remote areas. The development and improvement of RDTs is an area of intensive research in the last decade. To date, few parasite proteins have been targeted in RDTs which are known to have certain deficiencies and made the researchers to look for other promising candidates to address this problem. Plasmodium falciparum thioredoxin peroxidase 1 (PfTPx-1) is abundantly expressed in the cytoplasm of the parasite and well conserved across Plasmodium species, making this antigen a promising target for malaria diagnosis. Several monoclonal antibodies (mAbs) were produced against PfTPx-1. The binding affinities of mAbs were measured. Several immunochromatographic tests (ICTs) were developed using different combination of mAbs. All mAbs showed promising affinities to be used for diagnosis. The sensitivities of ICTs were evaluated using recombinant PfTPx-1 whose results lead us to the preparation of 4 different ICTs. These tests showed positive reaction with P. falciparum in vitro culture supernatant indicating the release of PfTPx-1 during schizont rupture. Altogether, these findings suggest that PfTPx-1 is a promising biomarker to diagnose P. falciparum infection. However, the diagnostic performance of this antigen should be further validated using clinical samples.

From Protease to Decarboxylase: THE MOLECULAR METAMORPHOSIS OF PHOSPHATIDYLSERINE DECARBOXYLASE.

Phosphatidylserine decarboxylase (PSDs) play a central role in the synthesis of phosphatidylethanolamine in numerous species of prokaryotes and eukaryotes. PSDs are unusual decarboxylase containing a pyruvoyl prosthetic group within the active site. The covalently attached pyruvoyl moiety is formed in a concerted reaction when the PSD proenzyme undergoes an endoproteolytic cleavage into a large ß-subunit, and a smaller α-subunit, which harbors the prosthetic group at its N terminus. The mechanism of PSD proenzyme cleavage has long been unclear. Using a coupled in vitro transcription/translation system with the soluble Plasmodium knowlesi enzyme (PkPSD), we demonstrate that the post-translational processing is inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride. Comparison of PSD sequences across multiple phyla reveals a uniquely conserved aspartic acid within an FFXRX6RX12PXD motif, two uniquely conserved histidine residues within a PXXYHXXHXP motif, and a uniquely conserved serine residue within a GS(S/T) motif, suggesting that PSDs belong to the D-H-S serine protease family. The function of the conserved D-H-S residues was probed using site-directed mutagenesis of PkPSD. The results from these mutagenesis experiments reveal that Asp-139, His-198, and Ser-308 are all essential for endoproteolytic processing of PkPSD, which occurs in cis. In addition, within the GS(S/T) motif found in all PSDs, the Gly-307 residue is also essential, but the Ser/Thr-309 is non-essential. These results define the mechanism whereby PSDs begin their biochemical existence as proteases that execute one autoendoproteolytic cleavage reaction to give rise to a mature PSD harboring a pyruvoyl prosthetic group.

Structure, function and inhibition of the phosphoethanolamine methyltransferases of the human malaria parasites Plasmodium vivax and Plasmodium knowlesi.

Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties of PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs.

Choline kinase active site provides features for designing versatile inhibitors.

Choline kinase (CK) is a homodimeric enzyme that catalyses the transfer of the ATP γ-phosphate to choline, generating phosphocholine and ADP in the presence of magnesium. Several isoforms of CK are present in humans but only the HsCKα has been associated with cancer and validated as a drug target to treat this disease. As a consequence a large number of compounds based on Hemicholinium (HC-3) have been described. Two compounds, previously reported to inhibit the human enzyme, have recently been shown to inhibit P. falciparum CK (PfCK) and therefore their potential applications might be anticipated to other pathogens. Herein, using molecular dynamic simulations, we have firstly observed that the ATP and the choline binding site of different CK in pathogens and human are conserved, suggesting that previous compounds inhibiting the human enzyme may also interact with CKs from different pathogens. We have substantiated such observation with experimental assays showing that HsCKα1, PfCK and CpCK bind to two compounds with distinct structural features in the low µM range. Collectively, these results uncover similarities among the choline kinase binding site from different pathogenic species and the human enzyme, highlighting the feasibility of designing novel inhibitors based on the choline binding pocket.

Plasmodium knowlesi thioredoxin peroxidase 1 binds to nucleic acids and has RNA chaperone activity.

Malaria parasites are under oxidative attack throughout their life cycle in human body and mosquito vector. Therefore, Plasmodium antioxidant defenses are crucial for its survival and being considered as interesting target for antimalarial drug design. Plasmodium knowlesi has emerged recently from its simian host to human in Southeast Asia and has been recognized as the fifth Plasmodium species that can cause human malaria. In this study, we cloned and characterized thioredoxin peroxidase 1 from P. knowlesi (PkTPx-1). PkTPx-1 gene was cloned, and recombinant protein was produced by heterologous overexpression in Escherichia coli. The recombinant protein was used for evaluation of enzymatic activity and polyclonal antibody production. Using the recombinant PkTPx-1 protein, its antioxidant activity was confirmed in a mixed-function oxidation assay where PkTPx-1 prevented nicking of DNA by hydroxyl radicals. PkTPx-1 was able to bind to double-strand DNA and RNA and had RNA chaperone activity in a nucleic acid melting assay indicating new function of PkTPx-1 other than antioxidant activity. Using specific polyclonal antibodies, it was indicated that PkTPx-1 is expressed in the cytoplasm of the parasite. Altogether, these results suggest that PkTPx-1 not only protects the parasite from the adverse effects of reactive oxygen species but also has RNA chaperone activity.