Inhibition of pyrimidine biosynthesis de novo in Plasmodium falciparum by 2-(4-t-butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone in vitro

The effects of the hydroxynaphthoquinone BW58C on some metabolite levels and the flux of H14CO3 through the de novo pyrimidine biosynthetic pathway of intact Plasmodium falciparum have been studied in vitro using HPLC techniques. 800 nM BW58C appeared to have no significant effect on the energy status of isolated P. falciparum, but at 0.1 nM it caused a dramatic decrease in the concentrations of pyrimidine nucleotides, specifically UTP, during 256 min of incubation. Although about one hour was required to achieve a significant decrease in pyrimidine nucleotide concentrations, a much more rapid inhibition of the flux of H14CO3 through the de novo pathway was found upon addition of 0.1 nM BW58C. This inhibition caused about a 10 fold increase in the radioactivity of carbamoyl-aspartate over a 64 min period, and an overall increase in the concentration of this metabolite of about 3 fold during 256 min of incubation. These effects of BW58C against P. falciparum in vitro are discussed in terms of inhibition of de novo pyrimidine biosynthesis at the site of dihydroorotate dehydrogenase.     

Cell cycle regulation and inhibition of de novo pyrimidine biosynthesis by leflunomide

Inflammation Research -     

In vitro biosynthesis and membrane translocation of the serine rich protein of Plasmodium falciparum

The serine-rich protein (SERP) of Plasmodium falciparum is found within the parasitophorous vacuole. Exons 1 and 2 of the SERP gene were combined to a continuous open reading frame and expressed in a cell free translation/translocation system to study translocation of the protein across membranes. The protein was found to be translocated co-translationally across canine pancreatic microsomes. This process required the presence of the signal recognition particle, and it was accompanied by cleavage of a signal peptide. We conclude that the authentic SERP is exported from the parasite cell via the endoplasmic reticulum.     

Plasmodium falciparum: in vitro growth inhibition by febrile temperatures

Febrile episodes are the hallmark of malarial infection. We determined the inhibitory effect of febrile temperatures on the in vitro growth of Plasmodium falciparum. Parasites were cultured at various temperatures between 37 degrees C and 40 degrees C for 4 days. A logistic decrease in parasitaemia as a function of temperature was observed for continuous cultures. Incubation of synchronized cultures for different lengths of time during the parasite cycle showed a strong increase of growth inhibition with the maturing of parasites. Febrile temperatures inhibit parasite growth and long, high fevers during malaria may be beneficial for parasite clearance.     

De novo and salvage biosynthesis of pteroylpentaglutamates in the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum was shown to synthesize pteroylpolyglutamate de novo from guanosine 5'-triphosphate (GTP), p-aminobenzoate (PABA), and L-glutamate (L-Glu). The parasite also had the capacity to synthesize pteroylpolyglutamate from both intact and degradation moieties (p-aminobenzoylglutamate and pterin-aldehyde) of exogenous folate added into the growth medium. The major product was identified as 5-methyl-tetrahydroteroylpentaglutamate following exposure to pteroylpolyglutamate hydrolase and oxidative degradation of the C9-N10 bond in the molecule and identification of products by reversed-phase high performance liquid chromatography. Inhibition of pteroylpentaglutamate synthesis from the radiolabelled metabolic precursors (GTP, PABA, L-Glu) and folate by the antifolate antimalarials, pyrimethamine and sulfadoxine at therapeutic concentrations, may suggest the existence of a unique biosynthetic pathway in the malaria parasite.     

Antibody-independent inhibition of Plasmodium falciparum in vitro cultures.

The sera of 100 Colombian individuals of African origin living in a malaria-endemic area of the Pacific coast were studied with regard to their capacity to inhibit Plasmodium falciparum cultures in vitro. Antimalarial antibody levels determined by indirect immunofluorescence were higher in the group of infected individuals than in the noninfected individuals, and inhibitory activity assessed by the inhibition of parasite incorporation of 3H-hypoxanthine in vitro was present in the sera of both the infected and noninfected patients. We believe that the noninfected patients were probably immune. The sera of some of the infected patients had high inhibitory capacities for the P. falciparum FCB-1 isolate. When the inhibitory effects of some of the sera were tested by using four parasite isolates from different regions of the world, striking differences among them were found.     

Cytokine-induced inhibition of Plasmodium falciparum erythrocytic growth in vitro.

The addition of recombinant cytokines to Plasmodium falciparum in vitro cultures retarded the growth of the parasite with the effect of recombinant IL-2 (rIL-2) > interferon-gamma (IFN-gamma) > tumour necrosis factor-beta (TNF-beta). The process was concentration dependent, being greatest at 30,000 U/ml and required a 72-h period of continuous exposure for maximum effect. Growth inhibition, as determined morphologically and radiometrically, was a consequence of defective schizont maturation rather than inhibition of merozoite invasion. It was cumulative and detectable within one erythrocytic (48 h) growth cycle.     

Enhancement or inhibition of Plasmodium falciparum erythrocyte reinvasion in vitro by antibodies to an asparagine rich protein

A clone encoding a recombinant protein which reacted strongly with human antibodies from a donor clinically immune to malaria, was isolated from a genomic Plasmodium falciparum library. Mice injected with this protein, designated 10b, produced antibodies which reacted with all developmental stages of erythrocytic asexual parasites in indirect immunofluorescence. In immunoblotting, the same antibodies recognized two P. falciparum polypeptides of 36 kDa and 33 kDa. Of three monoclonal antibodies raised against the 10b recombinant protein, two inhibited parasite reinvasion of erythrocytes in an isolate specific manner. Surprisingly, however, the third was found to significantly enhance reinvasion of erythrocytes and also to induce a more rapid maturation of intraerythrocytic parasites in all isolates tested. Nucleotide sequence analysis of the 1124 bp insert revealed that it encodes a protein which consists of 30% asparagine and contains three asparagine rich, imperfect tandem repeats: Lys-Lys-Asn-Asn (3x), Met-Asn-His/Gln-Pro-Asn-Asn (14x), and Lys-Asn-Asn-Asn-Asn (7x).     

Enhanced inhibition of in vitro multiplication of Plasmodium falciparum by stimulated human polymorphonuclear leucocytes

The effect of normal human peripheral blood polymorphonuclear leucocytes on in vitro multiplication of Plasmodium falciparum malaria parasites was investigated. It was shown that normal neutrophils were able to phagocytose parasitized erythrocytes and free parasites and thus inhibit in vitro multiplication of the parasite. Stimulation of the neutrophils by phorbol myristate acetate, a potent stimulus of leucocyte oxidative metabolism, resulted in enhanced inhibition of parasite growth. Superoxide dismutase, scavenger of superoxide anion, catalase, inhibitor of hydrogen peroxide, and sodium azide, inhibitor of myeloperoxidase, did not abrogate the inhibitory ability of the neutrophils. The results indicate that polymorphonuclear leucocytes play an important role in the defence against P. falciparum malaria.     

Leflunomide,a reversible inhibitor of pyrimidine biosynthesis?

Inflammation Research -     

In vitro cultivation of fields isolates of Plasmodium falciparum in Mali

Malaria immunology, molecular biology and pathogenicity studies often require the adaptation of Plasmodium falciparum field isolates to continuous in vitro cultivation. For this purpose we have established propagation protocols of asexual erythrocytic stages of P. falciparum samples from malaria patients or asymptomatic carriers in Mali. The parasites were grown in standard culture medium supplemented by human serum and in a culture medium without human serum but supplemented by AlbuMax 1. The candle jar environment and tissue culture flasks gassed with 5% CO2, 5% O2 and 90% N2 obtained from a portable gas mixer were used. Protocols for parasite cultivation in a resource-poor setting were developed. These protocols were successfully applied to fresh isolates in Mali as well as to blood samples frozen in liquid nitrogen and shipped to a laboratory in U.S.A.     

In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum

Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.     

Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum trophozoites were isolated by mechanical rupture of infected human erythrocytes followed by a series of differential centrifugation steps. After lysis with sonication, the 100 000 x g supernatant of parasites and uninfected host cells was used to determine the specific activities of a number of enzymes involved in purine and pyrimidine metabolism. P. falciparum possessed the purine salvage enzymes: adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyltransferase (PRTase), xanthine PRTase, adenine PRTase, adenosine kinase. The last two enzymes, however, were present at much lower activity levels. Hypoxanthine was converted (presumably via IMP) into adenine and guanine nucleotides only in the presence both of supernatant and membrane fractions of P. falciparum. Two enzymes involved in the de novo synthesis of pyrimidines, orotic acid PRTase, and orotidine 5'-phosphate decarboxylase, were present in parasite extracts as were the enzymes for pyrimidine nucleotide phosphorylation: UMP-CMP kinase, dTMP kinase, nucleoside diphosphate kinase. Xanthine oxidase, CTP synthetase, cytidine deaminase and several kinases for the salvage of pyrimidine nucleosides were not detected in the parasites. Both phosphoribosyl pyrophosphate synthetase and uracil PRTase were present but at low activity levels. Human erythrocytes displayed similar but not identical enzyme patterns. Enzyme specific activities, however, were generally much lower than those of the corresponding parasite enzymes.     

A role for rheumatoid factor enhancement of Plasmodium falciparum schizont inhibition in vitro.

Studies were undertaken to determine whether rheumatoid factor (RF) was present in immune human and Aotus trivirgatus monkey sera which inhibited Plasmodium falciparum schizonts in vitro and to determine whether RF could be responsible for or contribute to merozoite agglutination in the parasite inhibition test. Additional studies were conducted to determine the effect of exogenous RF on schizont inhibition when used alone or in conjunction with immune or normal sera. RF was not detected in any of the 11 immune monkey sera or the 3 immune human sera which were tested. However, when RF was added to immune human or Aotus sera, levels of schizont inhibition increased significantly over levels obtained with immune serum alone. When RF was used alone or in conjunction with normal sera, levels of schizont inhibition were comparable to those obtained with normal serum. Furthermore, adsorption of the RF with immunoglobulin G-coated erythrocytes removed the enhancing activity. The results of this study indicate that RF, which is sometimes produced during acute or chronic malarial infection, may contribute nonspecifically to the enhanced clearance of plasmodia in vivo.     

In vitro anti-plasmodial activity of some traditionally used medicinal plants against Plasmodium falciparum

The anti-plasmodial activity of different solvent extracts of Adhatoda vasica (root), Caesalpinia pulcherrima (leaf), Carica papaya (pulp), Erythroxylum monogynum (leaf), Lantana camara (whole plant), Ocimum sanctum (root) and Phyllanthus niruri (whole plant) were studied against Plasmodium falciparum. Of the 35 extracts tested, seven extracts showed good anti-plasmodial activity. Methanol extract of C. pulcherrima showed the lowest IC50 value (10.96 μg/mL) followed by methanol extract of A. vasica (IC(50)=11.1 μg/mL), chloroform extract of O. sanctum (IC(50)=11.47 μg/mL), methanol extract of E. monogynum (IC(50)=12.23 μg/mL), acetone extract of C. pulcherrima (IC(50)=12.49 μg/mL), methanol extract of O. sanctum and acetone extract of A. vasica (IC(50)=14.04 μg/mL). The results of the present study justify the use of these medicinal plants in traditional practice, and also, a further study on the isolation of anti-plasmodial molecules from their active crude extracts is in progress.     

TNF and inhibition of growth of Plasmodium falciparum

The mechanism of intra-erythrocyte death of Plasmodium chabaudi in vivo has not yet been elucidated. Here we summarise recent experiments in which serum from mice undergoing a successful immune response to this parasite did not inhibit Plasmodium falciparum in vivo unless the P. chabaudi infection and TNF levels were high enough to cause illness in the host. This was true for the 556KA and DS strains of P. chabaudi in intact mice, but not for 556KA in nude mice, which did not generate inhibitory activity at any parasitaemia. Tumour necrosis factor (TNF) inhibits malaria parasites via some undefined secondary mediator. 10 mg of r hu TNF generated this inhibitory activity, as measured against P. falciparum in vitro, in the serum of mice only if they were pretreated with Corynebacterium parvum, which activates macrophages and sensitises the mice to the toxic effects of TNF. This implies a role for activated macrophages downstream from TNF in the process involved in intra-erythrocytic death of parasites.     

In vitro,long-range sequence information for de novo genome assembly via transposase contiguity

We describe a method that exploits contiguity preserving transposase sequencing (CPT-seq) to facilitate the scaffolding of de novo genome assemblies. CPT-seq is an entirely in vitro means of generating libraries comprised of 9216 indexed pools, each of which contains thousands of sparsely sequenced long fragments ranging from 5 kilobases to >1 megabase. These pools are “subhaploid,” in that the lengths of fragments contained in each pool sums to ∼5% to 10% of the full genome. The scaffolding approach described here, termed fragScaff, leverages coincidences between the content of different pools as a source of contiguity information. Specifically, CPT-seq data is mapped to a de novo genome assembly, followed by the identification of pairs of contigs or scaffolds whose ends disproportionately co-occur in the same indexed pools, consistent with true adjacency in the genome. Such candidate “joins” are used to construct a graph, which is then resolved by a minimum spanning tree. As a proof-of-concept, we apply CPT-seq and fragScaff to substantially boost the contiguity of de novo assemblies of the human, mouse, and fly genomes, increasing the scaffold N50 of de novo assemblies by eight- to 57-fold with high accuracy. We also demonstrate that fragScaff is complementary to Hi-C-based contact probability maps, providing midrange contiguity to support robust, accurate chromosome-scale de novo genome assemblies without the need for laborious in vivo cloning steps. Finally, we demonstrate CPT-seq as a means of anchoring unplaced novel human contigs to the reference genome as well as for detecting misassembled sequences.The broad adoption of next-generation sequencing (NGS) has resulted in a proliferation of de novo genome assemblies (Pagani et al. 2012). For the most part, these assemblies are of far lower quality than the reference genomes produced by the International Human Genome Consortium (The International Human Genome Consortium 2001, 2004; Blanco-Ulate et al. 2013; Rong and McSpadden Gardener 2013; Shemesh et al. 2013; Wang et al. 2013), largely secondary to a dearth of readily accessible NGS-based methods for generating midrange and long-range contiguity information. Conventional NGS-based assemblies rely on deep sequencing of shotgun fragments and ∼3-kbp mate-pair libraries to provide short-range contiguity, both of which are generated via straightforward in vitro protocols. Dilution pools of PCR amplicons can provide long virtual reads, but these are limited to 10 kbp by the use of PCR, and the majority are ∼8 kbp (Voskoboynik et al. 2013). Midrange contiguity information requires still-expensive (but rapidly evolving) long sequencing reads (e.g., PacBio at ∼18 kbp) (Koren et al. 2012) or nanopore sequencing (Laszlo et al. 2014) or labor-intensive fosmid or BAC clone libraries (Gnerre et al. 2011; Zhang et al. 2012). Alternatively, long-range contiguity information can be generated in vitro via contact probability maps, wherein Hi-C read-pairs are used for chromosome-scale scaffolding (Burton et al. 2013). However, the performance of Hi-C-based scaffolding depends heavily on input assembly scaffold size, with optimal results requiring an input N50 of ∼200 kbp or greater, a level of contiguity that can be challenging to achieve with shotgun fragment and 3-kbp mate-pair libraries alone (Blanco-Ulate et al. 2013; Rong and McSpadden Gardener 2013; Shemesh et al. 2013; Wang et al. 2013). As such, there remains a strong need for robust in vitro methods to capture midrange contiguity information for de novo genome assembly.Transposase-mediated library construction, or “tagmentation,” utilizes a hyperactive Tn5 transposase to both fragment and append universal adaptors in a single enzymatic step (Goryshin and Reznikoff 1998; Adey et al. 2010). In recent years, tagmentation has been applied in diverse ways including DNA-seq (Adey et al. 2010), stranded RNA-seq (Gertz et al. 2012), whole-genome bisulfite sequencing (Adey and Shendure 2012), chromatin profiling (Buenrostro et al. 2013), and in situ mate-pair library preparation directly on a sequencing flowcell (Schwartz et al. 2012). We recently demonstrated a novel method, contiguity preserving transposase sequencing (CPT-seq) for haplotype-resolved genome sequencing (Amini et al. 2014). CPT-seq utilizes an inherent property of the Tn5 transposase in which the enzyme remains tightly bound to the target DNA after tagmentation, physically linking adjacent library molecules (Fig. 1A). Prior to PCR amplification, the high molecular weight products of linked templates are subjected to subhaploid dilution and compartmentalization, followed by protein denaturation in order to free the templates for amplification. To increase the effective number of compartments, a two-tiered indexing approach (Erlich et al. 2009) is applied (Fig. 1B). The initial tagmentation is performed using 96 uniquely indexed transposase-adaptor complexes. The high molecular weight linked templates are then pooled, followed by limiting dilution into 96 indexed PCR reactions such that each PCR well contains templates from 96 originating transposase reactions, thus producing 96 × 96 = 9216 distinct index combinations. Sequence reads corresponding to each of the 9216 “virtual compartments” (also referred to as indexed pools) provide sparse, shotgun representation of the high molecular weight genomic DNA fragments that were transposed within that indexed pool (Fig. 1C), analogous to fosmid (Kitzman et al. 2011) or in vitro dilution pools (Kaper et al. 2013) but with a ∼100× higher effective number of pools. With appropriate dilution of the input genomic DNA, these indexed pools are “subhaploid,” in that the lengths of fragments contained in each pool sums to ∼5% to 10% of full genome length. From an experimental perspective, the library preparation steps of CPT-seq are straightforward and scalable, with an overall processing time of <3 h, relying on readily available equipment and requiring minimal hands-on time (Amini et al. 2014).Open in a separate windowFigure 1.CPT-seq method and performance. (A) High molecular weight (HMW) genomic DNA reacted with hyperactive Tn5 transposase loaded with indexed adaptors. After the transposase complex fragments the DNA and appends the indexed adaptors, the enzyme remains tightly bound to the DNA, such that library molecules derived from the same HMW genomic DNA molecule remain physically linked. Once the transposase is removed by denaturation, PCR amplification of viable templates (gray boxes) can be performed. (B) Schematic of two tier indexing. A 96-plex indexed tagmentation is performed (but without removing the transposase), followed by pooling, mixing, and redistribution to 96 wells. These new pools are subjected to removal of the transposase, 96-plex indexed PCR and then pooling to a single sequencing library. Individual molecules within the final library have indices corresponding to both the pool in which their originating HMW genomic DNA fragment was present during tagmentation (96 indices) as well as during PCR (96 indices), such that there are effectively 96 × 96 = 9216 compartments. (C) Representation of coverage profiles for indexed fragment pools, i.e., compartments (top) and trimodal distribution of adjacently aligning reads within individual compartments. The first peak (∼100 bp; red) corresponds to simple read pairs; the second peak (∼3.2 kbp; green) corresponds to reads originating from the same HMW genomic DNA fragment; the third peak (∼1 Mbp; blue) corresponds to reads originating from different HMW genomic DNA fragments. (D) Distribution of estimated HMW genomic DNA fragment lengths for CPT-seq of GM12878. The mean fragment size is 33.9 kbp, but it is a broad distribution and nearly 1M fragments are >100 kbp.     

Effect of drugs inhibiting spermidine biosynthesis and metabolism on the in vitro development of Plasmodium falciparum

Treatment of Plasmodium falciparum with the potent inhibitor dicyclohexylamine completely arrests in vitro cell proliferation of the chloroquine-susceptible P. falciparum strain NF54 and the R strain, which shows less sensivity to chloroquine. The average inhibitory concentration (IC50) values determined for both strains revealed different inhibition profiles. The IC50 value for the chloroquine-sensitive NF54 strain was 97 microM and 501 microM for the R strain. Monitoring polyamine pools after treatment with dicyclohexylamine leads to a significant decrease in the intracellular spermidine content, which was nearly reversed by supplementation with spermidine. Since spermidine is an important precursor for the biosynthesis of hypusine and homospermidine in eukaryotes, we studied the developmental effect on both P. falciparum strains of 1,7-diaminoheptane as an inhibitor of deoxyhypusine synthase (EC 1.1.1.249) in mammalian cells, and agmatine as a moderate inhibitor of homospermidine synthase (EC 2.5.1.44). Inhibition profiles with 1,7-diaminoheptane resulted in an IC50 value of 466 microM for the NF54 strain and 319 microM for the R strain. Spermidine pools changed significantly. Inhibition with agmatine caused a strong decrease in parasitemia for the chloroquine-susceptible NF54 strain, with a determined IC50 value of 431 microM and an IC50 value of 340 microM for the less chloroquine-susceptible R strain. Spermidine was not detectable after inhibition. The uncommon triamine homospermidine occurred in both P. falciparum strains. To our knowledge this is the first evidence of homospermidine in P. falciparum. The use of specific inhibitors of spermidine metabolism might be a novel strategy for the design of new antimalarials, and suggests the occurrence of both enzymes in the parasite.     

In vitro biosynthesis and core glycosylation of the histidine-rich protein of Plasmodium lophurae

We have characterized the early biosynthetic forms of the histidine-rich protein (HisRP), a major, granule-bound protein (Mr 58 000) of the avian malarial parasite Plasmodium lophurae. We have translated poly(A)-containing, size-selected parasite mRNA in the wheat germ cell-free system in the presence of [3H]histidine. HisRP was synthesized as a larger precursor (Mr 63 000). When dog pancreas microsomal membranes were present in the cell-free system during translation, a still larger form of HisRP (Mr 66 000) was detected. This larger form was segregated into the dog pancreas microsomal vesicles and was core glycosylated. Presumably, it corresponds to an intermediate form located in the parasite rough endoplasmic reticulum (RER). The difference in the Mr of approx. 8 000 between this RER associated 'pro' form and the granule-bound, mature form of HisRP suggests that proteolytic processing occurs upon transport from the RER to the granule. Segregation and core glycosylation were strictly coupled to translation and were not observed upon posttranslational addition of microsomal membranes. Thus, the early events in the biosynthesis of HisRP are similar to those established for secretory and lysosomal proteins.     

In vitro sensitivity of Plasmodium falciparum isolates from Gabon to chloroquine and cycloguanil]

The in vitro susceptibility of 91 Plasmodium falciparum isolates obtained from malaria-infected children living near Libreville (Gabon) was evaluated against chloroquine and cycloguanil (biologically active metabolite of proguanil), using an isotopic micro-drug susceptibility test. In vitro resistance to chloroquine and cycloguanil was observed in 83% (35/42) and in 38% (30/78) of the patients, respectively. Our data showed that 41% (16/39) of Gabonese field isolates were resistant both to chloroquine and cycloguanil. These findings are of great importance because they might indicate imminent chloroquine-proguanil failure, and there are not many affordable antimalarial drugs to replace chloroquine-proguanil combination.