Maturation Of Trophozoites Research Articles

In vitro antiplasmodial and in vivo toxicity potential of Mentha piperita and Ocimum gratissimum essential oils and their synergistic effect with conventional antimalarial drugs against Plasmodium falciparum

The study aimed to investigate the in vitro antiplasmodial and in vivo toxicity potential of essential oils from Mentha piperita and Ocimum gratissimum against Plasmodium falciparum, singly and in combination with antimalarial drugs. The essential oils (EOs) were obtained by steam distillation and phytochemical screening conducted using gas chromatography equipped with a flame ionization detector. Blood samples of malaria positive patients were cultured and P. falciparum identified microscopically. Micro-technique of schizont counting was used to determine the antiplasmodial baseline of the EOs and in combination with antimalarial drugs. Hematological and serum biochemical indices for in vivo toxicity assay using Wister rats were determined following WHO test procedures. Dose-response assay determined after 48 h for in vitro inhibition of maturation of trophozoites into schizonts showed that EO of M. piperita had a higher antiplasmodial activity of 6.62 ± 0.20, 10.74 ± 1.64 and 14.21 ± 0.45 µg values than O. gratissimum for IC30, IC50 and IC90, respectively against P. falciparum. When the EOs were combined with conventional antimalarial agents, M. piperita with Sulfadoxine/Pyrimethamine had the highest antiplasmodial activity with inhibitory concentration values of 1.92 ± 1.00, 4.07 ± 1.34 and 6.85 ± 0.75 ݜ‡g/mL for IC30, IC50 and IC90, respectively than O. gratissimum. In vivo hematological and biochemical studies after 14 days oral treatment showed no toxicity as a result of treatment with the oil extracts and these were significantly different (P

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.

Protococcidian Eleutheroschizon duboscqi, an Unusual Apicomplexan Interconnecting Gregarines and Cryptosporidia.

This study focused on the attachment strategy, cell structure and the host-parasite interactions of the protococcidian Eleutheroschizon duboscqi, parasitising the polychaete Scoloplos armiger. The attached trophozoites and gamonts of E. duboscqi were detected at different development stages. The parasite develops epicellularly, covered by a host cell-derived, two-membrane parasitophorous sac forming a caudal tipped appendage. Staining with Evans blue suggests that this tail is protein-rich, supported by the presence of a fibrous substance in this area. Despite the ultrastructural evidence for long filaments in the tail, it stained only weakly for F-actin, while spectrin seemed to accumulate in this area. The attachment apparatus consists of lobes arranged in one (trophozoites) or two (gamonts) circles, crowned by a ring of filamentous fascicles. During trophozoite maturation, the internal space between the parasitophorous sac and parasite turns translucent, the parasite trilaminar pellicle seems to reorganise and is covered by a dense fibrous glycocalyx. The parasite surface is organised in broad folds with grooves in between. Micropores are situated at the bottom of the grooves. A layer of filaments organised in bands, underlying the folds and ending above the attachment fascicles, was detected just beneath the pellicle. Confocal microscopy, along with the application of cytoskeletal drugs (jasplakinolide, cytochalasin D, oryzalin) confirmed the presence of actin and tubulin polymerised forms in both the parasitophorous sac and the parasite, while myosin labelling was restricted to the sac. Despite positive tubulin labelling, no microtubules were detected in mature stages. The attachment strategy of E. duboscqi shares features with that of cryptosporidia and gregarines, i.e. the parasite itself conspicuously resembles an epicellularly located gregarine, while the parasitophorous sac develops in a similar manner to that in cryptosporidia. This study provides a re-evaluation of epicellular development in other apicomplexans and directly compares their niche with that of E. duboscqi.

Laboratory detection of artemisinin-resistant Plasmodium falciparum.

Conventional 48-h in vitro susceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivo parasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistant P. falciparum is prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P = 0.001). TMI IC50s correlated significantly with the in vivo responses to artesunate (parasite clearance time [r = 0.44, P = 0.001] and parasite clearance half-life [r = 0.46, P = 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility.

Plasmodium falciparum Antigen 332 Is a Resident Peripheral Membrane Protein of Maurer's Clefts

During the intraerythrocytic development of Plasmodium falciparum, the malaria parasite remodels the host cell cytosol by inducing membranous structures termed Maurer's clefts and inserting parasite proteins into the red blood cell cytoskeleton and plasma membrane. Pf332 is the largest known asexual malaria antigen that is exported into the red blood cell cytosol where it associates with Maurer's clefts. In the current work, we have utilized a set of different biochemical assays to analyze the solubility of the endogenous Pf332 molecule during its trafficking from the endoplasmic reticulum within the parasite to the host cell cytosol. Solubilization studies demonstrate that Pf332 is synthesized and trafficked within the parasite as a peripheral membrane protein, which after export into the host cell cytosol associates with the cytoplasmic side of Maurer's clefts in a peripheral manner. By immunofluorescence microscopy and flow cytometry, we show that Pf332 persists in close association with Maurer's clefts throughout trophozoite maturation and schizogony, and does not become exposed at the host cell surface. Our data also indicate that Pf332 interacts with the host cell cytoskeleton, but only in very mature parasite stages. Thus, the present study describes Pf332 as a resident peripheral membrane protein of Maurer's clefts and suggests that the antigen participates in host cytoskeleton modifications at completion of the intraerythrocytic developmental cycle.

Differential carbonylation of cytoskeletal proteins in blood group O erythrocytes: Potential role in protection against severe malaria

The molecular basis for the prevalence of blood group O in regions where malaria is endemic remains unclear. In some genetic backgrounds oxidative modifications have been linked to a reduced susceptibility to severe malaria disease. Through redox proteomics, we detected differences in carbonylated membrane proteins among the different blood groups, both in Plasmodium-infected and uninfected erythrocytes (RBC). Carbonylation profiles of RBC membrane proteins revealed that group O blood shows a reduced protein oxidation pattern compared to groups A, B and AB. Upon infection with Plasmodium falciparum Dd2, erythrocytes of all blood groups showed increased oxidation of membrane proteins. By examining 4-hydroxy-2-nonenal (4-HNE) modified proteins by LC–MS/MS (liquid chromatography/mass spectrometry) we observed that, upon malaria infection, the protein components of lipid rafts and cytoskeleton were the main targets of 4-HNE carbonylation in all blood groups. Ankyrins and protein bands 4.2 and 4.1 were differentially carbonylated in group O as compared to A and B groups. During trophozoite maturation in group O erythrocytes, a steady increase was observed in the number of 4-HNE-modified proteins, suggesting a parasite-driven 4-HNE-carbonylation process. Our findings indicate a possible correlation between the protection against severe malaria in blood group O individuals and a specific pattern of 4-HNE-carbonylation of cytoskeleton proteins.

Developmental Stage- and Cell Cycle Number-Dependent Changes in Characteristics of Plasmodium falciparum -Infected Erythrocyte Adherence to Placental Chondroitin-4-Sulfate Proteoglycan

The adherence of Plasmodium falciparum-infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin-4-sulfate (C4S). Although IRBC binding to C4S has been unequivocally established, the adherence characteristics of IRBCs at different stages of parasite development and through successive parasite generations after selection for C4S adherence are not known. Here we show that IRBCs acquire a significant capacity to bind to C4S at as early as 14 h and exhibit maximum binding at 22 to 26 h postinvasion. Surprisingly, the IRBC binding ability decreases by approximately 50% at the late trophozoite and schizont stages. The binding strength of the IRBCs also gradually decreases during successive generations after selection for C4S binding, and at the 32nd generation, the binding capacity was only approximately 31% of that of IRBCs at the 2nd generation, suggesting that IRBCs eventually lose their C4S-adherent capacity. We also tested the susceptibility of the adhesive protein(s) on the IRBC surface to trypsin treatment at different stages of parasite development. The data show that IRBCs with late trophozoites are more resistant to trypsin treatment than those containing early trophozoites, indicating that parasite proteins expressed on the IRBC surface during trophozoite maturation partially mask accessibility of adhesive protein for binding to C4S. These data provide important insights into the expression pattern of the C4S-adhesive protein(s) on the IRBC surface, emphasizing the need for understanding the regulation of genes involved in IRBC binding to C4S. Our data also define the parasite stage at which IRBCs are suitable for studying structural interactions with C4S.

Light and electron microscopical observations of the effects of high-density lipoprotein on growth of Plasmodium falciparum in vitro.

Human serum high-density lipoprotein (HDL) is necessary and sufficient for the short-term maintenance of Plasmodium falciparum in in vitro culture. However, at high concentrations it is toxic to the parasite. A heat-labile component is apparently responsible for the stage-specific toxicity to parasites within infected erythrocytes 12-42 h after invasion, i.e. during trophozoite maturation. The effects of HDL on parasite metabolism (as determined by nucleic acid synthesis) are evident at about 30 h after invasion. Parasites treated with HDL show gross abnormalities by light and electron microscopy.

Ankyrin Peptide Blocks Falcipain-2-mediated Malaria Parasite Release from Red Blood Cells

Falcipain-2 (FP-2) is a dual-function protease that cleaves hemoglobin at the early trophozoite stage and erythrocyte membrane ankyrin and protein 4.1 at the late stages of parasite development. FP-2-mediated cleavage of ankyrin and protein 4.1 is postulated to cause membrane instability facilitating parasite release in vivo. To test this hypothesis, here we have determined the precise peptide sequence at the hydrolysis site of ankyrin to develop specific inhibitor(s) of FP-2. Mass spectrometric analysis of the hydrolysis products showed that FP-2-mediated cleavage of ankyrin occurred immediately after arginine 1,210. A 10-mer peptide (ankyrin peptide, AnkP) containing the cleavage site completely inhibited the FP-2 enzyme activity in vitro and abolished all of the known functions of FP-2. To determine the effect of this peptide on the growth and development of P. falciparum, the peptide was delivered into intact parasite-infected red blood cells (RBCs) via the Antennapedia homeoprotein internalization domain. Growth and maturation of trophozoites and schizonts was markedly inhibited in the presence of the fused AnkP peptide. <10% of new ring-stage parasites were detected compared with the control sample. Together, our results identify a specific peptide derived from the spectrin-binding domain of ankyrin that blocks late-stage malaria parasite development in RBCs. Confocal microscopy with FP-2-specific antibodies demonstrated the proximity of the enzyme in apposition with the RBC membrane, further corroborating the proposed function of FP-2 in the cleavage of RBC skeletal proteins.

Glucosamine Inhibits Inositol Acylation of the Glycosylphosphatidylinositol Anchors in IntraerythrocyticPlasmodium falciparum

Glycosylphosphatidylinositol (GPI) anchors are crucial for the survival of the intraerythrocytic stage Plasmodium falciparum because of their role in membrane anchoring of merozoite surface proteins involved in parasite invasion of erythrocytes. Recently, we showed that mannosamine can prevent the growth of P. falciparum by inhibiting the GPI biosynthesis. Here, we investigated the effect of isomeric amino sugars glucosamine, galactosamine, and their N-acetyl derivatives on parasite growth and GPI biosynthesis. Glucosamine, but not galactosamine, N-acetylglucosamine, and N-acetylgalactosamine inhibited the growth of the parasite in a dose-dependent manner. Glucosamine specifically arrested the maturation of trophozoites, a stage at which the parasite synthesizes all of its GPI anchor pool and had no effect during the parasite growth from rings to early trophozoites and from late trophozoites to schizonts and merozoites. An analysis of GPI intermediates formed when parasites incubated with glucosamine indicated that the sugar interferes with the inositol acylation of glucosamine-phosphatidylinositol (GlcN-PI) to form GlcN-(acyl)PI. Consistent with the non-inhibitory effect on parasite growth, galactosamine, N-acetylglucosamine, and N-acetylgalactosamine had no significant effect on the parasite GPI biosynthesis. The results indicate that the enzyme that transfers the fatty acyl moiety to inositol residue of GlcN-PI discriminates the configuration at C-4 of hexosamines. An analysis of GPIs formed in a cell-free system in the presence and absence of glucosamine suggests that the effect of the sugar is because of direct inhibition of the enzyme activity and not gene repression. Because the fatty acid acylation of inositol is an obligatory step for the addition of the first mannosyl residue during the biosynthesis of GPIs, our results offer a strategy for the development of novel anti-malarial drugs. Furthermore, this is the first study to report the specific inhibition of GPI inositol acylation by glucosamine in eukaryotes.

The effect of atovaquone (566C80) on the maturation and viability of Plasmodium falciparum gametocytes in vitro

Atovaquone (566C80), a hydroxynaphthoquinone, was investigated for activity against Plasmodium falciparum gametocytes (NF54 strain) in vitro. After 96 h of continuous exposure to the drug at 1.4 × 10 −7M (a concentration achievable in humans 14 d after administration of a therapeutic dose of 10 mg/kg) reductions of 75%, 54% and 20% in the number of gametocyte stages 2, 3 and 4, respectively, were achieved. A small increase (14%) in stage 5 gametocytes was seen. At the same concentration, atovaquone showed greater activity against the asexual stages of P. falciparum, reductions of 93%, 96% and 43% in the number of rings, schizonts and trophozoites, respectively, being achieved. These data are consistent with inhibition of maturation of trophozoites. The observed effect on maturation of gametocytes is similarly consistent with blockade of gametocyte recruitment from merozoites produced by the preceding schizogony, or to stasis of intraerythrocytic sexual development before the formation of stage 2 gametocytes.

Plasmodium chabaudi-infected erythrocytes: Differential expression of trophozoite proteins in host cell plasma membranes

Neoproteins were compared in host cell plasma membranes isolated as ghosts from erythrocytes infected with early and late trophozoites of Plasmodium chabaudi. Ghosts isolated from early trophozoites-infected erythrocytes (= et-ghosts) contain seven neoproteins with apparent molecular masses of about 154, 145, 90, 72, 67, 52 and 33 kDa, respectively. Two additional neoproteins with masses of about 42 and 38 kDa are detected in ghosts isolated from late trophozoites-infected erythrocytes (= lt-ghosts). All neoproteins, except the 154, 145 and 38 kDa-proteins, can be metabolically labeled with both [(14)C]isoleucine and [(35)S]methionine. The 90 kDa-protein is predominantly labeled in et-ghosts, while the 42 kDa-protein is exclusively labeled in lt-ghosts. Both proteins, however, contain only minor radioactivity in the corresponding parasites. Moreover, the pI of the 90 kDa-neoprotein shifts from about 4.5 in et-ghosts to about 5.6 in lt-ghosts as detected by 2 D-gel electrophoresis and western blotting using a 90 kDa-protein specific monoclonal antibody. The data indicate a posttranslational modification of the 90 kDa-neoprotein and a sequential expression of the 90 kDa- and 42 kDa-neoproteins in host cell plasma membranes during intraerythrocytic maturation of trophozoites.

Enhanced transbilayer mobility of phospholipids in malaria-infected monkey erythrocytes: a spin-label study.

Using a recently described technique of electron spin resonance spectroscopy, we determined the rate of transbilayer mobility (flip) of the four major simian-erythrocyte phospholipids in the erythrocyte membrane after infection by Plasmodium knowlesi. The development of the malarial parasite induces a very large increase in the flip rate of these phospholipids (mainly during the ring stage and at the beginning of trophozoite maturation). The half flip time fell from 2 and 3 hr in the case of choline phospholipids in healthy erythrocytes to less than 15 min in erythrocytes infected with the last stage of the parasite.

In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo

The effect of pyrazofurin, an inhibitor of UMP synthesis, on Plasmodium falciparum growth in vitro has been studied. ID 50 values (concentration of compound causing 50% inhibition of [ 3H]hypoxanthine incorporation) for the FCQ-27, FCI-1 and K-1 (chloroquine-resistant) isolates were 10 ± 8.7, 6.4 ± 5.3 and 6.3 ± 0.5 μM, respectively. Comparative ID 50 values for chloroquine were 13.5 ± 4.2, 22.8 ± 7.6 and 343 ± 114 nM, respectively. Over the 48-h intraerythrocytic cycle of tightly synchronized parasites, pyrazofurin both reduced the parasitemia and retarded the maturation of trophozoites and schizonts. Addition of uracil or uridine to the in vitro culture did not decrease the anti-parasitic activity of pyrazofurin. Chloroquine reduced the parasitemia, but did not retard development of the remaining viable parasites. Pyrazofurin (20 μM) caused a 50% inhibition of parasite orotate phosphoribosyltransferase (E.C. 2.4.2.10) and, in the presence of adenosine kinase and ATP, a 73% inhibition of orotidine-5′-phosphate decarboxylase (E.C. 4.1.1.23).

Synthesis of DNA during the asexual cycle of Plasmodium falciparum in culture

DNA synthesis in cultures of tightly synchronized, developing, asexual forms of Plasmodium falciparum began during the early trophozoite stage between 29.5 and 31 h after parasite invasion and continued through most of the remainder of schizogony. DNA synthesis was inhibited by both hydroxyurea and aphidicolin. Aphidicolin had a biphasic inhibitory effect on DNA synthesis with a portion of the DNA synthesis being considerably more resistant to inhibition. These agents did not block the morphological maturation of ring forms to early trophozoites but did block further maturation of trophozoites and schizonts.

Parasite polypeptides lost during schizogony and erythrocyte invasion by the malaria parasites, Plasmodium chabaudi and Plasmodium knowlesi

By biosynthetically labelling Plasmodium chabaudi and P. knowlesi stage-specific polypeptides and allowing continued development, schizogony and reinvasion in vivo or in vitro, we have identified parasite polypeptides not taken into the erythrocyte by the invading mezozoite. Three major and two minor parasite polypeptides synthesized by rings or mid-stage trophozoites of P. chabaudi were either degraded preferentially during further development, or lost during schizogony and reinvasion. For both P. chabaudi and P. knowlesi, a 250 000 mol. wt. polypeptide synthesized during maturation of trophozoites to schizonts and merozoites was not taken into the erythrocyte by the invading merozoite. The late stage synthesis of this polypeptide by P. chabaudi and its loss at schizogony and reinvasion was confirmed by immunofluorescence staining with a monoclonal antibody to this antigen. The importance of these antigens in the erythrocyte invasion process and in the induction and expression of immunity to malaria is discussed.

Effects of Chloroquine, Quinine, and Cycloguanil upon the Maturation of Asexual Erythrocytic Forms of Two Strains of Plasmodium Falciparum in Vitro

Summary Maturation of trophozoites of Plasmodium falciparum into schizonts was observed during studies in which defibrinated parasitized blood was placed in vials containing glucose and incubated without agitation for 24 hours. Chloroquine or quinine added to this system in vitro inhibited maturation at successively earlier stages of development as parasites were exposed to increasing amounts of these two drugs. In contrast, the most striking morphologic effect detected after the addition of the dihydrotriazine metabolite of chlorguanide (DHT) reflected formation of abnormal-appearing schizonts, with uninhibited maturation of parasites to the preschizont stage. Studies of the effects of chloroquine, of quinine, and of DHT upon maturation of asexual parasites in vitro were performed with two strains of P. falciparum that had been studied in vivo: a chloroquine-sensitive strain from Uganda and a chloroquine-resistant strain from Malaya. Chloroquine, quinine, and DHT inhibited the maturation of trophozoites of the Ugandan strain to a greater extent than the maturation of trophozoites of the Malayan strain. The system employed to assess the effects of drugs in vitro may prove to be of practical value in a number of ways, including the differentiation of drug-sensitive from drug-resistant strains of P. falciparum and the preliminary appraisal of blood schizonticidal effects of prospective antimalarial agents.