Different Stages Of Parasite Development Research Articles

Vaccination against malaria

Vaccination against malaria is an old dream that reemerged in 2015 with the European Medicines Agency's favourable opinion on a first antimalarial vaccine, RTS,S/ AS01. Six years later, the World Health Organization (WHO) is advising a wide deployment of this vaccine in sub-Saharan Africa and in regions with high and moderate transmission where Plasmodium falciparum circulates. This follows favourable results from the pilot programme in Ghana, Kenya and Malawi involving over 800,000 children since 2019. This article addresses the objectives and main vaccine candidates targeting the different stages of parasite development, highlighting the progress and limitations of these different approaches. The RTS,S saga has been a milestone in vaccine development, with a first-generation vaccine recommended by the WHO for use in children over 5 months of age in sub-Saharan Africa and other areas of moderate to high transmission of P. falciparum malaria, in combination with other prevention measures. Research efforts continue to better understand the correlates of protection. With advances in vaccine platforms, new multi-antigen, multi-stage, and even multi-species approaches might emerge and brighten the horizon for malaria control.

Identification of Plasmodiumdipeptidyl aminopeptidase allosteric inhibitors by high throughput screening.

Dipeptidyl aminopeptidases (DPAPs) are cysteine proteases that cleave dipeptides from the N-terminus of protein substrates and have been shown to play important roles in many pathologies including parasitic diseases such as malaria, toxoplasmosis and Chagas’s disease. Inhibitors of the mammalian homologue cathepsin C have been used in clinical trials as potential drugs to treat chronic inflammatory disorders, thus proving that these enzymes are druggable. In Plasmodium species, DPAPs play important functions at different stages of parasite development, thus making them potential antimalarial targets. Most DPAP inhibitors developed to date are peptide-based or peptidomimetic competitive inhibitors. Here, we used a high throughput screening approach to identify novel inhibitor scaffolds that block the activity of Plasmodium falciparum DPAP1. Most of the hits identified in this screen also inhibit Plasmodium falciparum DPAP3, cathepsin C, and to a lesser extent other malarial clan CA proteases, indicating that these might be general DPAP inhibitors. Interestingly, our mechanism of inhibition studies indicate that most hits are allosteric inhibitors, which opens a completely new strategy to inhibit these enzymes, study their biological function, and potentially develop new inhibitors as starting points for drug development.

Combination of quercetin and ivermectin: In vitro and in vivo effects against Haemonchus contortus

The aim of the present study was to evaluate the in vitro effect of quercetin combined with ivermectin (IVM) on Haemonchus contortus larvae and adults with different resistance profiles and demonstrate the in vivo anthelmintic action of this combination when used in sheep naturally infected. The effect of combination was evaluated based on the analysis of the mean effective concentration (EC50) obtained for larvae using the larval migration inhibition test and for adults using the motility test on females. The tests with larvae and adults were conducted using isolates with different degrees of susceptibility to IVM (sensitive, intermediate and highly resistant). The in vivo effect was evaluated based on the reduction in the egg count (FEC) and reduction in the count of adult helminths recovered after parasitological necropsy. Using the combination of quercetin with IVM, it was observed that in larvae, quercetin did not significantly reduce the EC50 for IVM in the sensitive and highly resistant isolates, but led to a significant reduction in the EC50 for IVM in the intermediate isolate. In adults, quercetin did not significantly reduce the EC50 for IVM in any of the isolates. No significant effect of the combination was found regarding the reduction in FEC or total count of parasites. The results of the in vitro and in vivo tests performed in the present study on quercetin activity underscore the importance of evaluating resistance-reversing agents among different stages of parasite development as well as among isolates with different resistance profiles. The action of quercetin combined with IVM on the motility of H. contortus larvae and adults was influenced by the degree of resistance and development stage of the parasite. The combination was effective only on intermediate resistant larvae. No action of the combination against adults was found. Moreover, this combination, when administered through the intra-abomasal route, was not effective at reducing the FEC and parasite load of naturally infected sheep.

Immune cell kinetics in the ovine abomasal mucosa following hyperimmunization and challenge withHaemonchus contortus

Sheep were sensitized by repeated infection with Haemonchus contortus L3, followed by a 12 week rest period, and an abomasal cannula was surgically implanted in all sheep. Seven of the sensitized sheep were subsequently challenged with 50 000 H. contortus L3 while 4 control sheep were challenged with saline. Biopsy samples were taken using a fibreoptic endoscope on days 0, 1, 2, 3, 5, 7 and 28 after challenge and leukocyte subpopulations quantified by (immuno)histology. Differential blood cell counts were performed on the same days. At the end of the trial, sheep showed significantly reduced worm burdens compared to unsensitized control sheep, confirming their resistance status. Both blood and tissue eosinophils, as well as tissue γδ TCR+ cells were rapidly elevated by day 1 post L3 challenge (pc), peaking at day 3 pc. There was a slight increase in tissue CD4 T cells at day 2 pc, peaking at day 3 pc while no significant changes in CD8 T cells were observed. B cells (CD45R+) increased later into challenged tissues with a peak at 5 days pc. All tissue lymphocyte subpopulations as well as tissue and blood eosinophils were reduced by day 7 pc before increasing again at day 28 pc, suggesting separate responses to larval and adult antigens. In contrast, globule leukocytes and mucosal mast cells only showed one peak at day 5 pc and 28 pc, respectively. Unexpectedly, globule leukocytes correlated significantly with tissue eosinophils but not mucosal mast cells. The results are consistent with an early eosinophil-mediated killing of L3, possibly recruited by IL-5 produced by γδ T cells. In contrast to post-mortem studies, abomasal cannulation allowed sequential analysis of both early and late time points in the same animal, providing a more complete picture of cellular interactions at both peripheral and local sites, and their correlation with the different stages of parasite development.

Characteristics of Plasmodium falciparum -Infected-Erythrocyte Adhesion to Chondroitin Sulfate

In a recent article in Infection and Immunity, Madhunapantula et al. stated that the characteristics of adhesion of Plasmodium falciparum-infected erythrocytes (IEs) to chondroitin-4-sulfate (C4S) at different stages of parasite development were not known (6), and they presented findings that examined these properties. However, those authors have overlooked earlier reports that documented the effects of parasite development on IE adhesion to C4S. Previously, we studied P. falciparum over its full intraerythrocytic life cycle and reported that adhesion to C4S (also known as chondroitin sulfate A) varies with the developmental stage of the parasite (3). Adhesion to immobilized C4S occurred at low levels when parasites were at the immature ring stage and increased with parasite age until adhesion was maximal at the mature pigmented trophozoite stage (24 to 30 h). The levels of adhesion then declined as parasite age increased, and the adhesion of mature schizont-stage parasites was 38% of the maximal level of adhesion observed (3). Madhunapantula et al. now report similar findings, also demonstrating that adhesion declined as parasites matured into schizonts (6). Others reported that C4S-mediated adhesion of IEs to Saimiri brain endothelial cells did not significantly change from 24 to 44 h postinvasion (7), suggesting that adhesion to intact cells may differ from adhesion to purified receptors. The levels of adhesion to C4S across developmental stages are broadly consistent with the developmental stages of parasites that appear to be sequestered in the placenta (1). Of further relevance is the fact that others have reported that the adhesion of IEs to uninfected erythrocytes (rosette formation) and the binding of specific immunoglobulin G to IE surface antigens were lower among schizonts (8). Madhunapantula et al. also incorrectly stated that we found that the adhesion of the parasite line 3D7-CSA to C4S was “completely unaffected” by trypsin treatment (100 μg/ml) of the IE surface (6). Instead, we found that the adhesion of 3D7-CSA was inhibited by 57% at 10 μg/ml and >90% at 100 μg/ml (4) after a 15-min incubation, which is not dissimilar to results now reported by Madhunapantula et al. (although different concentrations of trypsin and incubation conditions were used). We did find that the parasite line CS2 was largely resistant to trypsin treatment at 100 μg/ml, even though the surface P. falciparum erythrocyte membrane protein 1 (PfEMP1) was cleaved (4). Although the sensitivity of IE adhesion to trypsin varied between parasite lines, trypsin cleaved PfEMP1 in all lines we studied (4). Differences between parasite lines in the levels of sensitivity of C4S adhesion to trypsin might be explained by polymorphisms in var2csa that affect the sites at which cleavage occurs (4). Interestingly, polymorphisms in var2csa also correspond with differences between isolates in the fine specificity of interactions with structural motifs of C4S for IE adhesion (2). It should be noted that the conditions used for the trypsin treatment of IEs has varied among different reports. We used short incubation times (10 to 15 min), consistent with conditions used in the original characterization of PfEMP1 (5). An awareness of these differences may be important when comparing results of studies.

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.

Observations on the internal and surface morphology of malaria infected blood cells using optical and atomic force microscopy

We describe a simple and fast method to probe the morphological changes on the exterior and interior of a malaria infected erythrocyte at different stages of parasite development. This involves the imaging and scanning of Giemsa stained malaria infected erythrocytes using optical microscopy and atomic force microscopy, respectively.

Merozoite surface protein 1-specific immune response is protective against exoerythrocytic forms of Plasmodium yoelii.

One of the difficulties in developing an effective malaria vaccine is the antigenic change of the parasite during the life cycle. It is desirable that vaccine-induced protective immunity be effective at different stages of parasite development. Merozoite surface protein 1 (MSP1) is a candidate vaccine antigen against blood-stage malaria, but it is also expressed in the exoerythrocytic forms. It was not known, however, whether the anti-MSP1 immune response is effective against the liver-stage malaria parasite. We generated a recombinant protein of MSP1 fused to heat-shock cognate protein 70 (hsc70) and studied its vaccination effect. When C57BL/6 mice were immunized with the fusion protein prior to challenge infection with Plasmodium yoelii sporozoites, the onset of parasitemia was delayed or no parasitemia was observed. To determine whether this was due to the protective immunity against liver-stage parasites, P. yoelii-specific rRNA in the infected liver was quantitated by real-time reverse transcription-PCR analysis. The level of parasite-specific rRNA was reduced in mice immunized with the fusion protein of MSP1 and hsc70 but not with hsc70 alone, indicating that MSP1-specific immunity can be protective against the exoerythrocytic form of the parasite. Furthermore, the adoptive transfer experiments of immune lymphocytes and serum into naive mice suggested that the protective immunity was dependent on cellular and not humoral immunity. Finally, the vaccine-induced protection was also observed in A/J, C3H, and BALB/c mice, suggesting that MSP1-specific protective immunity at the exoerythrocytic stage can be induced in animals over a wide range of genetic backgrounds.

A Plasmodium chabaudi chabaudi high molecular mass glycoprotein translocated to the host cell membrane by a non-classical secretory pathway

We have purified and characterized a novel high molecular mass glycoprotein of P. chabaudi chabaudi (Pc550gp) that is transported to the erythrocyte membrane during the intraerythrocytic cycle. Immuno fluorescence assays with polyclonal monospecific antibodies against Pc550gp show that the protein to be localized in the periphery of young trophozoite stages i.e., on the plasma membrane or parasitophorous vacuole membrane. However, in late trophozoites and schizonts the antigen is distributed in both parasite and host cell membranes. These results were confirmed by immunoblotting of isolated parasites and infected host cell membranes at different stages of parasite development. Moreover, alkali extraction of purified infected erythrocyte membranes at mature stages of parasite development does not solubilize Pc550gp, suggesting that it is an integral membrane protein. In addition proteinase K digestion of intact infected host cells induced the disappearance of Pc550gp. Further indicating its transmembrane nature and that it presents extracellular domains susceptible to proteolysis. Brefeldin A or low temperature (15 degrees C) treatment did not affect the translocation of Pc550gp from the parasite compartments to the erythrocyte membrane, indicating that the secretion of Pc550gp does not follow the classical transport pathway described in most eukaryotic cells.

Representation of Differential Expression: A New Approach to Study Differential Gene Expression in Trypanosomatids

During the past five years, several methods have been described that allow the isolation and cloning of stage-specific or cell-specific genes. The characterization of genes expressed at different stages of parasite development is of the utmost importance for the understanding of the mechanisms involved in the regulation of gene expression. Here, Samuel Goldenberg and Marco Aurelio Krieger describe a method for the amplification and cloning of Trypanosoma cruzi genes expressed specifically at different times of the metacyclogenesis process. This method, representation of differential expression (RDE), should be useful for the isolation and cloning of any trypanosomatid gene transcribing differentially expressed messenger RNA.

Plasmodium falciparum:Differential Parasite Growth Inhibition Mediated by Antibodies to the Antigens Pf332 and Pf155/RESA

Plasmodium falciparum-reactive antibodies can inhibit growth of parasite blood stagesin vitroby interfering at different stages of parasite development. Antibodies reactive with repeat sequences in Pf155/ring-infected surface antigen (Pf155/RESA) or in antigen Pf332 inhibit parasite growth as determined by a reduction of newly infected erythrocytes. Antibodies to Pf155/RESA have been implicated in merozoite invasion inhibition. Since it was considered unlikely that antibodies to the late stage antigen Pf332 act on merozoite invasion, we investigated at what developmental stage this growth inhibition takes place. Parasites were cultured in the presence of antibodies to repeat sequences of either Pf332 or Pf155/RESA and were examined with regard to the distribution of different stages and their morphology. As expected, antibodies to both antigens decreased the number of ring stages after reinfection. They did not affect the intraerythrocytic development of ring stages or trophozoites. However, antibodies to Pf332 induced high levels of schizonts displaying an abnormal morphology while antibodies to Pf155/RESA induced considerably lower levels of degenerated schizonts. Thus, Pf332-reactive antibodies seem to interfere with the schizont development by blocking the rupture of mature schizonts or, alternatively, by interfering intraerythrocytically with late stage parasites.

The pharmacokinetics of chloroquine in healthy and Plasmodium chabaudi-infected mice: implications for chronotherapy.

The schizogony of malarial parasite is a typical cyclic phenomenon where the different stages of parasite development appear at regular time intervals. Each of the stages is specifically sensitive to different antimalarial drugs. Knowledge of the details of the cycle, drug susceptibility and the pharmacokinetics of drugs, could allow the improvement of drug action by the chronotherapeutic approach: treatment at the time of appearance of the drug sensitive stage with a drug that displays rapid pharmacokinetics. Since murine malarias serve as preferable models for in vivo drug testing, the pharmacokinetics of subcutaneously (sc) administered chloroquine (CQ) were tested in the whole blood of healthy mice and in animals slightly (1.5-3.5% parasitemia) or heavily infected (21-25% parasitemia) with Plasmodium chabaudi chabaudi. The half-time of absorption was around 5 min and almost independent of parasitemia. The apparent half-time of drug concentration decay was around 40 min in healthy animals, about 90 min at low parasitemia and about 410 min in heavy infection, indicating that the concentration of CQ is a typical spike, that is prolonged by asymptomatic disease, and considerably more by the active accumulation of CQ in infected cells. The latter is confirmed by the 3-fold higher peak blood [CQ] at the trophozoite stage and < 1.5-fold increase during schizogony. In conjunction with our previous experiments which showed that a single sc injection of 5 mg/kg CQ is sufficient to eliminate the drug susceptible mid-term trophozoite stage, the present results seem to justify to propose the chronotherapeutic approach for the treatment of malaria.

Digestion of the host erythrocyte by malaria parasites is the primary target for quinolinecontaining antimalarials

Intraerythrocytic malaria parasites feed on their host cell cytosol. We show that human red blood cells infected with the malaria parasite Plasmodium falciparum, produce free amino adds the composition of which resembles that of globin, the most abundant red blood cell protein. The rate of amino acid production is almost equal to the rate of efflux of these acids from the infected cell. Production of amino acids increases with parasite age: the rates of production at the young ring and the mature trophozoite stages were 3.3 and 13.5 nmol/10 8 infected cells per min at 37°, respectively, compared with 0.04 nmol/10 8 cells per min in uninfected cells. The quinoline-containing antimalarial drugs, chloroquine, quinine and mefloquine, inhibit amino acid production at the same concentrations at which they inhibit parasite growth, but have no effect on the endogenous parasite protein degradation. We suggest that parasite feeding on host cell cytosol is the primary target for the antimalarial action of these drugs. Chloroquine accumulation, the rate of amino add production by infected cells and the inhibitory effect of the drug, were determined simultaneously at the different stages of parasite development. At all stages the rate of amino add production and chloroquine accumulation were directly related and both were inversely related to the inhibitory effidency of the drug. The lysosomotropic agents methylamine and NH 4Cl at millimolar concentrations also inhibit amino add production, suggesting that the process is pH dependent and localized in the vacuole. Host cytosol degradation and drug accumulation both ake place in the parasite food vacuole. Our observations imply that the metabolically dependent acidification of this parasite organelle is involved in both processes.

On the parasitism of Alectra vogeliiBenth. (Scrophulariaceae) I. Early development of the primary haustorium and initiation of the stem

The development of the primary haustorium of the hemiparasite, Alectra vogelii , on the roots of one of its hosts, Voandzeia subterranea (Fabaceae), has been studied morphologically and anatomically. A simple method for cultivation was used in order to observe the different stages of parasite development. Upon contact between parasite seedling and host, localized swelling of host root tissue ensues. The front cells of the advancing primary haustorium are densely structured and directed towards the stele of the host root. Several lateral roots originate in the swollen area of contact. That part of the formerly rather thin root of the parasite that remains outside the host root thickens. The apical meristem of the stem then grows out of the seed coat. The nature of the connecting tissue within the haustorium is presently being investigated.