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Nucleic Acids Research Feb 1992We describe a chromosome translocation in a karyotype mutant of the rodent malarial parasite Plasmodium berghei. In this mutant (named EP) a small chromosome (chromosome...
We describe a chromosome translocation in a karyotype mutant of the rodent malarial parasite Plasmodium berghei. In this mutant (named EP) a small chromosome (chromosome 7), which has exhibited a size range between 0.9 and 1.4 Mb in other clones of P. berghei, is translocated to chromosome 13 or 14 with a size of about 3 Mb. By comparison of Apa-I restriction fragments of the chromosomes from mutant EP and from a reference clone (named HP) of P. berghei, we found evidence for a junction of subtelomeric chromosome 7 sequences and internal chromosome 13/14 sequences. In addition, a new chromosome of 1.4 Mb (named EP7) is present in mutant EP, which is (mainly) composed of sequences of chromosome 13/14. EP7 contains one telomeric region derived from chromosome 13/14. We found evidence that internal sequences of chromosome 13/14 are joined to telomeric sequences in the other telomeric region of EP7. The karyotype of mutant EP was stable during asexual and sexual multiplication and we found no indications for phenotypic changes.
Topics: Actins; Animals; Blotting, Southern; Chromosomes; Karyotyping; Plasmodium berghei; RNA, Protozoan; RNA, Ribosomal; Repetitive Sequences, Nucleic Acid; Tetrahydrofolate Dehydrogenase; Translocation, Genetic
PubMed: 1741291
DOI: 10.1093/nar/20.3.581 -
Malaria Journal Nov 2020The menace of resistance to anti-malarial drugs is a great challenge to malaria control, necessitating the search for new anti-malarial agents. This search has led to...
BACKGROUND
The menace of resistance to anti-malarial drugs is a great challenge to malaria control, necessitating the search for new anti-malarial agents. This search has led to the exploration of natural products for efficacy in malaria therapy. Omidun is the supernatant of fermenting maize (ogi) slurry that has been widely investigated and reported to possess several health benefits and it is used traditionally as solvent for preparing anti-malarial herbs. However, there is no information on the anti-malarial activity of omidun itself. This study was conducted to investigate the prophylactic, curative and suppressive anti-malarial potential of omidun.
METHODS
Experimental mice in the curative group were infected with 1 × 10 cells of Plasmodium berghei strain ANKA and treated with either 0.2 ml of omidun containing 3 × 10 cfu/ml of viable lactic acid bacteria or 0.2 ml of 5 mg/kg of chloroquine (positive control) or 0.2 ml of saline (negative control) for 4 days from day 3 post infection. The prophylactic group of mice were pre-treated with either omidun, chloroquine or saline for 4 days before infection with P. berghei, while the suppressive group was treated with omidun or chloroquine or saline and infected with P. berghei simultaneously. A group of mice were uninfected but treated (with omidun and control samples), while a final group was uninfected and untreated (controls). Parasitaemia and histopathology analysis were done in all groups.
RESULTS
The curative and suppressive groups showed a significant difference between the omidun-treated mice (100% parasitaemia reduction) and the untreated mice (54.5% parasitaemia increase). There was no significance difference between the omidun treatment and chloroquine (positive control) treatment in suppressive group as both treatment had 100% parasitaemia reduction. The omidun prophylactic treatment however did not show any parasitaemia suppression, but a significant difference was observed between the omidun treatment (85% increase) and the chloroquine (positive control) treatment (100% reduction) in the group. Omidun treatment is non-toxic to the kidney.
CONCLUSION
This study provides scientific evidence supporting omidun usage in the treatment of malaria. Consequently, further work may yield the specific component of omidun responsible for the anti-malarial activity.
Topics: Animals; Antimalarials; Fermentation; Malaria; Mice; Nigeria; Plasmodium berghei; Zea mays
PubMed: 33213477
DOI: 10.1186/s12936-020-03486-0 -
Cellular Physiology and Biochemistry :... 2008Plasmodia express a sphingomyelinase, which is apparently required for their development. On the other hand, the sphingomyelinase product ceramide has previously been...
Plasmodia express a sphingomyelinase, which is apparently required for their development. On the other hand, the sphingomyelinase product ceramide has previously been shown to delay parasite development. Moreover, ceramide triggers suicidal erythrocyte death or eryptosis, characterized by exposure of phosphatidylserine at the erythrocyte surface and cell shrinkage. Accelerated eryptosis of infected erythrocytes is considered to clear infected erythrocytes from circulating blood and, thus, to favourably influence the clinical course of malaria. The present experiments explored whether the sphingomyelinase inhibitor amitriptyline or genetic knockout of host acid sphingomyelinase influence in vitro parasite growth, eryptosis of Plasmodium falciparum-infected human erythrocytes, in vivo parasitemia and survival of P. berghei-infected mice. Phosphatidylserine exposure was determined by annexin V-binding and cell volume by forward scatter in FACS analysis. In vitro infection of human erythrocytes increased annexin- binding, an effect blunted in the presence of amitriptyline (>or=50 microM). Amitriptyline did not significantly alter intraerythrocytic parasite development but significantly (>or= 1 microM) delayed the increase in parasitemia in vitro. Most importantly, amitriptyline treatment (1 mM in drinking water) resulted in a significant delay of parasitemia and death of infected mice. However, upon infection, ceramide formation was stimulated in both, acid sphingomyelinase knockout mice (Smpd1(-/-)) and their wild type littermates (Smpd1(+/+)). Parasitemia following P. berghei infection was significantly lower in Smpd1(-/-) than in Smpd1(+/+) mice but did not significantly extend the life span of infected animals. In conclusion, mammalian and parasite sphingomyelinase contribute to ceramide formation during malaria, whereby the parasite sphingomyelinase ultimately determines the course of the infection. Amitriptyline presumably blocks both sphingomyelinases and, thus, its use might be a novel strategy to treat malaria.
Topics: Amitriptyline; Animals; Apoptosis; Ceramides; Erythrocytes; Female; Humans; Malaria; Male; Mice; Parasitemia; Phosphatidylserines; Plasmodium berghei; Survival Analysis
PubMed: 19088422
DOI: 10.1159/000185482 -
Cellular Microbiology Mar 2021Gametogenesis, the formation of gametes from gametocytes, an essential step for malaria parasite transmission, is targeted by transmission-blocking drugs and vaccines....
Gametogenesis, the formation of gametes from gametocytes, an essential step for malaria parasite transmission, is targeted by transmission-blocking drugs and vaccines. We identified a conserved protein (PBANKA_0305900) in Plasmodium berghei, which encodes a protein of 22 kDa (thus named Pb22) and is expressed in both asexual stages and gametocytes. Its homologues are present in all Plasmodium species and its closely related, Hepatocystis, but not in other apicomplexans. Pb22 protein was localised in the cytosols of schizonts, as well as male and female gametocytes. During gamete-to-ookinete development, Pb22 became localised on the plasma membranes of gametes and ookinetes. Compared to the wild-type (WT) parasites, P. berghei with pb22 knockout (KO) showed a significant reduction in exflagellation (~89%) of male gametocytes and ookinete number (~97%) during in vitro ookinete culture. Mosquito feeding assays showed that ookinete and oocyst formation of the pb22-KO line in mosquito midguts was almost completely abolished. These defects were rescued in parasites where pb22 was restored. Cross-fertilisation experiments with parasite lines defective in either male or female gametes confirmed that the defects in the pb22-KO line were restricted to the male gametes, whereas female gametes in the pb22-KO line were fertile at the WT level. Detailed analysis of male gametogenesis showed that 30% of the male gametocytes in the pb22-KO line failed to assemble the axonemes, whereas ~48.9% of the male gametocytes formed flagella but failed to egress from the host erythrocyte. To explore its transmission-blocking potential, recombinant Pb22 (rPb22) was expressed and used to immunise mice. in vitro assays showed that the rPb22-antisera significantly inhibited exflagellation by ~64.8% and ookinete formation by ~93.4%. Mosquitoes after feeding on rPb22-immunised mice also showed significant decreases in infection prevalence (83.3-93.3%) and oocyst density (93.5-99.6%). Further studies of the Pb22 orthologues in human malaria parasites are warranted.
Topics: Animals; Anopheles; Antibodies, Protozoan; Antigens, Protozoan; Apicomplexa; Cell Membrane; Gene Knockout Techniques; Malaria; Malaria Vaccines; Mice; Mice, Inbred BALB C; Plasmodium berghei; Protozoan Proteins
PubMed: 33222390
DOI: 10.1111/cmi.13294 -
Cell Chemical Biology Sep 2019Plasmodium parasites undergo an obligatory and asymptomatic developmental stage within the liver before infecting red blood cells to cause malaria. The hijacked host...
Plasmodium parasites undergo an obligatory and asymptomatic developmental stage within the liver before infecting red blood cells to cause malaria. The hijacked host pathways critical to parasite infection during this hepatic phase remain poorly understood. Here, we implemented a forward genetic screen to identify over 100 host factors within the human druggable genome that are critical to P. berghei infection in hepatoma cells. Notably, we found knockdown of genes involved in protein trafficking pathways to be detrimental to parasite infection. The disruption of protein trafficking modulators, including COPB2 and GGA1, decreases P. berghei parasite size, and an immunofluorescence study suggests that these proteins are recruited to the Plasmodium parasitophorous vacuole in infected hepatocytes. These findings reveal that various host intracellular protein trafficking pathways are subverted by Plasmodium parasites during the liver stage and provide new insights into their manipulation for growth and development.
Topics: Adaptor Proteins, Vesicular Transport; Animals; Carcinoma, Hepatocellular; Cell Line; Coatomer Protein; Communicable Diseases; Hep G2 Cells; Hepatocytes; Humans; Liver; Malaria; Mice; Parasites; Plasmodium; Plasmodium berghei; Protein Transport
PubMed: 31257182
DOI: 10.1016/j.chembiol.2019.05.011 -
FEBS Letters Jun 2018Plasmodium berghei is used as a rodent model for the study of malaria. However, multiple genetic manipulations are restricted by the paucity of selectable markers. The...
Plasmodium berghei is used as a rodent model for the study of malaria. However, multiple genetic manipulations are restricted by the paucity of selectable markers. The bsd-blasticidin selection system is widely used for eukaryotic cells; however, it could not previously be used for P. berghei due to toxicity to the rodent host. Here, we report the application of this selection system in P. berghei using an in vitro selection method. The desired bsd-integrated mutants are enriched by more than 90% within 2 weeks when using this system. Furthermore, the bsd marker can be used sequentially with established pyrimethamine- and puromycin-resistant markers. This system allows deeper understanding of malaria parasite biology through extensive genetic manipulation of P. berghei.
Topics: Aminohydrolases; Animals; Aspergillus; Drug Resistance; Fungal Proteins; Humans; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Plasmodium berghei; Puromycin; Pyrimethamine
PubMed: 29774536
DOI: 10.1002/1873-3468.13100 -
International Journal For Parasitology Aug 2019Sexual development in malaria parasites involves multiple signal transduction pathways mediated by reversible protein phosphorylation. Here, we functionally...
Sexual development in malaria parasites involves multiple signal transduction pathways mediated by reversible protein phosphorylation. Here, we functionally characterised a protein phosphatase, Ser/Thr protein phosphatase 5 (PbPP5), during sexual development of the rodent malaria parasite Plasmodium berghei. The recombinant protein phosphatase domain displayed obvious protein phosphatase activity and was sensitive to PP1/PP2A inhibitors including cantharidic acid (IC = 122.2 nM), cantharidin (IC = 74.3 nM), endothall (IC = 365.5 nM) and okadaic acid (IC = 1.3 nM). PbPP5 was expressed in both blood stages and ookinetes with more prominent expression during sexual development. PbPP5 was localised in the cytoplasm of the parasite and highly concentrated beneath the parasite plasma membrane in free merozoites and ookinetes. Targeted deletion of the pbpp5 gene had no influence on asexual blood-stage parasite multiplication or the survival curve of the infected hosts. However, male gamete formation and fertility were severely affected, resulting in almost complete blockade of ookinete conversion and oocyst development in the Δpbpp5 lines. This sexual development defect was rescued by crossing Δpbpp5 with the female defective Δpbs47 parasite line, but not with the male defective Δpbs48/45 line, thus confirming the essential function of the pbpp5 gene in male gamete fertility. Furthermore, the aforementioned PP1/PP2A inhibitors all had inhibitory effects on exflagellation of male gametocytes and ookinete conversion. In particular, endothall, a selective inhibitor of PP2A, completely blocked exflagellation and ookinete conversion at ∼548.3 nM. This study elucidated an essential function of PbPP5 during male gamete development and fertility.
Topics: Animals; Blotting, Western; Female; Fertility; Fluorescent Antibody Technique, Indirect; Inhibitory Concentration 50; Male; Mice; Mice, Inbred BALB C; Phosphoprotein Phosphatases; Plasmodium berghei
PubMed: 31202684
DOI: 10.1016/j.ijpara.2019.03.007 -
Malaria Journal Jan 2015The evolution of drug-resistant parasites is a major hindrance to malaria control, and thus understanding the behaviour of drug-resistant mutants is of clinical...
BACKGROUND
The evolution of drug-resistant parasites is a major hindrance to malaria control, and thus understanding the behaviour of drug-resistant mutants is of clinical relevance. The study aimed to investigate how resistance against lumefantrine (LU) and piperaquine (PQ), anti-malarials used as partner drugs in artemisinin-based combination therapy (ACT), impacts parasite fitness. This is important since resistance to ACT, the first-line anti-malarial regimen is increasingly being reported.
METHODS
The stability of Plasmodium berghei ANKA strain that was previously selected for LU and PQ resistance was evaluated using the 4-day assay and established infection test in mice. Fitness cost of resistance was determined by comparing parasites proliferation rates in absence of drug pressure for the drug-exposed parasites between day 4 and 7 post-infection (pi), relative to the wild-type. Statistical analysis of data to compare mean parasitaemia and growth rates of respective parasite lines was carried out using student's t-test and one-way analysis of variance, with significance level set at p<0.05.
RESULTS
During serial passaging in the absence of the drug, the PQ-resistant parasite maintained low growth rates at day 7 pi (mean parasitaemia, 5.6% ± 2.3) relative to the wild-type (28.4% ± 6.6), translating into a fitness cost of resistance of 80.3%. Whilst resistance phenotype for PQ was stable, that of LU was transient since after several serial passages in the absence of drug, the LU-exposed line assumed the growth patterns of the wild-type.
CONCLUSIONS
The contrasting behaviour of PQ- and LU-resistance phenotypes support similar findings which indicate that even for drugs within the same chemical class, resistance-conferred traits may vary on how they influence parasite fitness and virulence. Resistance-mediating polymorphisms have been associated with less fit malaria parasites. In the absence of drug pressure in the field, it is therefore likely that the wild-type parasite will out-compete the mutant form. This implies the possibility of reintroducing a drug previously lost to resistance, after a period of suspended use. Considering the recent reports of high failure rates associated with ACT, high fitness cost of resistance to PQ is therefore of clinical relevance as the drug is a partner in ACT.
Topics: Animals; Antimalarials; Disease Models, Animal; Drug Resistance; Ethanolamines; Fluorenes; Genetic Fitness; Lumefantrine; Malaria; Male; Mice; Plasmodium berghei; Quinolines
PubMed: 25627576
DOI: 10.1186/s12936-015-0550-5 -
Infection and Immunity Aug 1998Pbs21 is a surface protein of the ookinete of Plasmodium berghei, which can induce a potent transmission-blocking immune response. Pbs21 is normally expressed only by...
Pbs21 is a surface protein of the ookinete of Plasmodium berghei, which can induce a potent transmission-blocking immune response. Pbs21 is normally expressed only by parasite stages in the mosquito, i.e., female gametes/zygotes, ookinetes, and oocysts. However, the Pbs21 gene is transcribed in female gametocytes which circulate in the bloodstream of the host, where translation of the resulting mRNA is totally repressed. Episomal transfection has been used to investigate whether expression of Pbs21 protein could be achieved in blood stages of the parasite. By using plasmid pMD221, the complete mRNA-encoding region of Pbs21, flanked only by 218 nucleotides (nt) of its promoter region and 438 nt of its 3' region downstream from the polyadenylation site, was introduced into the blood stages of gametocyte-producing and non-gametocyte-producing clones of P. berghei. In both of these transformed parasite lines, Pbs21 protein was expressed in asexual trophozoites, schizonts, and, when present, in both male and female gametocytes. Hence, the flanking regions present are sufficient to allow transcription but lack the elements that exert natural control of sex- and stage-specific transcription. The mRNA and the protein expressed by transformed blood stages were indistinguishable from the wild-type forms by the criteria tested, and the protein was recognized by both conformation-dependent and conformation-independent monoclonal antibodies raised against native Pbs21. In mice infected with transformed non-gametocyte-producing parasites, a Pbs21-specific immune response was induced and characterized with respect to isotype (IgG2a/IgG2b) and quantity (11. 5 +/- 10 microg/ml) of antibody produced. However, as found in previous studies, these antibody levels were insufficient to inhibit development of the parasites in the mosquito. The ability to express mosquito midgut-stage antigens in blood-stage parasites will facilitate further investigations of molecular and immunological properties of these proteins.
Topics: Animals; Animals, Genetically Modified; Anopheles; Antigens, Protozoan; Antigens, Surface; Base Sequence; DNA, Protozoan; Female; Gene Expression; Genes, Protozoan; Male; Mice; Molecular Sequence Data; Open Reading Frames; Peptide Chain Initiation, Translational; Plasmids; Plasmodium berghei; Protein Biosynthesis; Protozoan Proteins; RNA, Messenger; Transcription, Genetic; Transformation, Genetic; Transgenes
PubMed: 9673276
DOI: 10.1128/IAI.66.8.3884-3891.1998 -
Malaria Journal Jun 2012DNA microarrays have been a valuable tool in malaria research for over a decade but remain in limited use in part due their relatively high cost, poor availability, and...
BACKGROUND
DNA microarrays have been a valuable tool in malaria research for over a decade but remain in limited use in part due their relatively high cost, poor availability, and technical difficulty. With the aim of alleviating some of these factors next-generation DNA microarrays for genome-wide transcriptome analysis for both Plasmodium falciparum and Plasmodium berghei using the Agilent 8 x 15 K platform were designed.
METHODS
Probe design was adapted from previously published methods and based on the most current transcript predictions available at the time for P. falciparum or P. berghei. Array performance and transcriptome analysis was determined using dye-coupled, aminoallyl-labelled cDNA and streamlined methods for hybridization, washing, and array analysis were developed.
RESULTS
The new array design marks a notable improvement in the number of transcripts covered and average number of probes per transcript. Array performance was excellent across a wide range of transcript abundance, with low inter-array and inter-probe variability for relative abundance measurements and it recapitulated previously observed transcriptional patterns. Additionally, improvements in sensitivity permitted a 20-fold reduction in necessary starting RNA amounts, further reducing experimental costs and widening the range of application.
CONCLUSIONS
DNA microarrays utilizing the Agilent 8 x 15 K platform for genome-wide transcript analysis in P. falciparum and P. berghei mark an improvement in coverage and sensitivity, increased availability to the research community, and simplification of the experimental methods.
Topics: Gene Expression Profiling; Oligonucleotide Array Sequence Analysis; Parasitology; Plasmodium berghei; Plasmodium falciparum
PubMed: 22681930
DOI: 10.1186/1475-2875-11-187