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Malaria Journal Jun 2017Aspartate, which is converted from oxaloacetate (OAA) by aspartate aminotransferase, is considered an important precursor for purine salvage and pyrimidine de novo...
BACKGROUND
Aspartate, which is converted from oxaloacetate (OAA) by aspartate aminotransferase, is considered an important precursor for purine salvage and pyrimidine de novo biosynthesis, and is thus indispensable for the growth of Plasmodium parasites at the asexual blood stages. OAA can be produced in malaria parasites via two routes: (i) from phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxylase (PEPC) in the cytosol, or (ii) from fumarate by consecutive reactions catalyzed by fumarate hydratase (FH) and malate:quinone oxidoreductase (MQO) in the mitochondria of malaria parasites. Although PEPC-deficient Plasmodium falciparum and Plasmodium berghei (rodent malaria) parasites show a growth defect, the mutant P. berghei can still cause experimental cerebral malaria (ECM) with similar dynamics to wild-type parasites. In contrast, the importance of FH and MQO for parasite viability, growth and virulence is not fully understood because no FH- and MQO-deficient P. falciparum has been established. In this study, the role of FH and MQO in the pathogenicity of asexual-blood-stage Plasmodium parasites causing cerebral malaria was examined.
RESULTS
First, FH- and MQO-deficient parasites were generated by inserting a luciferase-expressing cassette into the fh and mqo loci in the genome of P. berghei ANKA strain. Second, the viability of FH-deficient and MQO-deficient parasites that express luciferase was determined by measuring luciferase activity, and the effect of FH or MQO deficiency on the development of ECM was examined. While the viability of FH-deficient P. berghei was comparable to that of control parasites, MQO-deficient parasites exhibited considerably reduced viability. FH activity derived from erythrocytes was also detected. This result and the absence of phenotype in FH-deficient P. berghei parasites suggest that fumarate can be metabolized to malate by host or parasite FH in P. berghei-infected erythrocytes. Furthermore, although the growth of FH- and MQO-deficient parasites was impaired, the development of ECM was suppressed only in mice infected with MQO-deficient parasites.
CONCLUSIONS
These findings suggest that MQO-mediated mitochondrial functions are required for development of ECM of asexual-blood-stage Plasmodium parasites.
Topics: Animals; Blood-Brain Barrier; Erythrocytes; Female; Fumarate Hydratase; Fumarates; Malaria, Cerebral; Malates; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Oxaloacetic Acid; Oxidoreductases; Plasmodium berghei; Specific Pathogen-Free Organisms
PubMed: 28606087
DOI: 10.1186/s12936-017-1898-5 -
Asian Pacific Journal of Tropical... Jan 2011To search and analyze nitric oxide synthase (NOS) and similar proteins from Plasmodium berghei(Pb).
OBJECTIVE
To search and analyze nitric oxide synthase (NOS) and similar proteins from Plasmodium berghei(Pb).
METHODS
The structure and function of nitric oxide synthase and similar proteins from Plasmodium berghei were analyzed and predicted by bioinformatics.
RESULTS
PbNOS were not available, but nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium (NADPH)-cytochrome p450 reductase(CPR) were gained. PbCPR was in the nucleus of Plasmodium berghei, while 134aa-229aa domain was localize in nucleolar organizer. The amino acids sequence of PbCPR had the closest genetic relationship with Plasmodium vivax showing a 73% homology. The tertiary structure of PbCPR displayed the forcep-shape with wings, but no wings existed in the tertiary structure of its' host, Mus musculus(Mm). 137aa-200aa, 201aa-218aa, 220aa-230aa, 232aa-248, 269aa-323aa, 478aa-501aa and 592aa-606aa domains of PbCPR showed no homology with MmCPRs', and all domains were exposed on the surface of the protein.
CONCLUSIONS
NOS can't be found in Plasmodium berghei and other Plasmodium species. PbCPR may be a possible resistance site of antimalarial drug, and the targets of antimalarial drug and vaccine. It may be also one of the mechanisms of immune evasion. This study on Plasmodium berghei may be more suitable to Plasmodium vivax. And 137aa-200aa, 201aa-218aa, 220aa-230aa, 232aa-248, 269aa-323aa, 478aa-501aa and 592aa-606aa domains of PbCPR are more ideal targets of antimalarial drug and vaccine.
Topics: Animals; Cluster Analysis; Computational Biology; Mice; Models, Molecular; NADPH-Ferrihemoprotein Reductase; Nitric Oxide Synthase; Phylogeny; Plasmodium berghei; Plasmodium vivax; Protein Structure, Tertiary; Protozoan Proteins; Sequence Homology, Amino Acid
PubMed: 21771405
DOI: 10.1016/S1995-7645(11)60021-7 -
Malaria Journal Jan 2009The ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins. ABC proteins play key roles in cellular detoxification of...
BACKGROUND
The ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins. ABC proteins play key roles in cellular detoxification of endobiotics and xenobiotics. Overexpression of certain ABC proteins, among them the multidrug resistance associated protein (MRP), contributes to drug resistance in organisms ranging from human neoplastic cells to parasitic protozoa. In the present study, the Plasmodium berghei mrp gene (pbmrp) was partially characterized and the predicted protein was classified using bioinformatics in order to explore its putative involvement in drug resistance.
METHODS
The pbmrp gene from the P. berghei drug sensitive, N clone, was sequenced using a PCR strategy. Classification and domain organization of pbMRP were determined with bioinformatics. The Plasmodium spp. MRPs were aligned and analysed to study their conserved motifs and organization. Gene copy number and organization were determined via Southern blot analysis in both N clone and the chloroquine selected line, RC. Chromosomal Southern blots and RNase protection assays were employed to determine the chromosomal location and expression levels of pbmrp in blood stages.
RESULTS
The pbmrp gene is a single copy, intronless gene with a predicted open reading frame spanning 5820 nucleotides. Bioinformatic analyses show that this protein has distinctive features characteristic of the ABCC sub-family. Multiple sequence alignments reveal a high degree of conservation in the nucleotide binding and transmembrane domains within the MRPs from the Plasmodium spp. analysed. Expression of pbmrp was detected in asexual blood stages. Gene organization, copy number and mRNA expression was similar in both lines studied. A chromosomal translocation was observed in the chloroquine selected RC line, from chromosome 13/14 to chromosome 8, when compared to the drug sensitive N clone.
CONCLUSION
In this study, the pbmrp gene was sequenced and classified as a member of the ABCC sub-family. Multiple sequence alignments reveal that this gene is homologous to the Plasmodium y. yoelii and Plasmodium knowlesi mrp, and the Plasmodium vivax and Plasmodium falciparum mrp2 genes. There were no differences in gene organization, copy number, or mRNA expression between N clone and the RC line, but a chromosomal translocation of pbmrp from chromosome 13/14 to chromosome 8 was detected in RC.
Topics: ATP-Binding Cassette Transporters; Amino Acid Sequence; Animals; Antimalarials; Blotting, Southern; Chloroquine; Chromosome Mapping; Computational Biology; Drug Resistance, Multiple; Electrophoresis, Gel, Pulsed-Field; Malaria; Mice; Molecular Sequence Data; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Open Reading Frames; Plasmodium berghei; Polymerase Chain Reaction; Protozoan Proteins; RNA, Protozoan; Sequence Alignment
PubMed: 19118502
DOI: 10.1186/1475-2875-8-1 -
Malaria Journal May 2011The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This...
BACKGROUND
The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR) mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development.
METHODS
A system to identify mutations in Pfdhfr gene that confer antifolate drug resistance using an animal Plasmodium parasite model was developed. By using error-prone PCR and Plasmodium transfection technologies, libraries of Pfdhfr mutant were generated and then episomally transfected to Plasmodium berghei parasites, from which pyrimethamine-resistant PfDHFR mutants were selected.
RESULTS
The principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic P. berghei, in which endogenous Pbdhfr allele was replaced with the mutant PfdhfrS108N, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine.
CONCLUSION
This study demonstrated the power of the transgenic P. berghei system to predict drug-resistant Pfdhfr mutations in an in vivo parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.
Topics: Animals; Antimalarials; Drug Resistance; Female; Folic Acid Antagonists; Gene Expression Regulation, Enzymologic; Humans; Malaria, Falciparum; Mice; Mice, Inbred BALB C; Mutation; Organisms, Genetically Modified; Parasitemia; Plasmids; Plasmodium berghei; Plasmodium falciparum; Polymerase Chain Reaction; Protozoan Proteins; Pyrimethamine; Tetrahydrofolate Dehydrogenase; Transfection
PubMed: 21554743
DOI: 10.1186/1475-2875-10-119 -
Nature Communications Nov 2015The proteins of Plasmodium, the malaria parasite, are strikingly rich in asparagine. Plasmodium depends primarily on host haemoglobin degradation for amino acids and has...
The proteins of Plasmodium, the malaria parasite, are strikingly rich in asparagine. Plasmodium depends primarily on host haemoglobin degradation for amino acids and has a rudimentary pathway for amino acid biosynthesis, but retains a gene encoding asparagine synthetase (AS). Here we show that deletion of AS in Plasmodium berghei (Pb) delays the asexual- and liver-stage development with substantial reduction in the formation of ookinetes, oocysts and sporozoites in mosquitoes. In the absence of asparagine synthesis, extracellular asparagine supports suboptimal survival of PbAS knockout (KO) parasites. Depletion of blood asparagine levels by treating PbASKO-infected mice with asparaginase completely prevents the development of liver stages, exflagellation of male gametocytes and the subsequent formation of sexual stages. In vivo supplementation of asparagine in mice restores the exflagellation of PbASKO parasites. Thus, the parasite life cycle has an absolute requirement for asparagine, which we propose could be targeted to prevent malaria transmission and liver infections.
Topics: Animals; Anopheles; Asparaginase; Asparagine; Aspartate-Ammonia Ligase; Fluorescent Antibody Technique; Gene Knockout Techniques; Life Cycle Stages; Liver; Malaria; Mice; Organisms, Genetically Modified; Plasmodium berghei; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 26531182
DOI: 10.1038/ncomms9775 -
Infection and Immunity Mar 2007Macrophage migration inhibitory factor (MIF) is a mammalian cytokine that participates in innate and adaptive immune responses. Homologues of mammalian MIF have been...
Macrophage migration inhibitory factor (MIF) is a mammalian cytokine that participates in innate and adaptive immune responses. Homologues of mammalian MIF have been discovered in parasite species infecting mammalian hosts (nematodes and malaria parasites), which suggests that the parasites express MIF to modulate the host immune response upon infection. Here we report the first biochemical and genetic characterization of a Plasmodium MIF (PMIF). Like human MIF, histidine-tagged purified recombinant PMIF shows tautomerase and oxidoreductase activities (although the activities are reduced compared to those of histidine-tagged human MIF) and efficiently inhibits AP-1 activity in human embryonic kidney cells. Furthermore, we found that Plasmodium berghei MIF is expressed in both a mammalian host and a mosquito vector and that, in blood stages, it is secreted into the infected erythrocytes and released upon schizont rupture. Mutant P. berghei parasites lacking PMIF were able to complete the entire life cycle and exhibited no significant changes in growth characteristics or virulence features during blood stage infection. However, rodent hosts infected with knockout parasites had significantly higher numbers of circulating reticulocytes. Our results suggest that PMIF is produced by the parasite to influence host immune responses and the course of anemia upon infection.
Topics: Amino Acid Sequence; Animals; Erythrocytes; Host-Parasite Interactions; Humans; Macrophage Migration-Inhibitory Factors; Malaria, Falciparum; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Plasmodium berghei; Plasmodium falciparum; Sequence Homology, Amino Acid
PubMed: 17158894
DOI: 10.1128/IAI.00902-06 -
Frontiers in Cellular and Infection... 2019Thrombospondin type I repeat (TSR) domains are commonly -fucosylated by protein -fucosyltransferase 2 (PoFUT2), and this modification is required for optimal folding and...
Thrombospondin type I repeat (TSR) domains are commonly -fucosylated by protein -fucosyltransferase 2 (PoFUT2), and this modification is required for optimal folding and secretion of TSR-containing proteins. The human malaria parasite expresses proteins containing TSR domains, such as the thrombospondin-related anonymous protein (TRAP) and circumsporozoite surface protein (CSP), which are -fucosylated. TRAP and CSP are present on the surface of sporozoites and play essential roles in mosquito and human host invasion processes during the transmission stages. Here, we have generated PoFUT2 null-mutant and (rodent) malaria parasites and, by phenotyping them throughout their complete life cycle, we show that PoFUT2 disruption does not affect the growth through the mosquito stages for both species. However, contrary to what has been described previously by others, PoFUT2 null mutant sporozoites showed no deleterious motility phenotypes and successfully established blood stage infection in mice. This unexpected result indicates that the importance of -fucosylation of TSR domains may differ between human and RODENT malaria parasites; complicating our understanding of glycosylation modifications in malaria biology.
Topics: Animals; Cell Line; Culicidae; Disease Models, Animal; Fucosyltransferases; Glycosylation; Humans; Life Cycle Stages; Malaria; Malaria, Falciparum; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Oocysts; Plasmodium berghei; Plasmodium falciparum; Protozoan Proteins; Sporozoites
PubMed: 31334132
DOI: 10.3389/fcimb.2019.00238 -
Parasites & Vectors Nov 2019The transmissible forms of Plasmodium parasites result from a process of sporogony that takes place inside their obligatory mosquito vector and culminates in the...
BACKGROUND
The transmissible forms of Plasmodium parasites result from a process of sporogony that takes place inside their obligatory mosquito vector and culminates in the formation of mammalian-infective parasite forms. Ivermectin is a member of the avermectin family of endectocides, which has been proposed to inhibit malaria transmission due its insecticidal effect. However, it remains unclear whether ivermectin also exerts a direct action on the parasite's blood and transmission stages.
METHODS
We employed a rodent model of infection to assess the impact of ivermectin treatment on P. berghei asexual and sexual blood forms in vivo. We then made use of a newly established luminescence-based methodology to evaluate the activity of ivermectin and other avermectins against the sporogonic stages of P. berghei parasites in vitro independent of their role on mosquito physiology.
RESULTS
Our results show that whereas ivermectin does not affect the parasite's parasitemia, gametocytemia or exflagellation in the mammalian host, several members of the avermectin family of compounds exert a strong inhibitory effect on the generation and development of P. berghei oocysts.
CONCLUSIONS
Our results shed light on the action of avermectins against Plasmodium transmission stages and highlight the potential of these compounds to help prevent the spread of malaria.
Topics: Animals; Antiparasitic Agents; Disease Models, Animal; Disease Transmission, Infectious; Ivermectin; Malaria; Mice, Inbred BALB C; Oocysts; Plasmodium berghei
PubMed: 31752986
DOI: 10.1186/s13071-019-3805-0 -
Eukaryotic Cell Nov 2008
Review
Topics: Animals; Antibodies, Protozoan; Antigens, Protozoan; Disease Models, Animal; Humans; Malaria; Malaria Vaccines; Mice; Plasmodium berghei
PubMed: 18806208
DOI: 10.1128/EC.00242-08 -
Tropical Biomedicine Apr 2011Intracellular parasites manipulate host cell apoptosis in different ways either to increase their life span within infected cells or to spread infection. The present...
Intracellular parasites manipulate host cell apoptosis in different ways either to increase their life span within infected cells or to spread infection. The present data provided information on the cellular changes taking place in spleen and peripheral blood during Plasmodium berghei-infection and indicated apoptosis mediated host immune response during infection. Our results suggested a significant change in cellular composition and absolute number of white blood cells in spleen and peripheral blood of P. berghei-infected Balb/c mice. Plasmodium berghei-infection was associated with marked increase in percentage of apoptotic mononuclear cells compared to polymorphonuclear white blood cells.
Topics: Animals; Apoptosis; Blood; Female; Leukocytes; Malaria; Male; Mice; Mice, Inbred BALB C; Plasmodium berghei; Spleen
PubMed: 21602777
DOI: No ID Found