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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 -
IUBMB Life Sep 2019Proliferative cell nuclear antigen (PCNA) is the processivity factor for various DNA polymerases and it functions in response to DNA damage in eukaryotic system....
Proliferative cell nuclear antigen (PCNA) is the processivity factor for various DNA polymerases and it functions in response to DNA damage in eukaryotic system. Plasmodium falciparum contains two PCNAs, while PCNA1 has been attributed to DNA replication, the role of PCNA2 has been assigned to DNA damage response in erythrocytic developmental stages. Although a recent transposon mediated knockout strategy qualified pcna2 as a nonessential gene in Plasmodium berghei, a conventional homologous recombination-based knockout strategy has not been employed for this gene yet. Moreover, the cellular dynamics of PCNA2 in extraerythrocytic stages still remain elusive in Plasmodium. We attempted multiple times to knock out PbPCNA2 from the parasite genome using homologous recombination strategy without much success. However, we were able to generate PbPCNA2-GFP tagged transgenic parasites confirming that the pcna2 locus is amenable to genetic manipulation. The GFP-tagged parasites showed similar growth phenotype, compared to wild-type parasites, in both erythrocytic and sporogonic cycle, suggesting that tagging had no effect on parasite physiology. PbPCNA2 expression was also observed during the sporogonic cycle in midgut oocyst and salivary gland sporozoites. The PbPCNA2 expression was upregulated in the presence of DNA damaging agents like hydroxyurea and methyl methanesulphonate. Our inability to knock out PCNA2 suggested its essentiality in the parasite development and elevated expression during DNA damaging condition hint at a critical role of the protein in parasite physiology. © 2019 IUBMB Life, 71(9):1293-1301, 2019.
Topics: Animals; DNA Damage; DNA Replication; DNA-Directed DNA Polymerase; Gene Expression Regulation; Gene Knockout Techniques; Genome; Humans; Plasmodium berghei; Plasmodium falciparum; Proliferating Cell Nuclear Antigen; Protozoan Proteins
PubMed: 30865364
DOI: 10.1002/iub.2036 -
Antimicrobial Agents and Chemotherapy May 2016Glutathione plays a central role in maintaining cellular redox homeostasis, and modulations to this status may affect malaria parasite sensitivity to certain types of...
Glutathione plays a central role in maintaining cellular redox homeostasis, and modulations to this status may affect malaria parasite sensitivity to certain types of antimalarials. In this study, we demonstrate that inhibition of glutathione biosynthesis in the Plasmodium berghei ANKA strain through disruption of the γ-glutamylcysteine synthetase (γ-GCS) gene, which encodes the first and rate-limiting enzyme in the glutathione biosynthetic pathway, significantly sensitizes parasites in vivo to pyrimethamine and sulfadoxine, but not to chloroquine, artesunate, or primaquine, compared with control parasites containing the same pyrimethamine-resistant marker cassette. Treatment of mice infected with an antifolate-resistant P. berghei control line with a γ-GCS inhibitor, buthionine sulfoximine, could partially abrogate pyrimethamine and sulfadoxine resistance. The role of glutathione in modulating the malaria parasite's response to antifolates suggests that development of specific inhibitors against Plasmodium γ-GCS may offer a new approach to counter Plasmodium antifolate resistance.
Topics: Animals; Antimalarials; Artemisinins; Artesunate; Chloroquine; Drug Resistance; Female; Glutamate-Cysteine Ligase; Glutathione; Malaria; Mice; Mice, Inbred BALB C; Plasmodium berghei; Pyrimethamine; Sulfadoxine
PubMed: 26953195
DOI: 10.1128/AAC.01836-15 -
Molecular and Biochemical Parasitology Apr 2019Malaria parasite oocysts generate sporozoites by a process termed sporogony. Essential for successful sporogony of Plasmodium berghei in Anopheles stephensi mosquitoes...
Malaria parasite oocysts generate sporozoites by a process termed sporogony. Essential for successful sporogony of Plasmodium berghei in Anopheles stephensi mosquitoes is a complex of six LCCL lectin domain adhesive-like proteins (LAPs). LAP null mutant oocysts undergo growth and mitosis but fail to form sporozoites. At a cytological level, LAP null mutant oocyst development is indistinguishable from its wildtype counterparts for the first week, supporting the hypothesis that LAP null mutant oocysts develop normally before cytokinesis. We show here that LAP1 null mutant oocysts display highly reduced expression of sporozoite proteins and their transcription factors. Our findings indicate that events leading up to the cytokinesis defect in LAP null mutants occur early in oocyst development.
Topics: Animals; Female; Gene Expression Regulation; Humans; Malaria; Mice; Mutation; Oocysts; Plasmodium berghei; Protozoan Proteins; Sporozoites
PubMed: 30753856
DOI: 10.1016/j.molbiopara.2019.02.001 -
Molecular and Biochemical Parasitology May 2007The malaria parasite encodes a wide range of proteases necessary to facilitate its many developmental transitions in vertebrate and insect hosts. Amongst these is a...
The malaria parasite encodes a wide range of proteases necessary to facilitate its many developmental transitions in vertebrate and insect hosts. Amongst these is a predicted cysteine protease structurally related to caspases, named Plasmodium metacaspase 1 (PxMC1). We have generated Plasmodium berghei parasites in which the PbMC1coding sequence is removed and replaced with a green fluorescent reporter gene to investigate the expression of PbMC1, its contribution to parasite development, and its involvement in previously reported apoptosis-like cell death of P. berghei ookinetes. Our results show that the pbmc1 gene is expressed in female gametocytes and all downstream mosquito stages including sporozoites, but not in asexual blood stages. We failed to detect an apparent loss-of-function phenotype, suggesting that PbMC1 constitutes a functionally redundant gene. We discuss these findings in the context of two other putative Plasmodium metacaspases that we describe here.
Topics: Amino Acid Sequence; Animals; Animals, Genetically Modified; Apoptosis; Base Sequence; Caspases; DNA, Protozoan; Female; Gene Deletion; Genes, Protozoan; Genes, Reporter; Green Fluorescent Proteins; Molecular Sequence Data; Phenotype; Plasmodium berghei; Protozoan Proteins; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 17335919
DOI: 10.1016/j.molbiopara.2007.01.016 -
Malaria Journal Oct 2021Plasmodium sporozoites are the highly motile forms of malaria-causing parasites that are transmitted by the mosquito to the vertebrate host. Sporozoites need to enter...
BACKGROUND
Plasmodium sporozoites are the highly motile forms of malaria-causing parasites that are transmitted by the mosquito to the vertebrate host. Sporozoites need to enter and cross several cellular and tissue barriers for which they employ a set of surface proteins. Three of these proteins are members of the thrombospondin related anonymous protein (TRAP) family. Here, potential additive, synergistic or antagonistic roles of these adhesion proteins were investigated.
METHODS
Four transgenic Plasmodium berghei parasite lines that lacked two or all three of the TRAP family adhesins TRAP, TLP and TREP were generated using positive-negative selection. The parasite lines were investigated for their capacity to attach to and move on glass, their ability to egress from oocysts and their capacity to enter mosquito salivary glands. One strain was in addition interrogated for its capacity to infect mice.
RESULTS
The major phenotype of the TRAP single gene deletion dominates additional gene deletion phenotypes. All parasite lines including the one lacking all three proteins were able to conduct some form of active, if unproductive movement.
CONCLUSIONS
The individual TRAP-family adhesins appear to play functionally distinct roles during motility and infection. Other proteins must contribute to substrate adhesion and gliding motility.
Topics: Microorganisms, Genetically-Modified; Plasmodium berghei; Protozoan Proteins; Sporozoites
PubMed: 34717635
DOI: 10.1186/s12936-021-03960-3 -
Phosphoglycolate phosphatase is a metabolic proofreading enzyme essential for cellular function in .The Journal of Biological Chemistry Mar 2019(Pf) 4-nitrophenylphosphatase has been shown previously to be involved in vitamin B1 metabolism. Here, conducting a BLASTp search, we found that...
(Pf) 4-nitrophenylphosphatase has been shown previously to be involved in vitamin B1 metabolism. Here, conducting a BLASTp search, we found that 4-nitrophenylphosphatase from Pf has significant homology with phosphoglycolate phosphatase (PGP) from mouse, human, and yeast, prompting us to reinvestigate the biochemical properties of the enzyme. Because the recombinant PfPGP enzyme is insoluble, we performed an extended substrate screen and extensive biochemical characterization of the recombinantly expressed and purified homolog from (Pb), leading to the identification of 2-phosphoglycolate and 2-phospho-L-lactate as the relevant physiological substrates of PbPGP. 2-Phosphoglycolate is generated during repair of damaged DNA ends, 2-phospho-L-lactate is a product of pyruvate kinase side reaction, and both potently inhibit two key glycolytic enzymes, triosephosphate isomerase and phosphofructokinase. Hence, PGP-mediated clearance of these toxic metabolites is vital for cell survival and functioning. Our results differ significantly from those in a previous study, wherein the PfPGP enzyme has been inferred to act on 2-phospho-D-lactate and not on the L isomer. Apart from resolving the substrate specificity conflict through direct enzyme assays, we conducted PGP gene knockout studies in , confirming that this conserved metabolic proofreading enzyme is essential in In summary, our findings establish PbPGP as an essential enzyme for normal physiological function in and suggest that drugs that specifically inhibit PGP may hold promise for use in anti-malarial therapies.
Topics: Animals; Gene Knockout Techniques; Glycolates; Glycolysis; Humans; Lactates; Malaria; Mice; Molecular Sequence Data; Phosphoric Monoester Hydrolases; Plasmodium berghei; Protozoan Proteins; Sequence Alignment; Substrate Specificity
PubMed: 30700551
DOI: 10.1074/jbc.AC118.007143 -
Genome Biology Aug 2019In multicellular organisms, alternative splicing is central to tissue differentiation and identity. Unicellular protists lack multicellular tissue but differentiate into...
BACKGROUND
In multicellular organisms, alternative splicing is central to tissue differentiation and identity. Unicellular protists lack multicellular tissue but differentiate into variable cell types during their life cycles. The role of alternative splicing in transitions between cell types and establishing cellular identity is currently unknown in any unicellular organism.
RESULTS
To test whether alternative splicing in unicellular protists plays a role in cellular differentiation, we conduct RNA-seq to compare splicing in female and male sexual stages to asexual intraerythrocytic stages in the rodent malaria parasite Plasmodium berghei. We find extensive changes in alternative splicing between stages and a role for alternative splicing in sexual differentiation. Previously, general gametocyte differentiation was shown to be modulated by specific transcription factors. Here, we show that alternative splicing establishes a subsequent layer of regulation, controlling genes relating to consequent sex-specific differentiation of gametocytes.
CONCLUSIONS
We demonstrate that alternative splicing is reprogrammed during cellular differentiation of a unicellular protist. Disruption of an alternative splicing factor, PbSR-MG, perturbs sex-specific alternative splicing and decreases the ability of the parasites to differentiate into male gametes and oocysts, thereby reducing transmission between vertebrate and insect hosts. Our results reveal alternative splicing as an integral, stage-specific phenomenon in these protists and as a regulator of cellular differentiation that arose early in eukaryotic evolution.
Topics: Alternative Splicing; Animals; Germ Cells; Life Cycle Stages; Mice; Plasmodium berghei; Transcription, Genetic
PubMed: 31370870
DOI: 10.1186/s13059-019-1756-6