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Biochimica Et Biophysica Acta Jul 2016Plasmodium has a complex biology including the ability to interact with host signals modulating their function through cellular machinery. Tumor necrosis factor (TNF)...
BACKGROUND
Plasmodium has a complex biology including the ability to interact with host signals modulating their function through cellular machinery. Tumor necrosis factor (TNF) elicits diverse cellular responses including effects in malarial pathology and increased infected erythrocyte cytoadherence. As TNF levels are raised during Plasmodium falciparum infection we have investigated whether it has an effect on the parasite asexual stage.
METHODS
Flow cytometry, spectrofluorimetric determinations, confocal microscopy and PCR real time quantifications were employed for characterizing TNF induced effects and membrane integrity verified by wheat germ agglutinin staining.
RESULTS
TNF is able to decrease intracellular parasitemia, involving calcium as a second messenger of the pathway. Parasites incubated for 48 h with TNF showed reduced erythrocyte invasion. Thus, TNF induced rises in intracellular calcium concentration, which were blocked by prior addition of the purinergic receptor agonists KN62 and A438079, or interfering with intra- or extracellular calcium release by thapsigargin or EGTA (ethylene glycol tetraacetic acid). Importantly, expression of PfPCNA1 which encodes the Plasmodium falciparum Proliferating-Cell Nuclear Antigen 1, decreased after P. falciparum treatment of TNF (tumor necrosis factor) or 6-Bnz cAMP (N(6)-benzoyladenosine-3',5'-cyclic monophosphate sodium salt).
CONCLUSIONS
This is potentially interesting data showing the relevance of calcium in downregulating a gene involved in cellular proliferation, triggered by TNF.
GENERAL SIGNIFICANCE
The data show that Plasmodium may subvert the immunological system and use TNF for the control of its proliferation within the vertebrate host.
Topics: Antimalarials; Calcium Signaling; Cell Adhesion; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Cells; Erythrocytes; Host-Parasite Interactions; Humans; Plasmodium falciparum; Proliferating Cell Nuclear Antigen; Protozoan Proteins; Time Factors; Tumor Necrosis Factor-alpha
PubMed: 27080559
DOI: 10.1016/j.bbagen.2016.04.003 -
MBio Apr 2019The global spread of chloroquine resistance transporter (PfCRT) variant haplotypes earlier caused the widespread loss of chloroquine (CQ) efficacy. In Asia, novel PfCRT...
The global spread of chloroquine resistance transporter (PfCRT) variant haplotypes earlier caused the widespread loss of chloroquine (CQ) efficacy. In Asia, novel PfCRT mutations that emerged on the Dd2 allelic background have recently been implicated in high-level resistance to piperaquine, and N326S and I356T have been associated with genetic backgrounds in which resistance emerged to artemisinin derivatives. By analyzing large-scale genome sequencing data, we report that the predominant Asian CQ-resistant Dd2 haplotype is undetectable in Africa. Instead, the GB4 and previously unexplored Cam783 haplotypes predominate, along with wild-type, drug-sensitive PfCRT that has reemerged as the major haplotype. To interrogate how these alleles impact drug susceptibility, we generated -modified isogenic parasite lines spanning the mutational interval between GB4 and Dd2, which includes Cam783 and involves amino acid substitutions at residues 326 and 356. Relative to Dd2, the GB4 and Cam783 alleles were observed to mediate lower degrees of resistance to CQ and the first-line drug amodiaquine, while resulting in higher growth rates. These findings suggest that differences in growth rates, a surrogate of parasite fitness, influence selection in the context of African infections that are frequently characterized by high transmission rates, mixed infections, increased immunity, and less recourse to treatment. We also observe that the Asian Dd2 allele affords partial protection against piperaquine yet does not directly impact artemisinin efficacy. Our results can help inform the regional recommendations of antimalarials, whose activity is influenced by and, in certain cases, enhanced against select PfCRT variant haplotypes. Our study defines the allelic distribution of , an important mediator of multidrug resistance in , in Africa and Asia. We leveraged whole-genome sequence analysis and gene editing to demonstrate how current drug combinations can select different allelic variants of this gene and shape region-specific parasite population structures. We document the ability of PfCRT mutations to modulate parasite susceptibility to current antimalarials in dissimilar, allele-specific ways. This study underscores the importance of actively monitoring genotypes to identify emerging patterns of multidrug resistance and help guide region-specific treatment options.
Topics: Africa; Asia; Drug Resistance, Multiple; Gene Frequency; Genetic Fitness; Genetics, Population; Genotype; Malaria, Falciparum; Membrane Transport Proteins; Mutant Proteins; Plasmodium falciparum; Protozoan Proteins
PubMed: 31040246
DOI: 10.1128/mBio.02731-18 -
MSphere Apr 2018Sexual reproduction is an obligate step in the life cycle, with mature gametocytes being the only form of the parasite capable of human-to-mosquito transmission....
Sexual reproduction is an obligate step in the life cycle, with mature gametocytes being the only form of the parasite capable of human-to-mosquito transmission. Development of male and female gametocytes takes 9 to 12 days, and although more than 300 genes are thought to be specific to gametocytes, only a few have been postulated to be male or female specific. Because these genes are often expressed during late gametocyte stages and for some, male- or female-specific transcript expression is debated, the separation of male and female populations is technically challenging. To overcome these challenges, we have developed an unbiased single-cell approach to determine which transcripts are expressed in male versus female gametocytes. Using microfluidic technology, we isolated single mid- to late-stage gametocytes to compare the expression of 91 genes, including 87 gametocyte-specific genes, in 90 cells. Such analysis identified distinct gene clusters whose expression was associated with male, female, or all gametocytes. In addition, a small number of male gametocytes clustered separately from female gametocytes based on sex-specific expression independent of stage. Many female-enriched genes also exhibited stage-specific expression. RNA fluorescent hybridization of male and female markers validated the mutually exclusive expression pattern of male and female transcripts in gametocytes. These analyses uncovered novel male and female markers that are expressed as early as stage III gametocytogenesis, providing further insight into sex-specific differentiation previously masked in population analyses. Our single-cell approach reveals the most robust markers for sex-specific differentiation in gametocytes. Such single-cell expression assays can be generalized to all eukaryotic pathogens. Most human deaths that result from malaria are caused by the eukaryotic parasite The only form of this parasite that is transmitted to the mosquito is the sexual form, called the gametocyte. The production of mature gametocytes can take up to 2 weeks and results in phenotypically distinct males and females, although what causes this gender-specific differentiation remains largely unknown. Here, we demonstrate the first use of microfluidic technology to capture single gametocytes and determine their temporal sex-specific gene expression in an unbiased manner. We were able to determine male or female identity of single cells based on the upregulation of gender-specific genes as early as mid-stage gametocytes. This analysis has revealed strong markers for male and female gametocyte differentiation that were previously concealed in population analyses. Similar single-cell analyses in eukaryotic pathogens using this method may uncover rare cell types and heterogeneity previously masked in population studies.
Topics: Gene Expression; In Situ Hybridization, Fluorescence; Life Cycle Stages; Microfluidics; Plasmodium falciparum; RNA, Protozoan; Single-Cell Analysis
PubMed: 29643077
DOI: 10.1128/mSphere.00130-18 -
Malaria Journal Dec 2021The devastating public health impact of malaria has prompted the need for effective interventions. Malaria control gained traction after the introduction of... (Review)
Review
BACKGROUND
The devastating public health impact of malaria has prompted the need for effective interventions. Malaria control gained traction after the introduction of artemisinin-based combination therapy (ACT). However, the emergence of artemisinin (ART) partial resistance in Southeast Asia and emerging reports of delayed parasite sensitivity to ACT in African parasites signal a gradual trend towards treatment failure. Monitoring the prevalence of mutations associated with artemisinin resistance in African populations is necessary to stop resistance in its tracks. Mutations in Plasmodium falciparum genes pfk13, pfcoronin and pfatpase6 have been linked with ART partial resistance.
METHODS
Findings from published research articles on the prevalence of pfk13, pfcoronin and pfatpase6 polymorphisms in Africa were collated. PubMed, Embase and Google Scholar were searched for relevant articles reporting polymorphisms in these genes across Africa from 2014 to August 2021, for pfk13 and pfcoronin. For pfatpase6, relevant articles between 2003 and August 2021 were retrieved.
RESULTS
Eighty-seven studies passed the inclusion criteria for this analysis and reported 742 single nucleotide polymorphisms in 37,864 P. falciparum isolates from 29 African countries. Five validated-pfk13 partial resistance markers were identified in Africa: R561H in Rwanda and Tanzania, M476I in Tanzania, F446I in Mali, C580Y in Ghana, and P553L in an Angolan isolate. In Tanzania, three (L263E, E431K, S769N) of the four mutations (L263E, E431K, A623E, S769N) in pfatpase6 gene associated with high in vitro IC were reported. pfcoronin polymorphisms were reported in Senegal, Gabon, Ghana, Kenya, and Congo, with P76S being the most prevalent mutation.
CONCLUSIONS
This meta-analysis provides an overview of the prevalence and widespread distribution of pfk13, pfcoronin and pfatpase6 mutations in Africa. Understanding the phenotypic consequences of these mutations can provide information on the efficacy status of artemisinin-based treatment of malaria across the continent.
Topics: Adenosine Triphosphatases; Antimalarials; Artemisinins; Drug Resistance; Microfilament Proteins; Plasmodium falciparum; Protozoan Proteins
PubMed: 34856982
DOI: 10.1186/s12936-021-03987-6 -
Journal of Vector Borne Diseases 2017Malaria is a life-threatening disease caused by Plasmodium parasites. The life-cycle of Plasmodium species involves several stages both in mosquito and the vertebrate...
BACKGROUND & OBJECTIVES
Malaria is a life-threatening disease caused by Plasmodium parasites. The life-cycle of Plasmodium species involves several stages both in mosquito and the vertebrate host. In the erythrocytic stage, Plasmodium resides inside the red blood cells (RBCs), where it meets most of its nutritional requirement by degrad- ing host's haemoglobin. L-arginine is required for growth and division of cells. The present study was aimed to demonstrate the effect of supplementation of different concentrations of L-arginine and L-citrulline on the growth of parasite, and effect of the culture supernatant on the host's peripheral blood mononuclear cells (PBMCs).
METHODS
To examine the effect of supplementation of L-arginine and L-citrulline, Plasmodium falciparum (3D7 strain) was cultured in RPMI 1640, L-arginine deficient RPMI 1640, and in different concentrations of L-arginine, and L-citrulline supplemented in arginine deficient RPMI 1640 medium. To have a holistic view of in vivo cell activation, the PBMCs isolated from healthy human host were cultured in the supernatant collected from P. falciparum culture.
RESULTS
Growth of the parasite was greatly enhanced in L-arginine supplemented media and was found to be concentration dependent. However, parasite growth was compromised in L-citrulline supplemented and L-arginine deficient media. The supernatant collected from L-arginine supplemented parasite media (sArg) showed increased FOXP3 and interleukin-10 (IL-10) expression as compared to the supernatant collected from L-citrulline supple- mented parasite media (sCit).
INTERPRETATION & CONCLUSION
The in vitro culture results showed, decreased parasite growth, and decreased expression of programmed cell death-1 (PD-1) (a coinhibitory molecule) and IL-10 in the L-citrulline supplemented media as compared to L-arginine supplemented media. Hence, it was concluded that L-citrulline supplementation would be a better alternative than L-arginine to inhibit the parasite growth.
Topics: Arginine; Cells, Cultured; Citrulline; Culture Media, Conditioned; Humans; Leukocytes, Mononuclear; Plasmodium falciparum
PubMed: 28748834
DOI: No ID Found -
ChemMedChem Nov 2022A series of pyrrolo[2,3-d]pyrimidines were designed in silico as potential bumped kinase inhibitors targeting P. falciparum calcium dependent protein kinase 4 (PfCDPK4),...
A series of pyrrolo[2,3-d]pyrimidines were designed in silico as potential bumped kinase inhibitors targeting P. falciparum calcium dependent protein kinase 4 (PfCDPK4), with the potential to inhibit PfCDPK1 based on earlier studies of the two kinases. A small series of these compounds were prepared and assessed for inhibitory activity against PfCDPK4 and PfCDPK1 in vitro. Four of the compounds displayed promising inhibitory activity against either PfCDPK4 (IC =0.210-0.530 μM), or PfCDPK1 (IC =0.589 μM). These data will enable optimisation of the molecular model to better predict inhibitory activity against PfCDPK4.
Topics: Amines; Plasmodium falciparum; Protein Kinase Inhibitors; Protein Kinases; Pyrimidines; Structure-Activity Relationship; Antimalarials
PubMed: 36106757
DOI: 10.1002/cmdc.202200421 -
The Biochemical Journal Aug 2014Despite a century of control and eradication campaigns, malaria remains one of the world's most devastating diseases. Our once-powerful therapeutic weapons are losing... (Review)
Review
Despite a century of control and eradication campaigns, malaria remains one of the world's most devastating diseases. Our once-powerful therapeutic weapons are losing the war against the Plasmodium parasite, whose ability to rapidly develop and spread drug resistance hamper past and present malaria-control efforts. Finding new and effective treatments for malaria is now a top global health priority, fuelling an increase in funding and promoting open-source collaborations between researchers and pharmaceutical consortia around the world. The result of this is rapid advances in drug discovery approaches and technologies, with three major methods for antimalarial drug development emerging: (i) chemistry-based, (ii) target-based, and (iii) cell-based. Common to all three of these approaches is the unique ability of structural biology to inform and accelerate drug development. Where possible, SBDD (structure-based drug discovery) is a foundation for antimalarial drug development programmes, and has been invaluable to the development of a number of current pre-clinical and clinical candidates. However, as we expand our understanding of the malarial life cycle and mechanisms of resistance development, SBDD as a field must continue to evolve in order to develop compounds that adhere to the ideal characteristics for novel antimalarial therapeutics and to avoid high attrition rates pre- and post-clinic. In the present review, we aim to examine the contribution that SBDD has made to current antimalarial drug development efforts, covering hit discovery to lead optimization and prevention of parasite resistance. Finally, the potential for structural biology, particularly high-throughput structural genomics programmes, to identify future targets for drug discovery are discussed.
Topics: Animals; Antimalarials; Drug Discovery; Drug Resistance, Multiple; Humans; Life Cycle Stages; Models, Biological; Molecular Structure; Plasmodium falciparum; Protozoan Proteins
PubMed: 25008945
DOI: 10.1042/BJ20140240 -
Molecular and Biochemical Parasitology 2015The transmission of malaria parasites depends on the presence of sexual stages (gametocytes) in the blood, making the ratio and densities of female and male gametocytes...
The transmission of malaria parasites depends on the presence of sexual stages (gametocytes) in the blood, making the ratio and densities of female and male gametocytes important determinants of parasite fitness. This manuscript describes the development of reverse transcriptase quantitative PCR (RT-qPCR) assays to separately quantify mature female and male gametocytes of the human malaria parasite Plasmodium falciparum, and reveals that Pfs25 mRNA is expressed only in female gametocytes. The female (Pfs25) and male (Pfs230p) gametocyte specific RT-qPCR assays have lower detection limits of 0.3 female and 1.8 male gametocytes per microlitre of blood, respectively, making them more sensitive than microscopy. Accurate quantification of the ratio and densities of female and male gametocytes will increase understanding of P. falciparum transmission and improve the evaluation of transmission blocking interventions.
Topics: Antigens, Protozoan; Gene Expression Profiling; Parasitology; Plasmodium falciparum; Protozoan Proteins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 25827756
DOI: 10.1016/j.molbiopara.2015.03.006 -
Cells Apr 2022The global burden of malaria and toxoplasmosis has been limited by the use of efficacious anti-parasitic agents, however, emerging resistance in species and threatens...
The global burden of malaria and toxoplasmosis has been limited by the use of efficacious anti-parasitic agents, however, emerging resistance in species and threatens disease control worldwide, implying that new agents/therapeutic targets are urgently needed. Nuclear localization signal (NLS)-dependent transport into the nucleus, mediated by members of the importin (IMP) superfamily of nuclear transporters, has shown potential as a target for intervention to limit viral infection. Here, we show for the first time that IMPα from and have promise as targets for small molecule inhibitors. We use high-throughput screening to identify agents able to inhibit IMPα binding to a NLS, identifying a number of compounds that inhibit binding in the µM-nM range, through direct binding to IMPα, as shown in thermostability assays. Of these, BAY 11-7085 is shown to be a specific inhibitor of IMPα-NLS recognition. Importantly, a number of the inhibitors limited growth by both and . The results strengthen the hypothesis that apicomplexan IMPα proteins have potential as therapeutic targets to aid in identifying novel agents for two important, yet neglected, parasitic diseases.
Topics: High-Throughput Screening Assays; Nuclear Localization Signals; Plasmodium falciparum; Protein Binding; alpha Karyopherins
PubMed: 35406765
DOI: 10.3390/cells11071201 -
Malaria Journal Feb 2021Simultaneous infection with multiple malaria parasite strains is common in high transmission areas. Quantifying the number of strains per host, or the multiplicity of...
Development of a new barcode-based, multiplex-PCR, next-generation-sequencing assay and data processing and analytical pipeline for multiplicity of infection detection of Plasmodium falciparum.
BACKGROUND
Simultaneous infection with multiple malaria parasite strains is common in high transmission areas. Quantifying the number of strains per host, or the multiplicity of infection (MOI), provides additional parasite indices for assessing transmission levels but it is challenging to measure accurately with current tools. This paper presents new laboratory and analytical methods for estimating the MOI of Plasmodium falciparum.
METHODS
Based on 24 single nucleotide polymorphisms (SNPs) previously identified as stable, unlinked targets across 12 of the 14 chromosomes within P. falciparum genome, three multiplex PCRs of short target regions and subsequent next generation sequencing (NGS) of the amplicons were developed. A bioinformatics pipeline including B4Screening pathway removed spurious amplicons to ensure consistent frequency calls at each SNP location, compiled amplicons by SNP site diversity, and performed algorithmic haplotype and strain reconstruction. The pipeline was validated by 108 samples generated from cultured-laboratory strain mixtures in different proportions and concentrations, with and without pre-amplification, and using whole blood and dried blood spots (DBS). The pipeline was applied to 273 smear-positive samples from surveys conducted in western Kenya, then providing results into StrainRecon Thresholding for Infection Multiplicity (STIM), a novel MOI estimator.
RESULTS
The 24 barcode SNPs were successfully identified uniformly across the 12 chromosomes of P. falciparum in a sample using the pipeline. Pre-amplification and parasite concentration, while non-linearly associated with SNP read depth, did not influence the SNP frequency calls. Based on consistent SNP frequency calls at targeted locations, the algorithmic strain reconstruction for each laboratory-mixed sample had 98.5% accuracy in dominant strains. STIM detected up to 5 strains in field samples from western Kenya and showed declining MOI over time (q < 0.02), from 4.32 strains per infected person in 1996 to 4.01, 3.56 and 3.35 in 2001, 2007 and 2012, and a reduction in the proportion of samples with 5 strains from 57% in 1996 to 18% in 2012.
CONCLUSION
The combined approach of new multiplex PCRs and NGS, the unique bioinformatics pipeline and STIM could identify 24 barcode SNPs of P. falciparum correctly and consistently. The methodology could be applied to field samples to reliably measure temporal changes in MOI.
Topics: DNA Barcoding, Taxonomic; High-Throughput Nucleotide Sequencing; Malaria, Falciparum; Multiplex Polymerase Chain Reaction; Plasmodium falciparum
PubMed: 33593329
DOI: 10.1186/s12936-021-03624-2