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Biomolecules Jul 2019The survival of the human malaria parasite under the physiologically distinct environments associated with their development in the cold-blooded invertebrate mosquito... (Review)
Review
The survival of the human malaria parasite under the physiologically distinct environments associated with their development in the cold-blooded invertebrate mosquito vectors and the warm-blooded vertebrate human host requires a genome that caters to adaptability. To this end, a robust stress response system coupled to an efficient protein quality control system are essential features of the parasite. Heat shock proteins constitute the main molecular chaperone system of the cell, accounting for approximately two percent of the malaria genome. Some heat shock proteins of parasites constitute a large part (5%) of the 'exportome' (parasite proteins that are exported to the infected host erythrocyte) that modify the host cell, promoting its cyto-adherence. In light of their importance in protein folding and refolding, and thus the survival of the parasite, heat shock proteins of have been a major subject of study. Emerging evidence points to their role not only being cyto-protection of the parasite, as they are also implicated in regulating parasite virulence. In undertaking their roles, heat shock proteins operate in networks that involve not only partners of parasite origin, but also potentially functionally associate with human proteins to facilitate parasite survival and pathogenicity. This review seeks to highlight these interplays and their roles in parasite pathogenicity. We further discuss the prospects of targeting the parasite heat shock protein network towards the developments of alternative antimalarial chemotherapies.
Topics: Animals; Heat-Shock Proteins; Humans; Plasmodium falciparum; Virulence
PubMed: 31340488
DOI: 10.3390/biom9070295 -
Malaria Journal Dec 2016Plasmodium falciparum gametocytes are vital to sustaining malaria transmission. Parasite densities, multiplicity of infection as well as asexual genotype are features...
BACKGROUND
Plasmodium falciparum gametocytes are vital to sustaining malaria transmission. Parasite densities, multiplicity of infection as well as asexual genotype are features that have been found to influence gametocyte production. Measurements of the prevalence of Plasmodium sp. gametocytes may serve as a tool to monitor the success of malaria eradication efforts.
METHODS
Whole blood was collected from 112 children aged between 6 months and 13 years with uncomplicated P. falciparum malaria attending three health facilities in southern Ghana from June to August, 2014 before (day 0) and 4 days after completion of anti-malaria drug treatment (day 7). Malaria parasites were observed by microscopy and polymerase chain reaction (PCR); submicroscopic gametocyte carriage was measured by a Pfs25 (PF3D7_1031000) mRNA real time reverse transcriptase polymerase chain reaction (RT-PCR). Parasite genotyping was performed on gDNA extracted from dried filter paper blood blots by amplification of the polymorphic regions of msp1 (PF3D7_0930300) and msp2 (PF3D7_0206800) using PCR.
RESULTS
Microscopy estimated 3.1% (3/96) of the total population to carry gametocytes on day 0, which decreased to 2.1% (2/96) on day 7. In contrast, reverse transcriptase-real time PCR (RT-PCR) analysis of a subset of 35 samples estimated submicroscopic gametocyte carriage to be as high as 77% (27/35) using primers specific for Pfs25 (CT < 35) on day 0 and by day 7 this only declined to 60% (21/35). Genotyping the msp2 gene identified higher levels of MOI than the msp1 gene.
CONCLUSIONS
Although below detection by microscopy, gametocyte prevalence at submicroscopic levels are high in this region and emphasize the need for more effective elimination approaches like the development of transmission-blocking vaccines and safer gametocytocidal drugs.
Topics: Adolescent; Child; Child, Preschool; Female; Genetic Variation; Genotype; Genotyping Techniques; Ghana; Humans; Infant; Malaria, Falciparum; Male; Microscopy; Plasmodium falciparum; Polymerase Chain Reaction; Prevalence
PubMed: 27938356
DOI: 10.1186/s12936-016-1640-8 -
Current Topics in Microbiology and... 2019The global spread of artemisinin resistance brings with it the threat of incurable malaria. Already, this disease threatens over 219 million lives per year and causes... (Review)
Review
The global spread of artemisinin resistance brings with it the threat of incurable malaria. Already, this disease threatens over 219 million lives per year and causes 5-6% losses in GDP in endemic areas, even with current advances in prevention and treatment. This chapter discusses the currently tenuous position we are in globally, and the impact that could be seen if artemisinin treatment is lost, whether due to the unchecked spread of K13 mutations or poor global investment in treatment and prevention advances. Artemisinin is the backbone of current ACT treatment programs and severe malarial treatment; without it, the success of future malaria eradication programs will be in jeopardy.
Topics: Antimalarials; Artemisinins; Drug Resistance; Epidemiological Monitoring; Humans; Malaria, Falciparum; Plasmodium falciparum
PubMed: 31218504
DOI: 10.1007/82_2019_163 -
Genome Biology and Evolution Jun 2017Plasmodium falciparum in western Cambodia has developed resistance to artemisinin and its partner drugs, causing frequent treatment failure. Understanding this evolution...
Plasmodium falciparum in western Cambodia has developed resistance to artemisinin and its partner drugs, causing frequent treatment failure. Understanding this evolution can inform the deployment of new therapies. We investigated the genetic architecture of 78 falciparum isolates using whole-genome sequencing, correlating results to in vivo and ex vivo drug resistance and exploring the relationship between population structure, demographic history, and partner drug resistance. Principle component analysis, network analysis and demographic inference identified a diverse central population with three clusters of clonally expanding parasite populations, each associated with specific K13 artemisinin resistance alleles and partner drug resistance profiles which were consistent with the sequential deployment of artemisinin combination therapies in the region. One cluster displayed ex vivo piperaquine resistance and mefloquine sensitivity with a high rate of in vivo failure of dihydroartemisinin-piperaquine. Another cluster displayed ex vivo mefloquine resistance and piperaquine sensitivity with high in vivo efficacy of dihydroartemisinin-piperaquine. The final cluster was clonal and displayed intermediate sensitivity to both drugs. Variations in recently described piperaquine resistance markers did not explain the difference in mean IC90 or clinical failures between the high and intermediate piperaquine resistance groups, suggesting additional loci may be involved in resistance. The results highlight an important role for partner drug resistance in shaping the P. falciparum genetic landscape in Southeast Asia and suggest that further work is needed to evaluate for other mutations that drive piperaquine resistance.
Topics: Adult; Antipruritics; Artemisinins; Cambodia; Drug Resistance; Female; Humans; Malaria, Falciparum; Male; Mefloquine; Phylogeny; Plasmodium falciparum; Protozoan Proteins; Quinolines; Treatment Failure
PubMed: 28854635
DOI: 10.1093/gbe/evx126 -
FEMS Microbiology Reviews May 2021Studies of the susceptibility of Plasmodium falciparum to the artemisinin family of antimalarial drugs provide a complex picture of partial resistance (tolerance)... (Review)
Review
Studies of the susceptibility of Plasmodium falciparum to the artemisinin family of antimalarial drugs provide a complex picture of partial resistance (tolerance) associated with increased parasite survival in vitro and in vivo. We present an overview of the genetic loci that, in mutant form, can independently elicit parasite tolerance. These encode Kelch propeller domain protein PfK13, ubiquitin hydrolase UBP-1, actin filament-organising protein Coronin, also carrying a propeller domain, and the trafficking adaptor subunit AP-2μ. Detailed studies of these proteins and the functional basis of artemisinin tolerance in blood-stage parasites are enabling a new synthesis of our understanding to date. To guide further experimental work, we present two major conclusions. First, we propose a dual-component model of artemisinin tolerance in P. falciparum comprising suppression of artemisinin activation in early ring stage by reducing endocytic haemoglobin capture from host cytosol, coupled with enhancement of cellular healing mechanisms in surviving cells. Second, these two independent requirements limit the likelihood of development of complete artemisinin resistance by P. falciparum, favouring deployment of existing drugs in new schedules designed to exploit these biological limits, thus extending the useful life of current combination therapies.
Topics: Antimalarials; Artemisinins; Drug Resistance; Plasmodium falciparum; Protozoan Proteins
PubMed: 33095255
DOI: 10.1093/femsre/fuaa056 -
Malaria Journal Nov 2017Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been...
BACKGROUND
Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans.
METHODS
The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS).
RESULTS
While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins.
CONCLUSIONS
This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.
Topics: Acetylglucosamine; Glycosylation; Plasmodium falciparum; Protein Processing, Post-Translational; Proteome; Protozoan Proteins
PubMed: 29187233
DOI: 10.1186/s12936-017-2131-2 -
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 -
Microbiology Spectrum Sep 2021The malaria parasite has a complex life cycle exhibiting phenotypic and morphogenic variations in two different hosts by existing in heterogeneous developmental states....
The malaria parasite has a complex life cycle exhibiting phenotypic and morphogenic variations in two different hosts by existing in heterogeneous developmental states. To investigate this cellular heterogeneity of the parasite within the human host, we performed single-cell RNA sequencing of synchronized cells under control and temperature treatment conditions. Using the Malaria Cell Atlas (https://www.sanger.ac.uk/science/tools/mca) as a guide, we identified 9 subtypes of the parasite distributed across known intraerythrocytic stages. Interestingly, temperature treatment results in the upregulation of the gene, the master regulator of sexual development in a small subpopulation of the parasites. Moreover, we identified a heterogeneous stress-responsive subpopulation (clusters 5, 6, and 7 [∼10% of the total population]) that exhibits upregulation of stress response pathways under normal growth conditions. We also developed an online exploratory tool that will provide new insights into gene function under normal and temperature stress conditions. Thus, our study reveals important insights into cell-to-cell heterogeneity in the parasite population under temperature treatment that will be instrumental toward a mechanistic understanding of cellular adaptation and population dynamics in Plasmodium falciparum. The malaria parasite has a complex life cycle exhibiting phenotypic variations in two different hosts accompanied by cell-to-cell variability that is important for stress tolerance, immune evasion, and drug resistance. To investigate cellular heterogeneity determined by gene expression, we performed single-cell RNA sequencing (scRNA-seq) of about 12,000 synchronized cells under physiologically relevant normal (37°C) and temperature stress (40°C) conditions phenocopying the cyclic bouts of fever experienced during malarial infection. In this study, we found that parasites exhibit transcriptional heterogeneity in an otherwise morphologically synchronized culture. Also, a subset of parasites is continually committed to gametocytogenesis and stress-responsive pathways. These observations have important implications for understanding the mechanisms of drug resistance generation and vaccine development against the malaria parasite.
Topics: Base Sequence; Humans; Life Cycle Stages; Malaria, Falciparum; Plasmodium falciparum; Protozoan Proteins; Single-Cell Analysis; Stress, Physiological; Temperature
PubMed: 34232098
DOI: 10.1128/Spectrum.00008-21 -
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 -
Antimicrobial Agents and Chemotherapy Dec 2019Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of carrying variants of the locus and exhibiting reduced susceptibility to...
Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of carrying variants of the locus and exhibiting reduced susceptibility to artemisinin. Mutations in two other genes, and , are associated with artemisinin resistance in rodent malaria and with clinical failure of combination therapy in African malaria patients. Transgenic clones, each carrying orthologues of mutations in and associated with artemisinin resistance in , were derived by Cas9 gene editing. Susceptibility to artemisinin and other antimalarial drugs was determined. Following exposure to 700 nM dihydroartemisinin in the ring-stage survival assay, we found strong evidence that transgenic parasites expressing the I592T variant (11% survival), but not the S160N variant (1% survival), of the AP2μ adaptin subunit were significantly less susceptible than the parental wild-type parasite population. The V3275F variant of UBP1, but not the V3306F variant, also displayed reduced susceptibility to dihydroartemisinin (8.5% survival versus 0.5% survival). AP2μ and UBP1 variants did not elicit reduced susceptibility to 48 h of exposure to artemisinin or to other antimalarial drugs. Therefore, variants of the AP2 adaptor complex μ-subunit and of the ubiquitin hydrolase UBP1 reduce artemisinin susceptibility at the early ring stage in These findings confirm the existence of multiple pathways to perturbation of either the mode of action of artemisinin, the parasite's adaptive mechanisms of resistance, or both. The cellular role of UBP1 and AP2μ in parasites should now be elucidated.
Topics: Antimalarials; Artemisinins; Gene Editing; Plasmodium falciparum; Protozoan Proteins
PubMed: 31636063
DOI: 10.1128/AAC.01542-19