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MSphere Aug 2020We developed a flow-cytometry-based method to separate and collect cocultured male and female gametocytes responsible for malaria transmission. The purity of the...
We developed a flow-cytometry-based method to separate and collect cocultured male and female gametocytes responsible for malaria transmission. The purity of the collected cells was estimated at >97% using flow cytometry, and sorted cells were observed by Giemsa-stained thin-smear and live-cell fluorescence microscopy. The expression of validated sex-specific markers corroborated the sorting strategy. Collected male and female gametocytes were used to confirm three novel sex-specific markers by quantitative real-time PCR that were more enriched in sorted male and female gametocyte populations than existing sex-specific markers. We also applied the method as a proof-of-principle drug screen that allows the identification of drugs that kill gametocytes in a sex-specific manner. Since the developed method allowed for the separation of male and female parasites from the same culture, we observed for the first time a difference in development time between the sexes: females developed faster than males. Hence, the ability to separate male and female gametocytes opens the door to a new field of sex-specific gametocyte biology to further our understanding of malaria transmission. The protozoan causes the most severe form of human malaria. The development of sexual forms (so-called gametocytes) is crucial for disease transmission. However, knowledge of these forms is severely hampered by the paucity of sex-specific markers and the inability to extract single sex gametocytes in high purity. Moreover, the identification of compounds that specifically affect one sex is difficult due to the female bias of the gametocytes. We have developed a system that allows for the separation of male and female gametocytes from the same population. Applying our system, we show that male and female parasites mature at different rates, which might have implications for transmission. We also identified new sex-specific genes that can be used as sex markers or to unravel sex-specific functions. Our system will not only aid in the discovery of much needed gametocidal compounds, but it also represents a valuable tool for exploring malaria transmission biology.
Topics: Antimalarials; Drug Discovery; Erythrocytes; Flow Cytometry; Germ Cells; Humans; Microscopy, Fluorescence; Plasmodium falciparum; Protozoan Proteins
PubMed: 32817458
DOI: 10.1128/mSphere.00671-20 -
Trends in Parasitology Sep 2022The recent study by Campelo Morillo et al. has shown that one of the small number of non-ApiAP2 DNA-binding proteins in the Plasmodium falciparum genome acts as a...
The recent study by Campelo Morillo et al. has shown that one of the small number of non-ApiAP2 DNA-binding proteins in the Plasmodium falciparum genome acts as a transcription factor in the gametocytogenesis cascade and is responsible for the gametocyte's distinctive morphology.
Topics: Gene Expression Regulation; Plasmodium falciparum; Transcription Factors
PubMed: 35871979
DOI: 10.1016/j.pt.2022.07.004 -
The Journal of Infectious Diseases Jul 2018Falcipain-2a ([FP2a] PF3D7_1115700) is a Plasmodium falciparum cysteine protease and hemoglobinase. Functional FP2a is required for potent activity of artemisinin, and...
BACKGROUND
Falcipain-2a ([FP2a] PF3D7_1115700) is a Plasmodium falciparum cysteine protease and hemoglobinase. Functional FP2a is required for potent activity of artemisinin, and in vitro selection for artemisinin resistance selected for an FP2a nonsense mutation.
METHODS
To investigate associations between FP2a polymorphisms and artemisinin resistance and to characterize the diversity of the enzyme in parasites from the China-Myanmar border, we sequenced the full-length FP2a gene in 140 P falciparum isolates collected during 2004-2011.
RESULTS
The isolates were grouped into 8 different haplotype groups. Haplotype group I appeared in samples obtained after 2008, coinciding with implementation of artemisinin-based combination therapy in this region. In functional studies, compared with wild-type parasites, the FP2a haplotypes demonstrated increased ring survival, and all haplotype groups exhibited significantly reduced FP2a activity, with group I showing the slowest protease kinetics and reduced parasite fitness.
CONCLUSIONS
These results suggest that altered hemoglobin digestion due to FP2a mutations may contribute to artemisinin resistance.
Topics: Antimalarials; Artemisinins; China; Cysteine Endopeptidases; DNA, Protozoan; Drug Resistance; Genetic Variation; Haplotypes; Humans; Malaria, Falciparum; Myanmar; Plasmodium falciparum; Sequence Analysis, DNA
PubMed: 29659945
DOI: 10.1093/infdis/jiy188 -
Scientific Reports Sep 2019Given the number of global malaria cases and deaths, the need for a vaccine against Plasmodium falciparum (Pf) remains pressing. Administration of live,...
Given the number of global malaria cases and deaths, the need for a vaccine against Plasmodium falciparum (Pf) remains pressing. Administration of live, radiation-attenuated Pf sporozoites can fully protect malaria-naïve individuals. Despite the fact that motility of these attenuated parasites is key to their infectivity and ultimately protective efficacy, sporozoite motility in human tissue (e.g. skin) remains wholly uncharacterized to date. We show that the ability to quantitatively address the complexity of sporozoite motility in human tissue provides an additional tool in the development of attenuated sporozoite vaccines. We imaged Pf movement in the skin of its natural host and compared wild-type and radiation-attenuated GFP-expressing Pf sporozoites. Using custom image analysis software and human skin explants we were able to quantitatively study their key motility features. This head-to-head comparison revealed that radiation attenuation impaired the capacity of sporozoites to vary their movement angle, velocity and direction, promoting less refined movement patterns. Understanding and overcoming these changes in motility will contribute to the development of an efficacious attenuated parasite malaria vaccine.
Topics: Animals; Anopheles; Green Fluorescent Proteins; Host-Parasite Interactions; Humans; Image Processing, Computer-Assisted; Organisms, Genetically Modified; Plasmodium falciparum; Skin; Software; Sporozoites
PubMed: 31530862
DOI: 10.1038/s41598-019-49895-3 -
Nature Reviews. Microbiology Jun 2018
Topics: Antimalarials; Drug Resistance; Genome, Protozoan; Genomics; Mutation; Plasmodium falciparum
PubMed: 29703939
DOI: 10.1038/s41579-018-0008-1 -
ELife Oct 2020Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity, and ultimately pathogenesis of rely on a macromolecular complex,...
Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity, and ultimately pathogenesis of rely on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here, we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor's mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future anti-malarials targeting both the glideosome motor and its regulatory elements.
Topics: Antimalarials; Nonmuscle Myosin Type IIA; Plasmodium falciparum; Protozoan Proteins
PubMed: 33046215
DOI: 10.7554/eLife.60581 -
Malaria Journal Apr 2016Escalating drug resistance in malaria parasites and lack of vaccine entails the discovery of novel drug targets and inhibitor molecules. The multi-component protein... (Review)
Review
Escalating drug resistance in malaria parasites and lack of vaccine entails the discovery of novel drug targets and inhibitor molecules. The multi-component protein translation machinery is a rich source of such drug targets. Malaria parasites contain three translational compartments: the cytoplasm, apicoplast and mitochondrion, of which the latter two are of the prokaryotic type. Recent explorations by many groups into the malaria parasite protein translation enzymes, aminoacyl-tRNA synthetases (aaRSs), have yielded many promising inhibitors. The understanding of the biology of this unique set of 36 enzymes has become much clearer in recent times. Current review discusses the advances made in understanding of crucial aaRSs from Plasmodium and also the specific inhibitors found against malaria aaRSs.
Topics: Amino Acyl-tRNA Synthetases; Drug Delivery Systems; Plasmodium falciparum; Protozoan Proteins
PubMed: 27068331
DOI: 10.1186/s12936-016-1247-0 -
Current Opinion in Microbiology Aug 2014The eukaryotic unicellular pathogen Plasmodium falciparum tightly regulates gene expression, both during development and in adaptation to dynamic host environments. This... (Review)
Review
The eukaryotic unicellular pathogen Plasmodium falciparum tightly regulates gene expression, both during development and in adaptation to dynamic host environments. This regulation is evident in the mutually exclusive expression of members of clonally variant virulence multigene families. While epigenetic regulators have been selectively identified at active or repressed virulence genes, their specific recruitment remains a mystery. In recent years, noncoding RNAs (ncRNAs) have emerged as lynchpins of eukaryotic gene regulation; by binding to epigenetic regulators, they provide target specificity to otherwise non-specific enzyme complexes. Not surprisingly, there is great interest in understanding the role of ncRNA in P. falciparum, in particular, their contribution to the mutually exclusive expression of virulence genes. The current repertoire of P. falciparum ncRNAs includes, but is not limited to, subtelomeric ncRNAs, virulence gene-associated ncRNAs and natural antisense RNA transcripts. Continued improvement in high-throughput sequencing methods is sure to expand this repertoire. Here, we summarize recent advances in P. falciparum ncRNA biology, with an emphasis on ncRNA-mediated epigenetic modes of gene regulation.
Topics: Adaptation, Physiological; Epigenesis, Genetic; Gene Expression Regulation; Plasmodium falciparum; RNA, Untranslated; Virulence Factors
PubMed: 25022240
DOI: 10.1016/j.mib.2014.06.013 -
Biomolecules Aug 2020The indoleamine compound melatonin has been extensively studied in the regulation of the circadian rhythm in nearly all vertebrates. The effects of melatonin have also... (Review)
Review
The indoleamine compound melatonin has been extensively studied in the regulation of the circadian rhythm in nearly all vertebrates. The effects of melatonin have also been studied in Protozoan parasites, especially in the synchronization of the human malaria parasite via a complex downstream signalling pathway. Melatonin activates protein kinase A (PfPKA) and requires the activation of protein kinase 7 (PfPK7), PLC-IP, and a subset of genes from the ubiquitin-proteasome system. In other parasites, such as and , melatonin increases inflammatory components, thus amplifying the protective response of the host's immune system and affecting parasite load. The development of melatonin-related indole compounds exhibiting antiparasitic properties clearly suggests this new and effective approach as an alternative treatment. Therefore, it is critical to understand how melatonin confers stimulatory functions in host-parasite biology.
Topics: Animals; Antimalarials; Humans; Life Cycle Stages; Malaria, Falciparum; Melatonin; Plasmodium falciparum; Reproduction, Asexual; Signal Transduction
PubMed: 32867164
DOI: 10.3390/biom10091243 -
International Journal For Parasitology.... Aug 2019Mutations in the Kelch domain of the K13 gene (PF3D7_1343700) were previously associated with artemisinin resistance in Plasmodium falciparum. This study followed the...
Mutations in the Kelch domain of the K13 gene (PF3D7_1343700) were previously associated with artemisinin resistance in Plasmodium falciparum. This study followed the dynamics of the K13 polymorphisms in P. falciparum parasites from the China-Myanmar border area obtained in 2007-2016, and their in vitro sensitivities to artesunate (AS) and dihydroartemisinin (DHA). The 50% effective concentration (EC) values of 133 culture-adapted field isolates to AS and DHA, measured by the conventional 72 h SYBR Green I-based assay, varied significantly among the parasites from different years; all were significantly higher than that of the reference strain 3D7. Compared with parasites from 2007 to 2008, ring survival rates almost doubled in parasites obtained in later years. Sequencing the full-length K13 genes identified 11 point mutations present in 85 (63.9%) parasite isolates. F446I was the predominant (55/133) variant, and its frequency was increased from 17.6% (3/17) in 2007 to 55.9% (19/34) in 2014-2016. No wild-type (WT) Kelch domain sequences were found in the 34 samples obtained from 2014 to 2016. In the 2014-2016 samples, a new mutation (G533S) appeared and reached 44.1% (15/34). Collectively, parasites with the Kelch domain mutations (after amino acid 440) had significantly higher ring survival rates than the WT parasites. Individually, F446I, G533S and A676D showed significantly higher ring survival rates than the WT. Although the drug sensitivity phenotypes measured by the RSA and EC assays may be intrinsically linked to the in vivo clinical efficacy data, the values determined by these two assays were not significantly correlated. This study identified the trend of K13 mutations in parasite populations from the China-Myanmar border area, confirmed an overall correlation of Kelch domain mutations with elevated ring-stage survival rates, and emphasized the importance of monitoring the evolution and spread of parasites with reduced artemisinin sensitivity along the malaria elimination course.
Topics: Antiprotozoal Agents; Artemisinins; China; Humans; Malaria, Falciparum; Microfilament Proteins; Mutation; Myanmar; Plasmodium falciparum; Protozoan Proteins
PubMed: 31009824
DOI: 10.1016/j.ijpddr.2019.04.002