-
Current Opinion in Microbiology Dec 2020For malaria parasites regulating sexual commitment, the frequency with which asexual bloodstream forms differentiate into non-replicative male and female gametocytes, is... (Review)
Review
For malaria parasites regulating sexual commitment, the frequency with which asexual bloodstream forms differentiate into non-replicative male and female gametocytes, is critical because asexual replication is required to maintain a persistent infection of the human host while gametocytes are essential for infection of the mosquito vector and transmission. Here, we describe recent advances in understanding of the regulatory mechanisms controlling this key developmental decision. These include new insights into the mechanistic roles of the transcriptional master switch AP2-G and the epigenetic modulator GDV1, as well as the identification of defined metabolic signals that modulate their activity. Many of these metabolites are linked to parasite phospholipid biogenesis and we propose a model linking this pathway to the epigenetic regulation underlying sexual commitment in P. falciparum.
Topics: Animals; Epigenesis, Genetic; Germ Cells; Humans; Malaria, Falciparum; Plasmodium falciparum; Reproduction
PubMed: 33053503
DOI: 10.1016/j.mib.2020.09.004 -
PLoS Neglected Tropical Diseases Aug 2021Understanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria...
BACKGROUND
Understanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria human-to-mosquito transmission.
METHODOLOGY/PRINCIPAL FINDINGS
Plasmodium falciparum and P. vivax parasites and gametocytes were quantified by qPCR and RT-qPCR assays using the same methodologies in 5 cross-sectional surveys involving 16,493 individuals in Brazil, Thailand, Papua New Guinea, and Solomon Islands. The proportion of infections with detectable gametocytes per survey ranged from 44-94% for P. falciparum and from 23-72% for P. vivax. Blood-stage parasite density was the most important predictor of the probability to detect gametocytes. In moderate transmission settings (prevalence by qPCR>5%), parasite density decreased with age and the majority of gametocyte carriers were children. In low transmission settings (prevalence<5%), >65% of gametocyte carriers were adults. Per survey, 37-100% of all individuals positive for gametocytes by RT-qPCR were positive by light microscopy for asexual stages or gametocytes (overall: P. falciparum 178/348, P. vivax 235/398).
CONCLUSIONS/SIGNIFICANCE
Interventions to reduce human-to-mosquito malaria transmission in moderate-high endemicity settings will have the greatest impact when children are targeted. In contrast, all age groups need to be included in control activities in low endemicity settings to achieve elimination. Detection of infections by light microscopy is a valuable tool to identify asymptomatic blood stage infections that likely contribute most to ongoing transmission at the time of sampling.
Topics: Adolescent; Asymptomatic Diseases; Brazil; Child; Child, Preschool; Cross-Sectional Studies; Female; Humans; Infant; Malaria, Falciparum; Malaria, Vivax; Male; Papua New Guinea; Plasmodium falciparum; Plasmodium vivax; Thailand; Young Adult
PubMed: 34449764
DOI: 10.1371/journal.pntd.0009672 -
ELife Sep 2022the causative agent of malaria, remains a global health threat as parasites continue to develop resistance to antimalarial drugs used throughout the world. Accordingly,...
the causative agent of malaria, remains a global health threat as parasites continue to develop resistance to antimalarial drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. parasites infect human red blood cells where they digest the host's main protein constituent, hemoglobin. Leucine aminopeptidase A-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here, we use both reverse genetics and a compound specifically designed to inhibit the activity of A-M17 to show that A-M17 is essential for survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that loss of A-M17 results in parasites exhibiting multiple digestive vacuoles at the trophozoite stage. In contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate A-M17 as a potential novel drug target.
Topics: Aminopeptidases; Digestion; Hemoglobins; Humans; Malaria, Falciparum; Plasmodium falciparum; Protease Inhibitors; Protozoan Proteins
PubMed: 36097817
DOI: 10.7554/eLife.80813 -
Turkiye Parazitolojii Dergisi Dec 2020is a protozoan parasite that causes many deaths worldwide. It's cultivation in an in vitro culture setting contributes significantly to scientific studies. However,...
OBJECTIVE
is a protozoan parasite that causes many deaths worldwide. It's cultivation in an in vitro culture setting contributes significantly to scientific studies. However, there are no laboratories in Turkey that cultivate . Hence, the purpose of this study was to cultivate .
METHODS
Five strains were used in our study and were kept frozen in liquid nitrogen tanks. These parasite strains were then thawed in a 37 °C water bath and transferred to the Albumax-complete medium that was previously prepared. After that, the petri dishes were placed in the chamber. For 30 seconds, a special gas mixture containing 5% CO, 5% O and 90% N was added into the chamber which was placed in a 37 °C oven and left for incubation for 2 days. At the end of the incubation period, thin smear preparations were prepared from the medium, stained with Giemsa and examined using an immersion lens.
RESULTS
Examination of the smears revealed that trophozoite and schizont forms of all isolates were present at a rate of 2% in culture medium.
CONCLUSION
As a result of our study, the culture of was successfully developed. With this, several projects such as biological and chemical characteristics, pathogenicity, phenotypic and molecular-level drug sensitivities and parasite vaccination studies can be carried out more easily in our country.
Topics: Animals; Culture Media; Humans; In Vitro Techniques; Plasmodium falciparum; Schizonts; Trophozoites; Turkey
PubMed: 33269565
DOI: 10.4274/tpd.galenos.2020.7148 -
Frontiers in Cellular and Infection... 2020Extracellular vesicles (EVs) are minute particles secreted by the cells of living organisms. Although the functional role of EVs is not yet clear, recent work has... (Review)
Review
Extracellular vesicles (EVs) are minute particles secreted by the cells of living organisms. Although the functional role of EVs is not yet clear, recent work has highlighted their role in intercellular communication. Here, we expand on this view by suggesting that EVs can also mediate communication among interacting organisms such as hosts, pathogens and vectors. This inter-kingdom communication via EVs is likely to have important evolutionary consequences ranging from adaptation of parasites to specialized niches in the host, to host resistance and evolution and maintenance of parasite virulence and transmissibility. A potential system to explore these consequences is the interaction among the human host, the mosquito vector and parasite involved in the malaria disease. Indeed, recent studies have found that EVs derived from infected red blood cells in humans are likely mediating the parasite's transition from the asexual to sexual stage, which might facilitate transmission to the mosquito vector. However, more work is needed to establish the adaptive consequences of this EV signaling among different taxa. We suggest that an integrative molecular approach, including a comparative phylogenetic analysis of the molecules (e.g., proteins and nucleic acids) derived from the EVs of interacting organisms (and their closely-related species) in the malaria system will prove useful for understanding interkingdom communication. Such analyses will also shed light on the evolution and persistence of host, parasite and vector interactions, with implications for the control of vector borne infectious diseases.
Topics: Animals; Biological Evolution; Extracellular Vesicles; Host-Parasite Interactions; Humans; Malaria, Falciparum; Mosquito Vectors; Plasmodium falciparum
PubMed: 32195195
DOI: 10.3389/fcimb.2020.00076 -
Molecular Microbiology Apr 2021Extrachromosomal (ec) DNAs are genetic elements that exist separately from the genome. Since ecDNA can carry beneficial genes, they are a powerful adaptive mechanism in...
Extrachromosomal (ec) DNAs are genetic elements that exist separately from the genome. Since ecDNA can carry beneficial genes, they are a powerful adaptive mechanism in cancers and many pathogens. For the first time, we report ecDNA contributing to antimalarial resistance in Plasmodium falciparum, the most virulent human malaria parasite. Using pulse field gel electrophoresis combined with PCR-based copy number analysis, we detected two ecDNA elements that differ in migration and structure. Entrapment in the electrophoresis well and low susceptibility to exonucleases revealed that the biologically relevant ecDNA element is large and complex in structure. Using deep sequencing, we show that ecDNA originates from the chromosome and expansion of an ecDNA-specific sequence may improve its segregation or expression. We speculate that ecDNA is maintained using established mechanisms due to shared characteristics with the mitochondrial genome. Implications of ecDNA discovery in this organism are wide-reaching due to the potential for new strategies to target resistance development.
Topics: Adaptation, Physiological; Antimalarials; DNA, Protozoan; Drug Resistance; Gene Amplification; Genome, Protozoan; Humans; Malaria, Falciparum; Plasmodium falciparum; Pyrimidines
PubMed: 33053232
DOI: 10.1111/mmi.14624 -
Malaria Journal Jul 2020High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers...
BACKGROUND
High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 (pfpm2), exonuclease (pfexo) and chloroquine resistance transporter (pfcrt) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy.
METHODS
To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined.
RESULTS
The characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone-proguanil combinations revealed synergistic antimalarial activity.
CONCLUSIONS
Surveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.
Topics: Antimalarials; Cambodia; Drug Resistance; Genetic Markers; Genotype; Phenotype; Plasmodium falciparum; Quinolines
PubMed: 32711538
DOI: 10.1186/s12936-020-03339-w -
Parasitology Research Oct 2019Plasmodium falciparum (Pf) refurbishes the infected erythrocytes by exporting a myriad of parasite proteins to the host cell. A novel exported protein family 'Plasmodium... (Review)
Review
Plasmodium falciparum (Pf) refurbishes the infected erythrocytes by exporting a myriad of parasite proteins to the host cell. A novel exported protein family 'Plasmodium Helical Interspersed Subtelomeric' (PHIST) has gained attention for its significant roles in parasite biology. Here, we have collected and analysed available information on PHIST members to enhance understanding of their functions, varied localization and structure-function correlation. Functional diversity of PHIST proteins is highlighted by their involvement in PfEMP1 (Pf erythrocyte membrane protein 1) expression, trafficking and switching. This family also contributes to cytoadherence, gametocytogenesis, host cell modification and generation of extracellular vesicles. While the PHIST domain forms the hallmark of this family, existence and functions of additional domains (LyMP, TIGR01639) and the MEC motif underscores its diversity further. Since specific PHIST proteins seem to form pairs with PfEMP1 members, we have used in silico tools to predict such potential partners in Pf. This information and our analysis of structural data on a PHIST member provide important insights into their functioning. This review overall enables readers to view the PHIST family comprehensively, while highlighting key knowledge gaps in the field.
Topics: Animals; Erythrocytes; Humans; Malaria, Falciparum; Multigene Family; Plasmodium falciparum; Protein Transport; Protozoan Proteins
PubMed: 31418110
DOI: 10.1007/s00436-019-06420-9 -
Current Opinion in Microbiology Oct 2021The search for host factors that leads to malaria parasite synchronization has been the focus of several laboratories. The host hormone melatonin synchronizes Plasmodium... (Review)
Review
The search for host factors that leads to malaria parasite synchronization has been the focus of several laboratories. The host hormone melatonin synchronizes Plasmodium falciparum in culture by increasing the number of mature parasite stages through a PLC-IP3 activation. Melatonin signaling is linked to crosstalk between Ca-cAMP that results in PKA activation. Two other kinases, PfPK7 and PfeIK1, and the nuclear protein PfMORC that lacks melatonin sensitivity in the inducible knock-down parasites are also identified as part of the hormone-signal transduction pathways. Melatonin also modulates P. falciparum mitochondrial fission genes FIS1, DYN1, and DYN2 in a stage-specific manner. How these multiple molecular mechanisms are orchestrated to lead to parasite synchronization is a fascinating and opened biological question.
Topics: Animals; Erythrocytes; Malaria, Falciparum; Melatonin; Parasites; Plasmodium falciparum
PubMed: 34428626
DOI: 10.1016/j.mib.2021.08.002 -
Malaria Journal Aug 2020The emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and...
BACKGROUND
The emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and if unresolved would eventually result in cell death. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum (Pf) MAG was characterized for its potential for development as an anti-malarial candidate.
METHODS
Native PfMAG from crude extract of chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was partially purified using three chromatographic procedures. From bio-informatics analysis, primers were designed for amplification, insertion into pBAD202/D-TOPO and heterologous expression in Escherichia coli of recombinant PfMAG. Functional and biochemical properties of the recombinant enzyme were characterized.
RESULTS
PfMAG activity was most prominent in parasite schizont stages, with a specific activity of 147 U/mg (partially purified) protein. K1 PfMAG contained an insertion of AAT (coding for asparagine) compared to 3D7 strain and 16% similarity to the human enzyme. Recombinant PfMAG (74 kDa) was twice as large as the human enzyme, preferred double-stranded DNA substrate, and demonstrated glycosylase activity over a pH range of 4-9, optimal salt concentration of 100-200 mM NaCl but reduced activity at 250 mM NaCl, no requirement for divalent cations, which were inhibitory in a dose-dependent manner.
CONCLUSION
PfMAG activity increased with parasite development being highest in the schizont stages. K1 PfMAG contained an indel AAT (asparagine) not present in 3D7 strain and the recombinant enzyme was twice as large as the human enzyme. Recombinant PfMAG had a wide range of optimal pH activity, and was inhibited at high (250 mM) NaCl concentration as well as by divalent cations. The properties of PfMAG provide basic data that should be of assistance in developing anti-malarials against this potential parasite target.
Topics: DNA Glycosylases; Plasmodium falciparum; Protozoan Proteins
PubMed: 32762689
DOI: 10.1186/s12936-020-03355-w