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MBio Jun 2021During its complex life cycle, the malaria parasite survives dramatic environmental stresses, including large temperature shifts. Protein prenylation is required during...
During its complex life cycle, the malaria parasite survives dramatic environmental stresses, including large temperature shifts. Protein prenylation is required during asexual replication of Plasmodium falciparum, and the canonical heat shock protein 40 protein (HSP40; PF3D7_1437900) is posttranslationally modified with a 15-carbon farnesyl isoprenyl group. In other organisms, farnesylation of Hsp40 orthologs controls their localization and function in resisting environmental stress. In this work, we find that plastidial isopentenyl pyrophosphate (IPP) synthesis and protein farnesylation are required for malaria parasite survival after cold and heat shock. Furthermore, loss of HSP40 farnesylation alters its membrane attachment and interaction with proteins in essential pathways in the parasite. Together, this work reveals that farnesylation is essential for parasite survival during temperature stress. Farnesylation of HSP40 may promote thermotolerance by guiding distinct chaperone-client protein interactions.
Topics: Erythrocytes; HSP40 Heat-Shock Proteins; Heat-Shock Response; Hemiterpenes; Host-Parasite Interactions; Humans; Life Cycle Stages; Organophosphorus Compounds; Plasmodium falciparum; Protein Prenylation; Protozoan Proteins; Thermotolerance
PubMed: 34182772
DOI: 10.1128/mBio.00760-21 -
MBio Oct 2022The repeated emergence of antimalarial drug resistance in Plasmodium falciparum, including to the current frontline antimalarial artemisinin, is a perennial problem for...
The repeated emergence of antimalarial drug resistance in Plasmodium falciparum, including to the current frontline antimalarial artemisinin, is a perennial problem for malaria control. Next-generation sequencing has greatly accelerated the identification of polymorphisms in resistance-associated genes but has also highlighted the need for more sensitive and accurate laboratory tools to profile current and future antimalarials and to quantify the impact of drug resistance acquisition on parasite fitness. The interplay of fitness and drug response is of fundamental importance in understanding why particular genetic backgrounds are better at driving the evolution of drug resistance in natural populations, but the impact of parasite fitness landscapes on the epidemiology of drug resistance has typically been laborious to accurately quantify in the lab, with assays being limited in accuracy and throughput. Here we present a scalable method to profile fitness and drug response of genetically distinct P. falciparum strains with well-described sensitivities to several antimalarials. We leverage CRISPR/Cas9 genome-editing and barcode sequencing to track unique barcodes integrated into a nonessential gene (). We validate this approach in multiplex competitive growth assays of three strains with distinct geographical origins. Furthermore, we demonstrate that this method can be a powerful approach for tracking artemisinin response as it can identify an artemisinin resistant strain within a mix of multiple parasite lines, suggesting an approach for scaling the laborious ring-stage survival assay across libraries of barcoded parasite lines. Overall, we present a novel high-throughput method for multiplexed competitive growth assays to evaluate parasite fitness and drug response. The complex interplay between antimalarial resistance and parasite fitness has important implications for understanding the development and spread of drug resistance alleles and the impact of genetic background on transmission. One limitation with current methodologies to measure parasite fitness is the ability to scale this beyond simple head-to-head competition experiments between a wildtype control line and test line, with a need for a scalable approach that allows tracking of parasite growth in complex mixtures. In our study, we have used CRISPR editing to insert unique DNA barcodes into a safe-harbor genomic locus to tag multiple parasite strains and use next-generation sequencing to read out strain dynamics. We observe inherent fitness differences between the strains, as well as sensitive modulation of responses to challenge with clinically relevant antimalarials, including artemisinin.
Topics: Antimalarials; Artemisinins; Complex Mixtures; Drug Resistance; Plasmodium falciparum; Protozoan Proteins; Genetic Fitness
PubMed: 35972144
DOI: 10.1128/mbio.00937-22 -
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 -
Nature Communications Jul 2023The chloroquine resistance transporter, PfCRT, of the human malaria parasite Plasmodium falciparum is sensitive to acidic pH. Consequently, PfCRT operates at 60% of its...
The chloroquine resistance transporter, PfCRT, of the human malaria parasite Plasmodium falciparum is sensitive to acidic pH. Consequently, PfCRT operates at 60% of its maximal drug transport activity at the pH of 5.2 of the digestive vacuole, a proteolytic organelle from which PfCRT expels drugs interfering with heme detoxification. Here we show by alanine-scanning mutagenesis that E207 is critical for pH sensing. The E207A mutation abrogates pH-sensitivity, while preserving drug substrate specificity. Substituting E207 with Asp or His, but not other amino acids, restores pH-sensitivity. Molecular dynamics simulations and kinetics analyses suggest an allosteric binding model in which PfCRT can accept both protons and chloroquine in a partial noncompetitive manner, with increased proton concentrations decreasing drug transport. Further simulations reveal that E207 relocates from a peripheral to an engaged location during the transport cycle, forming a salt bridge with residue K80. We propose that the ionized carboxyl group of E207 acts as a hydrogen acceptor, facilitating transport cycle progression, with pH sensing as a by-product.
Topics: Humans; Antimalarials; Chloroquine; Membrane Transport Proteins; Protozoan Proteins; Plasmodium falciparum; Hydrogen-Ion Concentration; Drug Resistance; Malaria, Falciparum
PubMed: 37454114
DOI: 10.1038/s41467-023-39969-2 -
Experimental Biology and Medicine... Jan 2021malaria is a global health problem. Erythrocyte invasion by merozoites appears to be a promising target to curb malaria. We have identified and characterized a novel...
malaria is a global health problem. Erythrocyte invasion by merozoites appears to be a promising target to curb malaria. We have identified and characterized a novel protein that is involved in erythrocyte invasion. Our data on protein subcellular localization, stage-specific protein expression pattern, and merozoite invasion inhibition by α-peptide antibodies suggest a role for PF3D7_1459400 protein during erythrocyte invasion. Even more, the human immunoepidemiology data present PF3D7_1459400 protein as an immunogenic antigen which could be further exploited for the development of new anti-infective therapy against malaria.
Topics: Adult; Amino Acid Sequence; Animals; Antibodies, Protozoan; Conserved Sequence; Erythrocytes; Humans; Life Cycle Stages; Plasmodium falciparum; Protozoan Proteins; Rats; Recombinant Proteins; Subcellular Fractions
PubMed: 33019810
DOI: 10.1177/1535370220961764 -
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 -
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 -
Emerging Infectious Diseases Apr 2022Single-nucleotide polymorphisms at several loci have been correlated with Plasmodium falciparum drug resistance. We examined the prevalence of resistance markers in P....
Single-nucleotide polymorphisms at several loci have been correlated with Plasmodium falciparum drug resistance. We examined the prevalence of resistance markers in P. falciparum from imported malaria cases in Canada during 3 time periods, 2008-2009, 2013-2014, and 2017-2018. We evaluated single-nucleotide polymorphisms at atpase6 (pfATPase6), pfcrt (chloroquine resistance transporter), cytb (cytochrome b), dhfr (dihydrofolate reductase), dhps (dihydropteroate synthetase), mdr1 (multidrug resistance protein) and mdr1 copy number, and kelch13 (kelch protein gene on chromosome 13). Over time, we observed increasing mutant genotypes for dhfr S108N and dhps A613T and decreasing mutant genotypes for mdr1 N86Y, D1246Y, pfcrt K76T, and pfcrt 74-75; we identified no kelch13 mutations. We observed fewer mutations indicative of chloroquine resistance over time, which may reflect reduced chloroquine pressure in specimens from travelers to Africa. Mutations conferring proguanil resistance increased over time. Minor genotypes confirm the heterogeneous nature of infection and may affect treatment success.
Topics: Anti-Infective Agents; Antimalarials; Ontario; Plasmodium falciparum; Protozoan Proteins
PubMed: 35318914
DOI: 10.3201/eid2804.210533 -
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