Parasite Lines Research Articles

Mapping of Ligula plerocercoids in the freshwater bream Abramis brama in Lake Syamozero and some other lakes of Northwestern Russia

The tapeworm Ligula intestinalis (Linnaeus, 1758) is a broadly distributed parasite with high epidemiological significance. Its first intermediate hosts are crustaceans, its second intermediate hosts are fish, while the definitive hosts are various fish-eating birds and, as recently shown, ringed seals. The stages of Ligula intestinalis in fish are called plerocercoids. In this study we obtained new data on the occurrence of plerocercoids of L. intestinalis in freshwater bream Abramis brama in six lakes in Karelia (Northwestern Russia). Infection of freshwater bream with L. intestinalis in Lakes Onego, Ladoga, Svyatozero and Konchezero was found for the first time. In Lake Syamozero, where long-term parasitological monitoring has been conducted, infection indices of bream with L. intestinalis were similar to those recorded in 1975. Using two mitochondrial genes (cytb and COI), we examined the genetic structure of L. intestinalis from different localities. Phylogenetic analysis showed that all specimens of of L. intestinalis examined in our study belonged to “Lineage A” identified by Nazarizadeh et al. (2023). We found 40 haplotypes of concatenated cox1+ cytb in freshwater bream from different water bodies in Northwestern Russia and Europe. Plerocercoids from Karelian lakes were represented by 22 haplotypes, of which 21 were unique. Our molecular analysis of L. intestinalis from freshwater bream in European part of the Palearctic shows that this parasite lineage is genetically diverse and rapidly expanding.

Study on the Resonance Characteristics and Active Damping Suppression Strategies of Multi-Inverter Grid-Connected Systems Under Weak Grid Conditions

When a multi-inverter grid-connected system is influenced by the parasitic parameters of LCL-type inverters and the impedance of the connected system’s lines, its resonance characteristics become more complex and difficult to predict. For LCL-type multi-inverter grid-connected systems, a mathematical model that considers the effects of parasitic parameters and line impedance has been established, leading to the derivation of the system’s Norton equivalent circuit and a general expression for the inverter output current. The resonance characteristics of multi-inverter grid-connected systems composed of inverters with the same and different parameters were analyzed under the influence of parasitic parameters and line impedance. To suppress the resonance in multi-inverter grid-connected systems and address the issue of traditional PI control not meeting grid requirements for LCL-type grid-connected inverters, a strategy combining superhelical sliding mode control with active damping was adopted. To verify the practical performance of the adopted resonance suppression strategy in complex environments, a grid-connected system model containing two LCL-type inverters was constructed using the MATLAB/Simulink software platform, followed by simulation analysis. The simulation results strongly confirm the feasibility and effectiveness of the adopted resonance suppression strategy, considering the effects of parasitic parameters and line impedance. This strategy demonstrates significant suppression effects in addressing resonance issues caused by parasitic parameters and line impedance, effectively enhancing the quality of grid-connected electrical energy.

Triple artemisinin-based combination therapy (TACT): Efficacy of dihydroartemisinin-piperaquine plus chloroquine against Plasmodium berghei ANKA strains with different drug sensitivities in a murine malaria model

BackgroundEvident detection of artemisinin resistance markers in patient isolates of Plasmodium falciparum from East Africa threatens the efficacy of artemisinin-based combination therapies (ACTs) as first-line treatment of malaria in sub-Saharan Africa. Repositioning previously used antimalarials as complementary addition to ACTs has been suggested as a viable option to mitigating this threat. This study evaluated the potential benefit of chloroquine (CQ) as a complementary partner to dihydroartemisinin/piperaquine (DHA/PQ) in the treatment of malaria in a mice model. MethodsThe comparative efficacy of the combination of DHA/PQ/CQ and DHA/PQ against two strains of Plasmodium berghei ANKA (MRA 311 and 671) with different levels of sensitivities to chloroquine was evaluated in separate experiments. Parasitological activities including; parasite suppression time, parasite clearance time, recrudescence time, and parasite reduction ratio were evaluated in vivo. The mean survival time was also monitored throughout the duration of the study. ResultsIn both parasite lines, 99.99 % chemo-suppression was observed on day 4 in the drug treatment groups (CQ alone, DHA/PQ and DHA/PQ/CQ). In the curative test, there were significant differences between DHA/PQ/CQ and DHQ/PQ treatment, highlighted by reduced parasite clearance time (4.75 ± 0.3 Vs 5.5 ± 0.3 days, P < 0.05), significantly delayed recrudescence time (28.5 ± 1.04 Vs 13.3 ± 0.48 days, P < 0.01), a 1.5-fold change in parasite reduction ratio, and a prolonged mean survival time (34.5 ± 1.04 Vs 26.7 ± 0.48 days, P < 0.05). ConclusionThe addition of chloroquine to dihydroartemisinin-piperaquine may be beneficial in the treatment of malaria, especially in areas where malaria parasite sensitivity to chloroquine is predominant.

Detailing organelle division and segregation in Plasmodium falciparum.

The malaria-causing parasite, P. falciparum, replicates through schizogony, a tightly orchestrated process where numerous daughter parasites are formed simultaneously. Proper division and segregation of one-per-cell organelles, like the mitochondrion and apicoplast, are essential, yet remain poorly understood. We developed a new reporter parasite line that allows visualization of the mitochondrion in blood and mosquito stages. Using high-resolution 3D imaging, we found that the mitochondrion orients in a cartwheel structure, prior to stepwise, non-geometric division during last-stage schizogony. Analysis of focused ion beam scanning electron microscopy data confirmed these mitochondrial division stages. Furthermore, these data allowed us to elucidate apicoplast division steps, highlighted its close association with the mitochondrion, and showed putative roles of the centriolar plaques in apicoplast segregation. These observations form the foundation for a new detailed mechanistic model of mitochondrial and apicoplast division and segregation during P. falciparum schizogony and pave the way for future studies into the proteins and protein complexes involved in organelle division and segregation.

A novel 4-aminoquinoline chemotype with multistage antimalarial activity and lack of cross-resistance with PfCRT and PfMDR1 mutants

Artemisinin-based combination therapy (ACT) is the mainstay of effective treatment of Plasmodium falciparum malaria. However, the long-term utility of ACTs is imperiled by widespread partial artemisinin resistance in Southeast Asia and its recent emergence in parts of East Africa. This underscores the need to identify chemotypes with new modes of action (MoAs) to circumvent resistance to ACTs. In this study, we characterized the asexual blood stage antiplasmodial activity and resistance mechanisms of LDT-623, a 4-aminoquinoline (4-AQ). We also detected LDT-623 activity against multiple stages (liver schizonts, stage IV-V gametocytes, and ookinetes) of Plasmodium’s life cycle, a feature unlike other 4-AQs such as chloroquine (CQ) and piperaquine (PPQ). Using heme fractionation profiling and drug uptake studies in PfCRT-containing proteoliposomes, we observed inhibition of hemozoin formation and PfCRT-mediated transport, which constitute characteristic features of 4-AQs’ MoA. We also found minimal cross-resistance to LDT-623 in a panel of mutant pfcrt or pfmdr1 lines, but not the PfCRT F145I mutant that is highly resistant to PPQ resistance yet is very unfit. No P. falciparum parasites were recovered in an in vitro resistance selection study, suggesting a high barrier for resistance to emerge. Finally, a competitive growth assay comprising &gt;50 barcoded parasite lines with mutated resistance mediators or major drug targets found no evidence of cross-resistance. Our findings support further exploration of this promising 4-AQ.

Parasitic plants show striking convergence in host preference across angiosperm lineages.

The host specificity of a parasite underpins its ecology, distribution, invasive potential and adaptability, yet for most parasitic plants host ranges are poorly understood. We examine host-parasite relationships across lineages to infer how host specificity may have influenced the evolution of parasitism in plants. Host preference data for all plant holoparasite species were manually collected from literature and herbarium specimens, then analysed to investigate and visualise host diversity and specificity. We reveal a disproportionality in host preference across host lineages: the Asteraceae contains 10% of angiosperm diversity but is infected by 31% of parasite species; meanwhile Monocots comprise 23% but are infected by just 3.2%. Furthermore, we observe striking convergence in host preference: Asteraceae, Euphorbiaceae and Fabaceae are infected by six, five and four independent parasite lineages, respectively. We also demonstrate considerable variation in the degree of host specificity among closely related parasite species; a result that does not reflect the expectation of holoparasites - especially endoparasites - as host specialists. The marked pattern of convergence in preference across disparate lineages points to a common pathway in the evolution of parasitism of eudicots in preference to monocots, which may have in turn have been driven by a divergence in host root and vascular architecture. The unexpected variation in host specificity among closely related species suggests that even apparent generalists may comprise cryptic host-specific taxa. This highlights the value of host preference as an additional consideration in parasitic plant taxonomy. Together, our data point to a complex interplay between ecological and physiological factors driving the evolution of host-parasite interactions. Moreover, they emphasize how little is known about the ecology of most holoparasitic plants, a group of organisms that are especially vulnerable at a time of unprecedented biodiversity loss and extinction.

Elevated Atmospheric Co2 Levels Impact Soil Protist Functional Core Community Compositions

Protists, known as microeukaryotes, are a significant portion of soil microbial communities. They are crucial predators of bacteria and depend on bacterial community dynamics for the growth and evolution of protistan communities. In parallel, increased levels of atmospheric CO2 significantly impact bacterial metabolic activity in rhizosphere soils. In this study, we investigated the effect of elevated atmospheric CO2 levels on the metabolically active protist community composition and function and their co-occurrences with bacteria from bulk and rhizosphere soils from the Giessen Free-Air CO2 enrichment grassland experiment. Metabarcoding sequencing data analyses of partial 18S rRNA from total soil RNA showed that elevated CO2 concentrations stimulated only a few ASVs of phagotrophic predators of bacteria and other microeukaryotes, affecting protist community composition (P = 0.006, PERMANOVA). In parallel, phagotrophic and parasitic lineages appeared slightly favoured under ambient CO2 conditions, results that were corroborated by microbial signature analyses. Cross-comparisons of protist-bacteria co-occurrences showed mostly negative relations between prokaryotes and microeukaryotes, indicating that the ongoing increase in atmospheric CO2 will lead to changes in microbial soil communities and their interactions, potentially cascading to higher trophic levels in soil systems.

Generation of a new DiCre expressing parasite strain for functional characterization of Plasmodium falciparum genes in blood stages

Conditional regulation is a highly beneficial system for studying the function of essential genes in Plasmodium falciparum and dimerizable Cre recombinase (DiCre) is a recently adapted conditional regulation system suitable for this purpose. In the DiCre system, two inactive fragments of Cre are reconstituted to form a functionally active enzyme in the presence of rapamycin. Different loci have been targeted to generate parasite lines that express the DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate the DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid, and highly specific excision of exogenously encoded gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and morphology of the mutant parasites were comparable to that of the control parasites in Giemsa smears. The availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria.

Diversity and Host Specificity of Avian Haemosporidians in an Afrotropical Conservation Region.

Afrotropical regions have high bird diversity, yet few studies have attempted to unravel the prevalence of avian haemosporidia in conservation areas. The diversity and host specificity of parasites in biodiversity hotspots is crucial to understanding parasite distribution and potential disease emergence. We test the hypothesis that biodiverse regions are associated with highly diverse parasites. By targeting the cytochrome b (Cytb) gene, we molecularly screened 1035 blood samples from 55 bird species for avian haemosporidia infections to determine its prevalence and diversity on sites inside and adjacent to the Kruger National Park. Overall infection prevalence was 28.41%. Haemoproteus, Leucocytozoon, and Plasmodium presented prevalences of 17.39%, 9.24%, and 4.64%, respectively. One hundred distinct parasite lineages were detected, of which 56 were new lineages. Haemoproteus also presented the highest diversity compared to Leucocytozoon and Plasmodium with varying levels of specificity. Haemoproteus lineages were found to be specialists while Plasmodium and Leucocytozoon lineages were generalists. We also found a positive relationship between avian host diversity and parasite diversity, supporting an amplification effect. These findings provide insight data for host-parasite and co-evolutionary relationship models.

Onametostat, a PfPRMT5 inhibitor, exhibits antimalarial activity to Plasmodium falciparum.

Protein arginine methyltransferases (PRMTs) play critical roles in Plasmodium falciparum, a protozoan causing the deadliest form of malaria, making them potential targets for novel antimalarial drugs. Here, we screened 11 novel PRMT inhibitors against P. falciparum asexual growth and found that onametostat, an inhibitor for type II PRMTs, exhibited strong antimalarial activity with a half-maximal inhibitory concentration (IC50) value of 1.69 ± 0.04 µM. In vitro methyltransferase activities of purified PfPRMT5 were inhibited by onametostat, and a shift of IC50 to onametostat was found in the PfPRTM5 disruptant parasite line, indicating that PfPRTM5 is the primary target of onametostat. Consistent with the function of PfPRMT5 in mediating symmetric dimethylation of histone H3R2 (H3R2me2s) and in regulating invasion-related genes, onametostat treatment led to the reduction of H3R2me2s level in P. falciparum and caused the defects on the parasite's invasion of red blood cells. This study provides a starting point for identifying specific PRMT inhibitors with the potential to serve as novel antimalarial drugs.

Parasite Abundance‐Occupancy Relationships Across Biogeographic Regions: Joint Effects of Niche Breadth, Host Availability and Climate

ABSTRACTAimChanging biodiversity and environmental conditions may allow multi‐host pathogens to spread among host species and affect prevalence. There are several widely acknowledged theories about mechanisms that may influence variation in pathogen prevalence, including the controversially debated dilution effect and abundance‐occupancy relationship hypotheses. Here, we explore such abundance‐occupancy relationships for unique lineages of three vector‐borne avian blood parasite genera (the avian malaria parasite Plasmodium and the related haemosporidian parasites Parahaemoproteus and Leucocytozoon) across biogeographical regions.LocationNearctic‐Neotropical and Palearctic‐Afrotropical regions.MethodsWe compiled a cross‐continental dataset of 17,116 bird individuals surveyed from 46 bird assemblages across the Nearctic‐Neotropical and Palearctic‐Afrotropical regions and explored relationships between local parasite lineage prevalence and host assemblage metrics in a Bayesian random regression framework.ResultsMost lineages from these three genera infected ≥ 5 host species and exhibited clear phylogenetic or functional host specificity. Lineage prevalence from all three genera increased with host range, but also with higher degrees of specialisation to phylogenetically or functionally related host species. Local avian community features were also found to be important drivers of prevalence. For example, bird species richness was positively correlated with lineage prevalence for Plasmodium and Leucocytozoon, whereas higher relative abundances of the main host species were associated with lower prevalence for Plasmodium and Parahaemoproteus but higher prevalence for Leucocytozoon.ConclusionsOur results broadly support several of the leading hypotheses about mechanisms that influence pathogen prevalence, including the niche breadth hypothesis in that higher avian host species diversity and broader host range amplify prevalence through increasing ecological opportunities and the trade‐off hypotheses in that specialisation among subsets of available host species may increase prevalence. Furthermore, the three studied avian haemosporidian genera exhibited different abundance‐occupancy relationships across the major global climate gradients and in relation to host availability, emphasising that these relationships do not strictly follow common rules for vector‐borne parasites with different life histories.

Single cell transcriptional changes across the blood stages of artemisinin resistant K13580Y Plasmodium falciparum upon dihydroartemisinin exposure.

Artemisinins have been a cornerstone of malaria control, but resistance in Plasmodium falciparum, due to mutations in the Kelch13 (K13) protein, threaten these advances. Artemisinin exposure results in a dynamic transcriptional response across multiple pathways, but most work has focused on ring stages and ex vivo transcriptional analysis. We applied single cell RNAseq to two unsynchronized coisogenic parasite lines (K13C580 and K13580Y) over 6 hrs after a pulse exposure to dihydroartemisinin (DHA). Transcription was altered across all stages, with the greatest occurring at the trophozoite and ring stage in both lines. This response involved the arrest of metabolic processes, support for a dormancy phenomenon upon treatment, and the enhancement of protein trafficking and the unfolded protein response. While similar, the response was consistent across stages in K13580Y, with enhanced parasite survival to drug induced stress. Increased surface protein expression was seen in K13580Y parasites at baseline and upon drug exposure, highlighted by the increased expression of PfEMP1 and GARP, a potential therapeutic target. Antibody targeting GARP maintained anti-parasitic efficacy in K13580Y parasites. This work provides single cell insight of gene transcription across all life cycle stages revealing transcriptional changes that could initiate a dormancy state and mediate survival upon treatment.

Highly virulent avian brood-parasitic species show elevated embryonic metabolic rates at specific incubation stages compared to less virulent and non-parasitic species.

As the avian embryo grows and develops within the egg, its metabolic rate gradually increases. Obligate avian brood-parasitic birds lay their eggs in the nests of other species to avoid the costs of parental care, and all but one of these brood-parasitic species are altricial at hatching. Yet the chicks of some altricial brood-parasitic species perform the physically demanding task of evicting, stabbing or otherwise killing host progeny within days of hatching. This implies a need for high metabolic rates in the embryo, just as precocial species require. Using flow-through respirometry in situ, we investigated embryonic metabolic rates in diverse avian brood parasite lineages which either kill host offspring (high virulence) or share the nest with host young (low virulence). High-virulence brood parasite embryos exhibited higher overall metabolic rates than both non-parasitic (parental) species and low-virulence parasites. This was driven by significantly elevated metabolic rates around the halfway point of incubation. Additionally, a fine-scale analysis of the embryos of a host-parasitic pair showed faster increases in metabolic rates in the parasite. Together these results suggest that the metabolic patterns of the embryos of high-virulence parasites facilitate their early-life demands.

The genetically encoded calcium indicator GCaMP3 reveals spontaneous calcium oscillations at asexual stages of the human malaria parasite Plasmodium falciparum

Most protocols used to study the dynamics of calcium (Ca2+) in the malaria parasite are based on dyes, which are invasive and do not allow discrimination between the signal from the host cell and the parasite. To avoid this pitfall, we have generated a parasite line expressing the genetically encoded calcium sensor GCaMP3. The PfGCaMP3 parasite line is an innovative tool for studying spontaneous intracellular Ca2+ oscillations without external markers. Using this parasite line, we demonstrate the occurrence of spontaneous Ca2+ oscillations in the ring, trophozoite, and schizont stages in Plasmodium falciparum. Using the Fourier transform to fluorescence intensity data extracted from different experiments, we observe cytosolic Ca2+ fluctuations. These spontaneous cytosolic Ca2+ oscillations occur in the three intraerythrocytic stages of the parasite, with most oscillations occurring in the ring and trophozoite stages. A control parasite line expressing only a GFP control did not reveal such fluctuations, demonstrating the specificity of the observations. Our results clearly show dynamic, spontaneous Ca2+ oscillations during the asexual stage in P. falciparum, independent from external stimuli.

Peptidic Boronic Acid Plasmodium falciparum SUB1 Inhibitors with Improved Selectivity over Human Proteasome.

Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P3 amino acid side chains as well as N-capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.

Association study of Plasmodium falciparum Acetyl-CoA Synthetase Mutations S868G and V949I with antimalarial drugs.

Plasmodium falciparum acetyl-CoA synthetase (PfACAS) protein is an important source of acetyl-CoA. We detected the mutations S868G and V949I in PfACAS by whole-genome sequencing analysis in some recrudescent parasites after antimalarial treatment with artesunate and dihydroartemisinin-piperaquine, suggesting that they may confer drug resistance. Using CRISPR/Cas9 technology, we engineered parasite lines carrying the PfACAS S868G and V949I mutations in two genetic backgrounds and evaluated their susceptibility to antimalarial drugs in vitro. The results demonstrated that PfACAS S868G and V949I mutations alone or in combination were not enough to provide resistance to antimalarial drugs.

CRISPR-based functional profiling of the Toxoplasma gondii genome during acute murine infection.

Examining host-pathogen interactions in animals can capture aspects of infection that are obscured in cell culture. Using CRISPR-based screens, we functionally profile the entire genome of the apicomplexan parasite Toxoplasma gondii during murine infection. Barcoded gRNAs enabled bottleneck detection and mapping of population structures within parasite lineages. Over 300 genes with previously unknown roles in infection were found to modulate parasite fitness in mice. Candidates span multiple axes of host-parasite interaction. Rhoptry Apical Surface Protein 1 was characterized as a mediator of host-cell tropism that facilitates repeated invasion attempts. GTP cyclohydrolase I was also required for fitness in mice and druggable through a repurposed compound, 2,4-diamino-6-hydroxypyrimidine. This compound synergized with pyrimethamine against T. gondii and malaria-causing Plasmodium falciparum parasites. This work represents a complete survey of an apicomplexan genome during infection of an animal host and points to novel interfaces of host-parasite interaction.

Complete chloroplast genomes of the hemiparasitic genus Cymbaria: Insights into comparative analysis, development of molecular markers, and phylogenetic relationships.

The hemiparasitic tribe Cymbarieae (Orobanchaceae) plays a crucial role in elucidating the initial stage of the transition from autotrophism to heterotrophism. However, the complete chloroplast genome of the type genus Cymbaria has yet to be reported. In addition, the traditional Mongolian medicine Cymbaria daurica is frequently subjected to adulteration or substitution because of the minor morphological differences with Cymbaria mongolica. In this study, the complete chloroplast genomes of the two Cymbaria species were assembled and annotated, and those of other published 52 Orobanchaceae species were retrieved for comparative analyses. We found that the Cymbaria chloroplast genomes are characterized by pseudogenization or loss of stress-relevant genes (ndh) and a unique rbcL-matK inversion. Unlike the high variability observed in holoparasites, Cymbaria and other hemiparasites exhibit high similarity to autotrophs in genome size, guanine-cytosine (GC) content, and intact genes. Notably, four pairs of specific DNA barcodes were developed and validated to distinguish the medicinal herb from its adulterants. Phylogenetic analyses revealed that the genus Cymbaria and the Schwalbea-Siphonostegia clade are grouped into the tribe Cymbarieae, which forms a sister clade to the remaining Orobanchaceae parasitic lineages. Moreover, the diversification of monophyletic Cymbaria occurred during the late Miocene (6.72 Mya) in the Mongol-Chinese steppe region. Our findings provide valuable genetic resources for studying the phylogeny of Orobanchaceae and plant parasitism, and genetic tools to validate the authenticity of the traditional Mongolian medicine "Xinba.".

Mutation of the Plasmodium falciparum Flavokinase Confers Resistance to Roseoflavin and 8-Aminoriboflavin.

The riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilization. To determine their mechanism of action, we generated roseoflavin-resistant parasites by in vitro evolution. Relative to wild-type, these parasites were 4-fold resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Whole genome sequencing of the resistant parasites revealed a missense mutation leading to an amino acid change (L672H) in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene. The IC50 values for roseoflavin and 8-aminoriboflavin against the roseoflavin-resistant parasites created through in vitro evolution were indistinguishable from those against parasites in which the missense mutation was introduced into the native PfFK. We also generated two parasite lines episomally expressing GFP-tagged versions of either the wild-type or mutant forms of PfFK. We found that PfFK-GFP localizes to the parasite cytosol and that immunopurified PfFK-GFP phosphorylated riboflavin, roseoflavin, and 8-aminoriboflavin. The L672H mutation increased the KM for roseoflavin, explaining the resistance phenotype. Mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonized by increasing the extracellular concentration of riboflavin, consistent with it also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore, are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite proliferation by inhibiting riboflavin phosphorylation and via the generation of toxic flavin cofactor analogues.

A Plasmodium falciparum genetic cross reveals the contributions of pfcrt and plasmepsin II/III to piperaquine drug resistance.

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC50. We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt. A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage.