Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum.

Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.

The Crk4-Cyc4 complex regulates G2/M transition in Toxoplasma gondii.

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4-TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4-TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4-Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.

Characterization of the dual role of Plasmodium falciparum DNA methyltransferase in regulating transcription and translation.

DNA modifications are critical in fine-tuning the biological processes in model organisms. However, the presence of cytosine methylation (5mC) and the function of the putative DNA methyltransferase, PfDNMT2, in the human malaria pathogen, Plasmodium falciparum, remain controversial. Here, we revisited the 5mC in the parasite genome and the function of PfDNMT2. Low levels of genomic 5mC (0.1-0.2%) during asexual development were identified using a sensitive mass spectrometry procedure. Native PfDNMT2 displayed substantial DNA methylation activities, and disruption or overexpression of PfDNMT2 resulted in reduced or elevated genomic 5mC levels, respectively. PfDNMT2 disruption led to an increased proliferation phenotype, with the parasites having an extended schizont stage and producing a higher number of progenies. Consistent with PfDNMT2's interaction with an AP2 domain-containing transcription factor, transcriptomic analyses revealed that PfDNMT2 disruption led to a drastic alteration in the expression of many genes, some of which provided the molecular basis of enhanced proliferation after PfDNMT2 disruption. Furthermore, levels of tRNAAsp and its methylation rate at position C38, and the translation of a reporter containing an aspartate repeat were significantly reduced after PfDNMT2 disruption, while the levels of tRNAAsp and its C38 methylation were restored after complementation of PfDNMT2. Our study sheds new light on the dual function of PfDNMT2 during P. falciparum asexual development.

Effectiveness of unsupervised primaquine regimen for preventing Plasmodium vivax malaria relapses in northeast Myanmar, a single-arm non-randomized observational study.

BACKGROUND: Plasmodium vivax presents a significant challenge for malaria elimination in the Greater Mekong Subregion (GMS). We evaluated the effectiveness of primaquine (PQ) for reducing relapses of vivax malaria. METHODS: Patients with uncomplicated P. vivax malaria from eastern Myanmar received chloroquine (CQ, 25 mg base/kg given in 3 days) plus unsupervised PQ (0.25 mg/kg/day for 14 days) without screening for glucose-6-phosphate dehydrogenase deficiency and were followed for a year. RESULTS: Totally 556 patients were enrolled to receive the CQ/PQ treatment from February 2012 to August 2013. During the follow-up, 38 recurrences were detected, presenting a cumulative rate of recurrence of 9.1% (95% confidence interval, 4.1-14.1%). Genotyping at the pvmsp1 and pvmsp3α loci by Amplicon deep sequencing and model prediction indicated that 13 of the 27 recurrences with genotyping data were likely due to relapses. Notably, all confirmed relapses occurred within the first six months. CONCLUSIONS: The unsupervised standard dose of PQ was highly effective as a radical cure for P. vivax malaria in eastern Myanmar. The high presumed effectiveness might have benefited from the health messages delivered during the enrollment and follow-up activities. Six-month follow-ups in the GMS are sufficient for detecting most relapses.

Plasmodium falciparum GCN5 plays a key role in regulating artemisinin resistance-related stress responses.

Plasmodium falciparum causes the most severe malaria and is exposed to various environmental and physiological stresses in the human host. Given that GCN5 plays a critical role in regulating stress responses in model organisms, we aimed to elucidate PfGCN5's function in stress responses in P. falciparum. The protein level of PfGCN5 was substantially induced under three stress conditions [heat shock, low glucose starvation, and dihydroartemisinin, the active metabolite of artemisinin (ART)]. With a TetR-DOZI conditional knockdown (KD) system, we successfully down-regulated PfGCN5 to ~50% and found that KD parasites became more sensitive to all three stress conditions. Transcriptomic analysis via RNA-seq identified ~1,000 up- and down-regulated genes in the wild-type (WT) and KD parasites under these stress conditions. Importantly, DHA induced transcriptional alteration of many genes involved in many aspects of stress responses, which were heavily shared among the altered genes under heat shock and low glucose conditions, including ART-resistance-related genes such as K13 and coronin. Based on the expression pattern between WT and KD parasites under three stress conditions, ~300-400 genes were identified to be involved in PfGCN5-dependent, general, and stress-condition-specific responses with high levels of overlaps among three stress conditions. Notably, using ring-stage survival assay, we found that KD or inhibition of PfGCN5 could sensitize the ART-resistant parasites to the DHA treatment. All these indicate that PfGCN5 is pivotal in regulating general and ART-resistance-related stress responses in malaria parasites, implicating PfGCN5 as a potential target for malaria intervention.

In Vitro Susceptibility of Plasmodium falciparum Isolates from the China-Myanmar Border Area to Piperaquine and Association with Candidate Markers.

Plasmodium falciparum from the Greater Mekong subregion has evolved resistance to the artemisinin-based combination therapy dihydroartemisinin and the partner drug piperaquine. To monitor the potential westward spread or independent evolution of piperaquine resistance, we evaluated the in vitro susceptibility of 120 P. falciparum isolates collected at the China-Myanmar border during 2007-2016. The parasite isolates displayed a relatively wide range of piperaquine susceptibility estimates. While 56.7% of the parasites showed bimodal drug response curves, all but five generated area-under-the-curve (AUC) estimates consistent with a susceptible phenotype. Using the piperaquine survival assay (PSA), 5.6% parasites showed reduced susceptibility. Of note, parasites from 2014-2016 showed the highest AUC value and the highest proportion with a bimodal curve, suggesting falling effectiveness in these later years. Unsupervised K-mean analysis of the combined data assigned parasites into three clusters and identified significant correlations between IC50, IC90, and AUC values. No parasites carried the E415G mutation in a putative exo-nuclease, new mutations in PfCRT, or amplification of the plasmepsin 2/3 genes, suggesting mechanisms of reduced piperaquine susceptibility that differ from those described in other countries of the region. The association of increased AUC, IC50, and IC90 values with major PfK13 mutations (F446I and G533S) suggests that piperaquine resistance may evolve in these PfK13 genetic backgrounds. Additionally, the Pfmdr1 F1226Y mutation was associated with significantly higher PSA values. Further elucidation of piperaquine resistance mechanisms and continuous surveillance are warranted.

A unique GCN5 histone acetyltransferase complex controls erythrocyte invasion and virulence in the malaria parasite Plasmodium falciparum.

The histone acetyltransferase GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. In this study, we identified a divergent GCN5 complex in Plasmodium falciparum, which contains two plant homeodomain (PHD) proteins (PfPHD1 and PfPHD2) and a plant apetela2 (AP2)-domain transcription factor (PfAP2-LT). To dissect the functions of the PfGCN5 complex, we generated parasite lines with either the bromodomain in PfGCN5 or the PHD domain in PfPHD1 deleted. The two deletion mutants closely phenocopied each other, exhibiting significantly reduced merozoite invasion of erythrocytes and elevated sexual conversion. These domain deletions caused dramatic decreases not only in histone H3K9 acetylation but also in H3K4 trimethylation, indicating synergistic crosstalk between the two euchromatin marks. Domain deletion in either PfGCN5 or PfPHD1 profoundly disturbed the global transcription pattern, causing altered expression of more than 60% of the genes. At the schizont stage, these domain deletions were linked to specific down-regulation of merozoite genes involved in erythrocyte invasion, many of which contain the AP2-LT binding motif and are also regulated by AP2-I and BDP1, suggesting targeted recruitment of the PfGCN5 complex to the invasion genes by these specific factors. Conversely, at the ring stage, PfGCN5 or PfPHD1 domain deletions disrupted the mutually exclusive expression pattern of the entire var gene family, which encodes the virulent factor PfEMP1. Correlation analysis between the chromatin state and alteration of gene expression demonstrated that up- and down-regulated genes in these mutants are highly correlated with the silent and active chromatin states in the wild-type parasite, respectively. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including an AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.

Risk of hemolysis in Plasmodium vivax malaria patients receiving standard primaquine treatment in a population with high prevalence of G6PD deficiency.

BACKGROUND: Primaquine is essential for the radical cure of Plasmodium vivax malaria, but it poses a potential danger of severe hemolysis in G6PD-deficient (G6PDd) patients. This study aimed to determine whether primaquine is safe in a population with high G6PD prevalence but lacking G6PD diagnosis capacity. METHODS: In Myanmar, 152 vivax patients were gender- and age-matched at 1:3 for G6PDd versus G6PD-normal (G6PDn). Their risk of acute hemolysis was followed for 28 days after treatment with the standard chloroquine and 14-day primaquine (0.25 mg/kg/day) regimen. RESULTS: Patients anemic and non-anemic at enrollment showed a rising and declining trend in the mean hemoglobin level, respectively. In males, the G6PDd group showed substantially larger magnitudes of hemoglobin reduction and lower hemoglobin nadir levels than the G6PDn group, but this trend was not evident in females. Almost 1/3 of the patients experienced clinically concerning declines in hemoglobin, with five requiring blood transfusion. CONCLUSIONS: The standard 14-day primaquine regimen carries a significant risk of acute hemolytic anemia (AHA) in vivax patients without G6PD testing in a population with a high prevalence of G6PD deficiency and anemia. G6PD testing would avoid most of the clinically significant Hb reductions and AHA in male patients.

Epigenetic Potential and DNA Methylation in an Ongoing House Sparrow (Passer domesticus) Range Expansion.

AbstractDuring range expansions, organisms can use epigenetic mechanisms to adjust to conditions in novel areas by altering gene expression and enabling phenotypic plasticity. Here, we predicted that the number of CpG sites within the genome, one form of epigenetic potential, would be important for successful range expansions because DNA methylation can modulate gene expression and, consequently, plasticity. We asked how the number of CpG sites and DNA methylation varied across five locations in the ∼70-year-old Kenyan house sparrow (Passer domesticus) range expansion. We found that the number of CpG sites was highest toward the vanguard of the invasion and decreased toward the range core. Analysis suggests that this pattern may have been driven by selection, favoring birds with more CpG sites at the range edge. However, we cannot rule out other processes, including nonrandom gene flow. Additionally, DNA methylation did not change across the range expansion, nor was it more variable. We hypothesize that as new areas are colonized, epigenetic potential may be selectively advantageous early but eventually be replaced by less plastic and perhaps genetically canalized traits as populations adapt to local conditions. Although further work is needed on epigenetic potential, this form (CpG number) appears to be a promising mechanism to investigate as a driver of expansions via capacitated phenotypic plasticity in other natural and anthropogenic range expansions.

Lineage-Specific Expansion of Plasmodium falciparum Parasites With pfhrp2 Deletion in the Greater Mekong Subregion.

Deletion of the pfhrp2 gene in Plasmodium falciparum can lead to false-negative rapid diagnostic test (RDT) results, constituting a major challenge for evidence-based malaria treatment. Here we analyzed the whole genome sequences of 138 P. falciparum clinical samples collected from the China-Myanmar boarder for pfhrp2 and pfhrp3 gene deletions. We found pfhrp2 and pfhrp3 deletions in 9.4% and 3.6% of samples, respectively, with no samples harboring deletions of both genes. The pfhrp2 deletions showed 2 distinct breakpoints, representing 2 different chromosomal deletion events. A phylogenetic analysis performed using genome-wide single-nucleotide polymorphisms revealed that the 2 pfhrp2 breakpoint groups as well as all the pfhrp3-negative parasites formed separate clades, suggesting they might have resulted from clonal expansion of pfhrp2- and pfhrp3-negative parasites. These findings highlight the need for urgent surveys to determine the prevalence of pfhrp2-negative parasites causing false-negative RDT results and a plan for switching of RDTs pending the survey results.

Quantitative insertion-site sequencing (QIseq) for high throughput phenotyping of transposon mutants.

Genetic screening using random transposon insertions has been a powerful tool for uncovering biology in prokaryotes, where whole-genome saturating screens have been performed in multiple organisms. In eukaryotes, such screens have proven more problematic, in part because of the lack of a sensitive and robust system for identifying transposon insertion sites. We here describe quantitative insertion-site sequencing, or QIseq, which uses custom library preparation and Illumina sequencing technology and is able to identify insertion sites from both the 5' and 3' ends of the transposon, providing an inbuilt level of validation. The approach was developed using piggyBac mutants in the human malaria parasite Plasmodium falciparum but should be applicable to many other eukaryotic genomes. QIseq proved accurate, confirming known sites in >100 mutants, and sensitive, identifying and monitoring sites over a >10,000-fold dynamic range of sequence counts. Applying QIseq to uncloned parasites shortly after transfections revealed multiple insertions in mixed populations and suggests that >4000 independent mutants could be generated from relatively modest scales of transfection, providing a clear pathway to genome-scale screens in P. falciparum QIseq was also used to monitor the growth of pools of previously cloned mutants and reproducibly differentiated between deleterious and neutral mutations in competitive growth. Among the mutants with fitness defects was a mutant with a piggyBac insertion immediately upstream of the kelch protein K13 gene associated with artemisinin resistance, implying mutants in this gene may have competitive fitness costs. QIseq has the potential to enable the scale-up of piggyBac-mediated genetics across multiple eukaryotic systems.

Impact of genetic and clinical factors on warfarin therapy in patients early after heart valve replacement surgery.

PURPOSE: Factors influencing responsiveness to warfarin at treatment onset time were not well identified in Chinese patients undergoing heart valve replacement. We sought to select the most relevant factors that associated with patient response to warfarin early after heart valve surgery. METHODS: In this observational study, 289 patients starting warfarin therapy early after heart valve replacement surgery were enrolled. CYP2C9 *1, *2, *3, and *5; VKORC1-1639 G>A, CYP4F2 V433M, and GGCX rs11676382 genotypes; clinical characteristics, response to therapy, and bleeding and thrombosis events were collected. The primary outcomes were the time to the first INR equal to or more than lower limit of therapeutic range and the warfarin dose requirements. Stepwise multiple linear regression was performed to develop a dosing algorithm to predict the warfarin dose requirements. RESULTS: The results of univariate analysis showed lone VKORC1-1639 G>A, CYP2C9 *1/*3, cefazolin, cefoperazone-sulbactam, increased BMI, Δhemoglobin, and white blood cell count could significantly affect patient responsiveness to warfarin in the initial period of anticoagulation. Multivariate analysis resulted in an equation: Accumulated warfarin doses (mg) = 17.068 VKORC1-1639 G>A - 4.261 hypertension + 0.593 BMI - 0.115 age - 4.852 CYP2C9 *1/*3 - 2.617 cefazolin - 4.902 cefoperazone-sulbactam - 4.537, which could explain 40.2% of the variability in warfarin dose needed to reach the first INR equal to or more than lower limit of therapeutic range. CONCLUSIONS: Both genetic and clinical factors contributed to anticoagulation effect of warfarin in the initial period of treatment. Our findings could provide a basis for the personalized management of warfarin use in the early stage of anticoagulation in northern Chinese patients.

In-depth phenotypic characterization of reticulocyte maturation using mass cytometry.

Progress towards an in-depth understanding of the final steps of the erythroid lineage development is paramount for many hematological diseases. We have characterized the final stages of reticulocyte maturation from bone marrow to peripheral blood using for the first time single-cell Mass Cytometry (CyTOF). We were able to measure the expression of 31 surface markers within a single red blood cell (RBC). We demonstrate the validity of CyTOF for RBC phenotyping by confirming the progressive reduction of transferrin receptor 1 (CD71) during reticulocyte maturation to mature RBC. We highlight the high-dimensional nature of mass cytometry data by correlating the expression of multiple proteins on individual RBCs. We further describe a more drastic reduction pattern for a component of the alpha4/beta1 integrin CD49d at the very early steps of reticulocyte maturation in bone marrow and directly linked with the mitochondria remnants clearance pattern. The enhanced and accurate RBC phenotyping potential of CyTOF described herein could be beneficial to decipher RBC preferences, as well as still not well understood receptor-ligand interaction of some hemotropic parasites such as the malaria causing agent Plasmodium vivax.

Punctuated chromatin states regulate Plasmodium falciparum antigenic variation at the intron and 2 kb upstream regions.

BACKGROUND: Understanding the regulation mechanism of var gene expression is crucial for explaining antigenic variation in Plasmodium falciparum. Recent work observed that while all var genes produce transcripts, only a few var genes exhibit high expression levels. However, the global regulation of var expression and the relationship between epigenetic and genetic control remains to be established. RESULT: We have systematically reanalyzed the existing genomic data including chromatin configurations and gene expressions; and for the first time used robust statistical methods to show that the intron and 2 kb upstream regions of each endogenous var gene always maintain high chromatin accessibility, with high potential to bind transcription factors (TFs). The levels of transcripts for different var gene family members are associated with this chromatin accessibility. Any given var gene thus shows punctuated chromatin states throughout the asexual life cycle. This is demonstrated by three independent transcript datasets. Chromatin accessibility in the var intron and 2 kb upstream regions are also positively correlated with their GC content, suggesting the level of var genes silencing might be encoded in their intron sequences. Interestingly, both var intron and 2 kb upstream regions exhibit higher chromatin accessibility when the genes have relatively lower transcription levels, suggesting a punctuated repressive function for these regulatory elements. CONCLUSION: By integrating and analyzing epigenomic, genomic and transcriptomic data, our work reveals a novel distal element in var control. We found dynamic modulations of specific epigenetic marks around the var intron and distal upstream regions are involved in the general var gene expression patterns in malarial antigenic variation.

Synthesis of Mesoionic Isoquinolines by Rhodium(III)-Catalyzed C-H Activation.

Hydroxyl-substituted benzaldimines underwent a Rh(III) -catalyzed C-H activation and annulation with alkynes to provide novel mesoionic isoquinoline derivatives in moderate to excellent yields using oxygen as an internal anion source. This simple and efficient approach has a broad substrate scope.

Rhodium(III)-catalyzed C-C coupling of 7-azaindoles with vinyl acetates and allyl acetates.

The behaviour of electron-rich alkenes with 7-azaindoles in rhodium(III)-catalyzed C-H activation is investigated. Various substituted vinyl acetates and allyl acetates as coupling partners reacted smoothly providing a wide variety of 7-azaindole derivatives, and the selectivity of the coupling reaction is alkene-dependent. In addition, the approaches of rhodium(III)-catalyzed dehydrogenative Heck-type reaction (DHR) and carbonylation reaction were quite novel and simple.

Functional diversity of CTCFs is encoded in their binding motifs.

BACKGROUND: The CCCTC-binding factor (CTCF) has diverse regulatory functions. However, the definitive characteristics of the CTCF binding motif required for its functional diversity still remains elusive. RESULTS: Here, we describe a new motif discovery workflow by which we have identified three CTCF binding motif variations with highly divergent functionalities. Supported by transcriptomic, epigenomic and chromatin-interactomic data, we show that the functional diversity of the CTCF binding motifs is strongly associated with their GC content, CpG dinucleotide coverage and relative DNA methylation level at the 12th position of the motifs. Further analysis suggested that the co-localization of cohesin, the key factor in cohesion of sister chromatids, is negatively correlated with the CpG coverage and the relative DNA methylation level at the 12th position. Finally, we present evidences for a hypothetical model in which chromatin interactions between promoters and distal regulatory regions are likely mediated by CTCFs binding to sequences with high CpG. CONCLUSION: These results demonstrate the existence of definitive CTCF binding motifs corresponding to CTCF's diverse functions, and that the functional diversity of the motifs is strongly associated with genetic and epigenetic features at the 12th position of the motifs.

Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum.

It is a significant challenge to assess the functions of many uncharacterized genes in human malaria parasites. Here, we present a genetic screening tool to assess the contribution of essential genes from Plasmodium falciparum by the conditional CRISPR-/deadCas9-based interference and activation (i/a) systems. We screened both CRISPRi and CRISPRa sets, consisting of nine parasite lines per set targeting nine genes via their respective gRNAs. By conducting amplicon sequencing of gRNA loci, we identified the contribution of each targeted gene to parasite fitness upon drug (artemisinin, chloroquine) and stress (starvation, heat shock) treatment. The screening was highly reproducible, and the screening libraries were easily generated by transfection of mixed plasmids expressing different gRNAs. We demonstrated that this screening is straightforward, robust, and can provide a fast and efficient tool to study essential genes that have long presented a bottleneck in assessing their functions using existing genetic tools.

MYST regulates DNA repair and forms a NuA4-like complex in the malaria parasite Plasmodium falciparum.

Histone lysine acetyltransferase MYST-associated NuA4 complex is conserved from yeast to humans and plays key roles in cell cycle regulation, gene transcription, and DNA replication/repair. Here, we identified a Plasmodium falciparum MYST-associated complex, PfNuA4, which contains 11 of the 13 conserved NuA4 subunits. Reciprocal pulldowns using PfEAF2, a shared component between the NuA4 and SWR1 complexes, not only confirmed the PfNuA4 complex but also identified the PfSWR1 complex, a histone remodeling complex, although their identities are low compared to the homologs in yeast or humans. Notably, both H2A.Z/H2B.Z were associated with the PfSWR1 complex, indicating that this complex is involved in the deposition of H2A.Z/H2B.Z, the variant histone pair that is enriched in the activated promoters. Overexpression of PfMYST resulted in earlier expression of genes involved in cell cycle regulation, DNA replication, and merozoite invasion, and upregulation of the genes related to antigenic variation and DNA repair. Consistently, PfMYST overexpression led to high basal phosphorylated PfH2A (γ-PfH2A), the mark of DNA double-strand breaks, and conferred protection against genotoxic agent methyl methanesulfonate (MMS), X-rays, and artemisinin, the first-line antimalarial drug. In contrast, the knockdown of PfMYST caused a delayed parasite recovery upon MMS treatment. MMS induced the gradual disappearance of PfMYST in the cytoplasm and concomitant accumulation of PfMYST in the nucleus, suggesting cytoplasm-nucleus shuttling of PfMYST. Meanwhile, PfMYST colocalized with the γ-PfH2A, indicating PfMYST was recruited to the DNA damage sites. Collectively, PfMYST plays critical roles in cell cycle regulation, gene transcription, and DNA replication/DNA repair in this low-branching parasitic protist.IMPORTANCEUnderstanding gene regulation and DNA repair in malaria parasites is critical for identifying targets for antimalarials. This study found PfNuA4, a PfMYST-associated, histone modifier complex, and PfSWR1, a chromatin remodeling complex in malaria parasite Plasmodium falciparum. These complexes are divergent due to the low identities compared to their homologs from yeast and humans. Furthermore, overexpression of PfMYST resulted in substantial transcriptomic changes, indicating that PfMYST is involved in regulating the cell cycle, antigenic variation, and DNA replication/repair. Consistently, PfMYST was found to protect against DNA damage caused by the genotoxic agent methyl methanesulfonate, X-rays, and artemisinin, the first-line antimalarial drug. Additionally, DNA damage led to the relocation of cytoplasmic PfMYST to the nucleus and colocalization of PfMYST with γ-PfH2A, the mark of DNA damage. In summary, this study demonstrated that the PfMYST complex has critical functions in regulating cell cycle, antigenic variation, and DNA replication/DNA repair in P. falciparum.

The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum.

In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.