PfHDAC1 is an essential regulator of P. falciparum asexual proliferation and host cell invasion genes with a dynamic genomic occupancy responsive to artemisinin stress.

Plasmodium falciparum, the deadly protozoan parasite responsible for malaria, has a tightly regulated gene expression profile closely linked to its intraerythrocytic development cycle. Epigenetic modifiers of the histone acetylation code have been identified as key regulators of the parasite's transcriptome but require further investigation. In this study, we map the genomic distribution of Plasmodium falciparum histone deacetylase 1 (PfHDAC1) across the erythrocytic asexual development cycle and find it has a dynamic occupancy over a wide array of developmentally relevant genes. Overexpression of PfHDAC1 results in a progressive increment in parasite load over consecutive rounds of the asexual infection cycle and is associated with enhanced gene expression of multiple families of host cell invasion factors (merozoite surface proteins, rhoptry proteins, etc.) and with increased merozoite invasion efficiency. With the use of class-specific inhibitors, we demonstrate that PfHDAC1 activity in parasites is crucial for timely intraerythrocytic development. Interestingly, overexpression of PfHDAC1 results in decreased sensitivity to frontline-drug dihydroartemisinin in parasites. Furthermore, we identify that artemisinin exposure can interfere with PfHDAC1 abundance and chromatin occupancy, resulting in enrichment over genes implicated in response/resistance to artemisinin. Finally, we identify that dihydroartemisinin exposure can interrupt the in vitro catalytic deacetylase activity and post-translational phosphorylation of PfHDAC1, aspects that are crucial for its genomic function. Collectively, our results demonstrate PfHDAC1 to be a regulator of critical functions in asexual parasite development and host invasion, which is responsive to artemisinin exposure stress and deterministic of resistance to it. IMPORTANCE: Malaria is a major public health problem, with the parasite Plasmodium falciparum causing most of the malaria-associated mortality. It is spread by the bite of infected mosquitoes and results in symptoms such as cyclic fever, chills, and headache. However, if left untreated, it can quickly progress to a more severe and life-threatening form. The World Health Organization currently recommends the use of artemisinin combination therapy, and it has worked as a gold standard for many years. Unfortunately, certain countries in southeast Asia and Africa, burdened with a high prevalence of malaria, have reported cases of drug-resistant infections. One of the major problems in controlling malaria is the emergence of artemisinin resistance. Population genomic studies have identified mutations in the Kelch13 gene as a molecular marker for artemisinin resistance. However, several reports thereafter indicated that Kelch13 is not the main mediator but rather hinted at transcriptional deregulation as a major determinant of drug resistance. Earlier, we identified PfGCN5 as a global regulator of stress-responsive genes, which are known to play a central role in artemisinin resistance generation. In this study, we have identified PfHDAC1, a histone deacetylase as a cell cycle regulator, playing an important role in artemisinin resistance generation. Taken together, our study identified key transcriptional regulators that play an important role in artemisinin resistance generation.

Awakening the sleeping giant: Epstein-Barr virus reactivation by biological agents.

Epstein-Barr virus (EBV) may cause harm in immunocompromised conditions or on stress stimuli. Various chemical agents have been utilized to induce the lytic cycle in EBV-infected cells. However, apart from chemical agents and external stress stimuli, certain infectious agents may reactivate the EBV. In addition, the acute infection of other pathogens may provide suitable conditions for EBV to thrive more and planting the roots for EBV-associated pathologies. Various bacteria such as periodontal pathogens like Aggregatibacter, Helicobacter pylori, etc. have shown to induce EBV reactivation either by triggering host cells directly or indirectly. Viruses such as Human simplex virus-1 (HSV) induce EBV reactivation by HSV US3 kinase while other viruses such as HIV, hepatitis virus, and even novel SARS-CoV-2 have also been reported to cause EBV reactivation. The eukaryotic pathogens such as Plasmodium falciparum and Aspergillus flavus can also reactivate EBV either by surface protein interaction or as an impact of aflatoxin, respectively. To highlight the underexplored niche of EBV reactivation by biological agents, we have comprehensively presented the related information in this review. This may help to shedding the light on the research gaps as well as to unveil yet unexplored mechanisms of EBV reactivation.

Genomic surveillance reveals early detection and transition of delta to omicron lineages of SARS-CoV-2 variants in wastewater treatment plants of Pune, India.

The COVID-19 pandemic has emphasized the urgency for rapid public health surveillance methods to detect and monitor the transmission of infectious diseases. The wastewater-based epidemiology (WBE) has emerged as a promising tool for proactive analysis and quantification of infectious pathogens within a population before clinical cases emerge. In the present study, we aimed to assess the trend and dynamics of SARS-CoV-2 variants using a longitudinal approach. Our objective included early detection and monitoring of these variants to enhance our understanding of their prevalence and potential impact. To achieve our goals, we conducted real-time quantitative polymerase chain reaction (RT-qPCR) and Illumina sequencing on 442 wastewater (WW) samples collected from 10 sewage treatment plants (STPs) in Pune city, India, spanning from November 2021 to April 2022. Our comprehensive analysis identified 426 distinct lineages representing 17 highly transmissible variants of SARS-CoV-2. Notably, fragments of Omicron variant were detected in WW samples prior to its first clinical detection in Botswana. Furthermore, we observed highly contagious sub-lineages of the Omicron variant, including BA.1 (~28%), BA.1.X (1.0-72%), BA.2 (1.0-18%), BA.2.X (1.0-97.4%) BA.2.12 (0.8-0.25%), BA.2.38 (0.8-1.0%), BA.2.75 (0.01-0.02%), BA.3 (0.09-6.3%), BA.4 (0.24-0.29%), and XBB (0.01-21.83%), with varying prevalence rates. Overall, the present study demonstrated the practicality of WBE in the early detection of SARS-CoV-2 variants, which could help track future outbreaks of SARS-CoV-2. Such approaches could be implicated in monitoring infectious agents before they appear in clinical cases.

The many paths to artemisinin resistance in Plasmodium falciparum.

Emerging resistance against artemisinin (ART) poses a major challenge in controlling malaria. Parasites with mutations in PfKelch13, the major marker for ART resistance, are known to reduce hemoglobin endocytosis, induce unfolded protein response (UPR), elevate phosphatidylinositol-3-phosphate (PI3P) levels, and stimulate autophagy. Nonetheless, PfKelch13-independent resistance is also reported, indicating extensive complementation by reconfiguration in the parasite metabolome and transcriptome. These findings implicate that there may not be a single 'universal identifier' of ART resistance. This review sheds light on the molecular, transcriptional, and metabolic pathways associated with ART resistance, while also highlighting the interplay between cellular heterogeneity, environmental stress, and ART sensitivity.

RNA Polymerase II evolution and adaptations: Insights from Plasmodium and other parasitic protists.

The C-terminal domain (CTD) of RNA polymerase II plays a crucial role in regulating transcription dynamics in eukaryotes. The phosphorylation of serine residues within the CTD controls transcription initiation, elongation, and termination. While the CTD is highly conserved across eukaryotes, lower eukaryotes like protists, including Plasmodium, exhibit some differences. In this study, we performed a comparative analysis of CTD in eukaryotic systems to understand why the parasites evolved in this particular manner. The Plasmodium falciparum RPB1 is exceptionally large and feature a gap between the first and second heptad repeats, resulting in fifteen canonical heptad repeats excluding the initial repeat. Analysis of this intervening sequence revealed sub motifs of heptads where two serine residues occupy the first and fourth positions (S1X2X3S4). These motifs lie in the intrinsically disordered region of RPB1, a characteristic feature of the CTD. Interestingly, the S1X2X3S4 sub-motif was also observed in early-divergingeukaryotes like Leishmania major, which lack canonical heptad repeats. Furthermore, eukaryotes across the phylogenetic tree revealed a sigmoid pattern of increasing serine frequency in the CTD, indicating that serine enrichment is a significant step in the evolution of heptad-rich RPB1. Based on these observations and analysis, we proposed an evolutionary model for RNA Polymerase II CTD, encompassing organisms previously deemed exceptions, notably Plasmodium species. Thus, our study provides novel insights into the evolution of the CTD and will prompt further investigations into the differences exhibited by Plasmodium RNA Pol II and determine if they confer a survival advantage to the parasite.

Chasing SARS-CoV-2 XBB.1.16 Recombinant Lineage in India and the Clinical Profile of XBB.1.16 Cases in Maharashtra, India.

Background SARS-CoV-2 has evolved rapidly, resulting in the emergence of lineages with a competitive advantage over one another. Co-infections with different SARS-CoV-2 lineages can give rise to recombinant lineages. To date, the XBB lineage is the most widespread recombinant lineage worldwide, with the recently named XBB.1.16 lineage causing a surge in the number of COVID-19 cases in India. Methodology The present study involved retrieval of SARS-CoV-2 genome sequences from India (between December 1, 2022 and April 8, 2023) through GISAID; sequences were curated, followed by lineage and phylogenetic analysis. Demographic and clinical data from Maharashtra, India were collected telephonically, recorded in Microsoft® Excel, and analyzed using IBM® SPSS statistics, version 29.0.0.0 (241). Results A total of 2,944 sequences were downloaded from the GISAID database, of which 2,856 were included in the study following data curation. The sequences from India were dominated by the XBB.1.16* lineage (36.17%) followed by XBB.2.3* (12.11%) and XBB.1.5* (10.36%). Of the 2,856 cases, 693 were from Maharashtra; 386 of these were included in the clinical study. The clinical features of COVID-19 cases with XBB.1.16* infection (XBB.1.16* cases, 276 in number) showed that 92% of those had a symptomatic disease, with fever (67%), cough (42%), rhinorrhea (33.7%), body ache (14.5%) and fatigue (14.1%) being the most common symptoms. The presence of comorbidity was found in 17.7% of the XBB.1.16* cases. Among the XBB.1.16* cases, 91.7% were vaccinated with at least one dose of vaccine against COVID-19. While 74.3% of XBB.1.16* cases were home-isolated; 25.7% needed hospitalization/institutional quarantine, of these, 33.8% needed oxygen therapy. Out of 276 XBB.1.16* cases, seven (2.5%) cases succumbed to the disease. The majority of XBB.1.16* cases who died belonged to an elderly age group (60 years and above), had underlying comorbid condition/s, and needed supplemental oxygen therapy. The clinical features of COVID-19 cases infected with other co-circulating Omicron variants were similar to XBB.1.16* cases. Conclusion The study reveals that XBB.1.16* lineage has become the most predominant SARS-CoV-2 lineage in India. The study also shows that the clinical features and outcome of XBB.1.16* cases were similar to those of other co-circulating Omicron lineage infected cases in Maharashtra, India.

A tale of two waves: Delineating diverse genomic and transmission landscapes driving the COVID-19 pandemic in Pune, India.

BACKGROUND: Modern response to pandemics, critical for effective public health measures, is shaped by the availability and integration of diverse epidemiological outbreak data. Tracking variants of concern (VOC) is integral to understanding the evolution of SARS-CoV-2 in space and time, both at the local level and global context. This potentially generates actionable information when integrated with epidemiological outbreak data. METHODS: A city-wide network of researchers, clinicians, and pathology diagnostic laboratories was formed for genome surveillance of COVID-19 in Pune, India. The genomic landscapes of 10,496 sequenced samples of SARS-CoV-2 driving peaks of infection in Pune between December-2020 to March-2022, were determined. As a modern response to the pandemic, a "band of five" outbreak data analytics approach was used. This integrated the genomic data (Band 1) of the virus through molecular phylogenetics with key outbreak data including sample collection dates and case numbers (Band 2), demographics like age and gender (Band 3-4), and geospatial mapping (Band 5). RESULTS: The transmission dynamics of VOCs in 10,496 sequenced samples identified B.1.617.2 (Delta) and BA(x) (Omicron formerly known as B.1.1.529) variants as drivers of the second and third peaks of infection in Pune. Spike Protein mutational profiling during pre and post-Omicron VOCs indicated differential rank ordering of high-frequency mutations in specific domains that increased the charge and binding properties of the protein. Time-resolved phylogenetic analysis of Omicron sub-lineages identified a highly divergent BA.1 from Pune in addition to recombinant X lineages, XZ, XQ, and XM. CONCLUSIONS: The band of five outbreak data analytics approach, which integrates five different types of data, highlights the importance of a strong surveillance system with high-quality meta-data for understanding the spatiotemporal evolution of the SARS-CoV-2 genome in Pune. These findings have important implications for pandemic preparedness and could be critical tools for understanding and responding to future outbreaks.

Genomic analysis of Indian isolates of Plasmodium falciparum: Implications for drug resistance and virulence factors.

The emergence of drug resistance to frontline treatments such as Artemisinin-based combination therapy (ACT) is a major obstacle to the control and eradication of malaria. This problem is compounded by the inherent genetic variability of the parasites, as many established markers of resistance do not accurately predict the drug-resistant status. There have been reports of declining effectiveness of ACT in the West Bengal and Northeast regions of India, which have traditionally been areas of drug resistance emergence in the country. Monitoring the genetic makeup of a population can help to identify the potential for drug resistance markers associated with it and evaluate the effectiveness of interventions aimed at reducing the spread of malaria. In this study, we performed whole genome sequencing of 53 isolates of Plasmodium falciparum from West Bengal and compared their genetic makeup to isolates from Southeast Asia (SEA) and Africa. We found that the Indian isolates had a distinct genetic makeup compared to those from SEA and Africa, and were more similar to African isolates, with a high prevalence of mutations associated with antigenic variation genes. The Indian isolates also showed a high prevalence of markers of chloroquine resistance (mutations in Pfcrt) and multidrug resistance (mutations in Pfmdr1), but no known mutations associated with artemisinin resistance in the PfKelch13 gene. Interestingly, we observed a novel L152V mutation in PfKelch13 gene and other novel mutations in genes involved in ubiquitination and vesicular transport that have been reported to support artemisinin resistance in the early stages of ACT resistance in the absence of PfKelch13 polymorphisms. Thus, our study highlights the importance of region-specific genomic surveillance for artemisinin resistance and the need for continued monitoring of resistance to artemisinin and its partner drugs.

Clinical Characteristics of SARS-CoV-2 Omicron Cases in Pune, Maharashtra, India.

Background The SARS-CoV-2 Omicron variant, within two months of its detection, replaced the Delta variant to become the dominant circulating variant globally. Therefore, it is essential to understand the characteristics of the disease caused by the variant and its impact on vaccination. Methods A total of 165 confirmed Omicron cases attending a tertiary care hospital in Pune, Maharashtra, between December 2021 to February 2022 were studied. Their demographic, clinical, and immunization history was recorded. Results Among the 165 cases, 7.88% were B.1.1.529 Omicron cases, 25.45% were BA.1 Omicron cases, and 66.67% were BA.2 Omicron cases. Of these 165 patients, 146 (88.48%) were discharged after treatment, 12 (7.27%) died during hospitalization, and seven (4.24%) were brought dead. The presence of one or more comorbid conditions was seen in 15.15%, of which diabetes mellitus and hypertension (28% each) were the most common conditions. Older age (greater than 60 years), an important risk factor for poor outcomes, was present in 9.1% of cases. Among the 165 cases, vaccination with at least one dose of vaccine was found in 80.61% of cases. Out of 165 cases, clinical data was available for 158 cases. Of these 158 cases, 86.71% had symptoms, and 13.29% were asymptomatic. Fever, followed by cough, myalgia, runny nose, and headache, were the most common presenting symptoms. The mean duration of illness was 2.69 days, with 91.14% of cases having the illness for less than five days, and 89.24% of cases had a National Early Warning Score (NEWS) of 1-4, suggesting a good prognosis. In 93.90% of cases, the chest X-ray findings were normal. Of the 158 cases, 92.41% of cases recovered with supportive treatment, and only 7.59% of cases required oxygen therapy. Conclusion The current study shows that the Omicron variant caused mild disease with reduced need for hospital admission and oxygen therapy in India.

Clinical Characteristics and Outcomes of Laboratory-Confirmed SARS-CoV-2 Cases Infected With Omicron Subvariants and the XBB Recombinant Variant.

Background SARS-CoV-2 has evolved to produce new variants causing successive waves of infection. Currently, six variants are being monitored by the World Health Organization that are replacing BA.5. These include BF.7 (BA.5 + R346T in spike), BQ.1 (and BQ.1.1, with BA.5 + R346T, K444T, N460K mutations in spike), BA.2.75 (including BA.2.75.2 and CH.1.1), and XBB (including XBB.1.5). BQ.1 and XBB variants are more immune evasive and have spread quickly throughout the world. Concerning the potential severity of infections caused by these variants, the present study describes the clinical characteristics and outcomes of these major variants in Maharashtra. Methodology A total of 1,141 reverse transcriptase-polymerase chain reaction (RT-PCR)-positive SARS-CoV-2 samples, with a cycle threshold (Ct) value of less than 25, were processed for SARS-CoV-2 whole genome sequencing between July 10, 2022, and January 12, 2023. All corresponding demographic and clinical data were recorded and analyzed using Microsoft® Excel and Epi Info™. Results Out of the 1,141 samples sequenced, BA.2.75* (63.78%) was the predominant Omicron variant, followed by the XBB* (18.88%), BA.2.38* (4.94%), BA.5* (4.06%), BA.2.10* (3.51%), and BQ.1* (1.65%). A total of 540 cases were contacted telephonically, of whom 494 (91.48%) were symptomatic with mild symptoms. Fever (77.73%) was the most common symptom, followed by cold (47.98%), cough (42.31%), and myalgia and fatigue (18.83%). Of the 540 cases, 414 (76.67%) cases recovered at home, and 126 (23.33%) were institutionally quarantined/hospitalized. Among the home-isolated and hospitalized cases, 416 (99.76%) and 108 (87.80%), respectively, recovered with symptomatic treatment, while one (0.24%) and 15 (12.20%), respectively, succumbed to the disease. Out of the 540 cases, 491 (90.93%) were vaccinated with at least one dose of the COVID-19 vaccine, 41 (7.59%) were unvaccinated, and for eight (1.48%) cases, vaccination data was not available. Conclusions The current study indicates that the XBB* variant is causing mild disease in India. However, as XBB* possesses both immune-escape and infectivity-enhancing mutations, it has the potential to spread to other parts of the world rapidly. Further, anti-SARS-CoV-2 vaccination improves survival rates in COVID-19.

Histidinal-Based Potent Antimalarial Agents.

Herein we report the synthesis and evaluation of peptide-histidinal conjugated drug scaffolds, which have the potential to target the hemoglobin-degrading proteases falcipain-2/3 from the human malaria parasite. Scaffolds with various substitutions were tested for antimalarial activity, and compounds 8 g, 8 h, and 15 exhibited EC50 values of ∼0.018 µM, ∼0.069 µM, and ∼0.02 µM, respectively. Structure-based docking studies on falcipain-2/3 proteases (PDB:2GHU and PDB:3BWK) revealed that compounds 8 g, 8 h, and 15 interact strongly with binding sites of falcipain-2/3 in a substrate-like manner. In silico ADME studies revealed that the molecules of interest showed no or minimal violations of drug-likeness parameters. Further, phenotypic assays revealed that compound 8 g and its biotinylated version inhibit hemoglobin degradation in the parasite food vacuole. The identification of falcipain-2/3 targeting potent inhibitors of the malaria parasite can serve as a starting point for the development of lead compounds as future antimalarial drug candidates.

Pervasive sequence-level variation in the transcriptome of Plasmodium falciparum.

Single-nucleotide variations (SNVs) in RNA, arising from co- and post-transcriptional phenomena including transcription errors and RNA-editing, are well studied in a range of organisms. In the malaria parasite Plasmodium falciparum, stage-specific and non-specific gene-expression variations accompany the parasite's array of developmental and morphological phenotypes over the course of its complex life cycle. However, the extent, rate and effect of sequence-level variation in the parasite's transcriptome are unknown. Here, we report the presence of pervasive, non-specific SNVs in the P. falciparum transcriptome. SNV rates for a gene were correlated to gene length (r[Formula: see text]0.65-0.7) but not to the AT-content of that gene. Global SNV rates for the P. falciparum lines we used, and for publicly available P. vivax and P. falciparum clinical isolate datasets, were of the order of 10-3 per base, ∼10נhigher than rates we calculated for bacterial datasets. These variations may reflect an intrinsic transcriptional error rate in the parasite, and RNA editing may be responsible for a subset of them. This seemingly characteristic property of the parasite may have implications for clinical outcomes and the basic biology and evolution of P. falciparum and parasite biology more broadly. We anticipate that our study will prompt further investigations into the exact sources, consequences and possible adaptive roles of these SNVs.

Identification of Co-Existing Mutations and Gene Expression Trends Associated With K13-Mediated Artemisinin Resistance in Plasmodium falciparum.

Plasmodium falciparum infects millions and kills thousands of people annually the world over. With the emergence of artemisinin and/or multidrug resistant strains of the pathogen, it has become even more challenging to control and eliminate the disease. Multiomics studies of the parasite have started to provide a glimpse into the confounding genetics and mechanisms of artemisinin resistance and identified mutations in Kelch13 (K13) as a molecular marker of resistance. Over the years, thousands of genomes and transcriptomes of artemisinin-resistant/sensitive isolates have been documented, supplementing the search for new genes/pathways to target artemisinin-resistant isolates. This meta-analysis seeks to recap the genetic landscape and the transcriptional deregulation that demarcate artemisinin resistance in the field. To explore the genetic territory of artemisinin resistance, we use genomic single-nucleotide polymorphism (SNP) datasets from 2,517 isolates from 15 countries from the MalariaGEN Network (The Pf3K project, pilot data release 4, 2015) to dissect the prevalence, geographical distribution, and co-existing patterns of genetic markers associated with/enabling artemisinin resistance. We have identified several mutations which co-exist with the established markers of artemisinin resistance. Interestingly, K13-resistant parasites harbor α-ß hydrolase and putative HECT domain-containing protein genes with the maximum number of SNPs. We have also explored the multiple, publicly available transcriptomic datasets to identify genes from key biological pathways whose consistent deregulation may be contributing to the biology of resistant parasites. Surprisingly, glycolytic and pentose phosphate pathways were consistently downregulated in artemisinin-resistant parasites. Thus, this meta-analysis highlights the genetic and transcriptomic features of resistant parasites to propel further exploratory studies in the community to tackle artemisinin resistance.

Chromodomain Protein Interacts with H3K9me3 and Controls RBC Rosette Formation by Regulating the Expression of a Subset of RIFINs in the Malaria Parasite.

Plasmodium falciparum expresses clonally variant proteins on the surface of infected erythrocytes to evade the host immune system. The clonally variant multigene families include var, rifin, and stevor, which express Erythrocyte Membrane Protein 1 (EMP1), Repetitive Interspersed Families of polypeptides (RIFINs), and Sub-telomeric Variable Open Reading frame (STEVOR) proteins, respectively. The rifins are the largest multigene family and are essentially involved in the RBC rosetting, the hallmark of severe malaria. The molecular regulators that control the RIFINs expression in Plasmodium spp. have not been reported so far. This study reports a chromodomain-containing protein (PfCDP) that binds to H3K9me3 modification on P. falciparum chromatin. Conditional deletion of the chromodomain (CD) gene in P. falciparum using an inducible DiCre-LoxP system leads to selective up-regulation of a subset of virulence genes, including rifins, a few var, and stevor genes. Further, we show that PfCDP conditional knockout (PfΔCDP) promotes RBC rosette formation. This study provides the first evidence of an epigenetic regulator mediated control on a subset of RIFINs expression and RBC rosetting by P. falciparum.

Autophagy Underlies the Proteostasis Mechanisms of Artemisinin Resistance in P. falciparum Malaria.

Emerging resistance to artemisinin (ART) has become a challenge for reducing worldwide malaria mortality and morbidity. The C580Y mutation in Plasmodium falciparum Kelch13 has been identified as the major determinant for ART resistance in the background of other mutations, which include the T38I mutation in autophagy-related protein PfATG18. Increased endoplasmic reticulum phosphatidylinositol-3-phosphate (ER-PI3P) vesiculation, unfolded protein response (UPR), and oxidative stress are the proteostasis mechanisms proposed to cause ART resistance. While UPR and PI3P are known to stimulate autophagy in higher organisms to clear misfolded proteins, participation of the parasite autophagy machinery in these mechanisms of ART resistance has not yet been experimentally demonstrated. Our study establishes that ART-induced ER stress leads to increased expression of P. falciparum autophagy proteins through induction of the UPR. Furthermore, the ART-resistant K13C580Y isolate shows higher basal expression levels of autophagy proteins than those of its isogenic counterpart, and this magnifies under starvation conditions. The copresence of PfK13 with PfATG18 and PI3P on parasite hemoglobin-trafficking vesicles demonstrate interactions between the autophagy and hemoglobin endocytosis pathways proposed to be involved in ART resistance. Analysis of PfK13 mutations in 2,517 field isolates, revealing an impressive >85% coassociation between PfK13 C580Y and PfATG18 T38I, together with our experimental studies with an ART-resistant P. falciparum strain establishes that parasite autophagy underpins various mechanisms of ART resistance and is a starting point to further explore this pathway for developing antimalarials. IMPORTANCE There is an urgent need to clearly understand the mechanisms of ART resistance as it is emerging in the Greater Mekong Subregion (GMS) and other parts of the world, such as Africa. Deciphering the mechanisms of the parasite's stress response pathways of ART resistance will provide insights to identify novel drug targets for developing new antimalarial regimens.

Nup93 and CTCF modulate spatiotemporal dynamics and function of the HOXA gene locus during differentiation.

Nucleoporins regulate nuclear transport and are also involved in DNA damage, repair, cell cycle, chromatin organization and gene expression. Here, we studied the role of nucleoporin Nup93 and the chromatin organizer CTCF in regulating expression of the HOXA gene locus during differentiation. ChIP sequencing revealed a significant overlap between Nup93 and CTCF peaks. Interestingly, Nup93 and CTCF are associated with the 3' and 5' HOXA genes, respectively. Depletions of Nup93 and CTCF antagonistically modulate expression levels of 3' and 5' HOXA genes in the undifferentiated human NT2/D1 cell line. Nup93 also regulates the localization of the HOXA gene locus, which disengages from the nuclear periphery upon Nup93 but not CTCF depletion, consistent with its upregulation. The dynamic association of Nup93 and CTCF with the HOXA locus during differentiation correlates with its spatial positioning and expression. Whereas Nup93 tethers the HOXA locus to the nuclear periphery, CTCF potentially regulates looping of the HOXA gene cluster in a temporal manner. In summary, Nup93 and CTCF complement one another in modulating the spatiotemporal dynamics and function of the HOXA gene locus during differentiation. This article has an associated First Person interview with the first authors of the paper.

Analysis of drug resistance marker genes of Plasmodium falciparum after implementation of artemisinin-based combination therapy in Pune district, India.

The global emergence and spread of malaria parasites resistant to antimalarial drugs is a major problem in malaria control and elimination. In this study, samples from Pune district were characterized to determine prevalence of molecular markers of resistance to chloroquine (pfcrt codons C72S, M74I, N75E, K76T and pfmdr-1 N86Y, Y184F), pyrimethamine (pfdhfr C50R, N51I, C59R, S108N), sulfadoxine (pfdhps, S436A, A437G, K540E, A581G), and artemisinin (pfkelch13, C580Y, R539T). The pfcrt K76T mutation was found in 78% samples as CVMNT, SVMNT and CVIET haplotype. The pfmdr-1 N86Y and Y184F mutations were found in 54% of samples. The pfdhfr double mutation C59R + S108N was present in 67% of samples, while the pfdhfr triple mutation (N51I + C59R + S108N) was not detected. The pfdhps mutations A437G and K540E were found in 67% of samples. Single mutants of pfdhps were rare, with K540E detected in only 6 patient samples. Similarly, pfdhps A581G was found in 13 of the isolates. The molecular markers associated with artemisinin resistance (mutations in pfkelch13 C580Y, R539T) were not detected in any of the isolates. These results suggest an emerging problem with multidrug-resistant P. falciparum. Though the genotype conventionally associated with artemisinin resistance was not observed, chloroquine-resistant genotype has reached complete fixation in the population. Moreover, the prevalence of mutations in both pfdhfr and pfdhps, with the presence of the quadruple mutant, indicates that continued monitoring is required to assess whether sulfadoxine-pyrimethamine can be used efficiently as a partner drug for artemisinin for the treatment of P. falciparum.

Single-Cell RNA Sequencing Reveals Cellular Heterogeneity and Stage Transition under Temperature Stress in Synchronized Plasmodium falciparum Cells.

The malaria parasite has a complex life cycle exhibiting phenotypic and morphogenic variations in two different hosts by existing in heterogeneous developmental states. To investigate this cellular heterogeneity of the parasite within the human host, we performed single-cell RNA sequencing of synchronized Plasmodium cells under control and temperature treatment conditions. Using the Malaria Cell Atlas (https://www.sanger.ac.uk/science/tools/mca) as a guide, we identified 9 subtypes of the parasite distributed across known intraerythrocytic stages. Interestingly, temperature treatment results in the upregulation of the AP2-G gene, the master regulator of sexual development in a small subpopulation of the parasites. Moreover, we identified a heterogeneous stress-responsive subpopulation (clusters 5, 6, and 7 [∼10% of the total population]) that exhibits upregulation of stress response pathways under normal growth conditions. We also developed an online exploratory tool that will provide new insights into gene function under normal and temperature stress conditions. Thus, our study reveals important insights into cell-to-cell heterogeneity in the parasite population under temperature treatment that will be instrumental toward a mechanistic understanding of cellular adaptation and population dynamics in Plasmodium falciparum. IMPORTANCE The malaria parasite has a complex life cycle exhibiting phenotypic variations in two different hosts accompanied by cell-to-cell variability that is important for stress tolerance, immune evasion, and drug resistance. To investigate cellular heterogeneity determined by gene expression, we performed single-cell RNA sequencing (scRNA-seq) of about 12,000 synchronized Plasmodium cells under physiologically relevant normal (37°C) and temperature stress (40°C) conditions phenocopying the cyclic bouts of fever experienced during malarial infection. In this study, we found that parasites exhibit transcriptional heterogeneity in an otherwise morphologically synchronized culture. Also, a subset of parasites is continually committed to gametocytogenesis and stress-responsive pathways. These observations have important implications for understanding the mechanisms of drug resistance generation and vaccine development against the malaria parasite.

Dynamic association of the H3K64 trimethylation mark with genes encoding exported proteins in Plasmodium falciparum.

Epigenetic modifications have emerged as critical regulators of virulence genes and stage-specific gene expression in Plasmodium falciparum. However, the specific roles of histone core epigenetic modifications in regulating the stage-specific gene expression are not well understood. In this study, we report an unconventional trimethylation at lysine 64 on histone 3 (H3K64me3) and characterize its functional relevance in P. falciparum. We show that PfSET4 and PfSET5 proteins of P. falciparum methylate H3K64 and that they prefer the nucleosome as a substrate over free histone 3 proteins. Structural analysis of PfSET5 revealed that it interacts with the nucleosome as a dimer. The H3K64me3 mark is dynamic, being enriched in the ring and trophozoite stages and drastically reduced in the schizont stages. Stage-specific global chromatin immunoprecipitation -sequencing analysis of the H3K64me3 mark revealed the selective enrichment of this methyl mark on the genes of exported family proteins in the ring and trophozoite stages and a significant reduction of the same in the schizont stages. Collectively, our data identify a novel epigenetic mark that is associated with the subset of genes encoding for exported proteins, which may regulate their expression in different stages of P. falciparum.

Histone acetyltransferase PfGCN5 regulates stress responsive and artemisinin resistance related genes in Plasmodium falciparum.

Plasmodium falciparum has evolved resistance to almost all front-line drugs including artemisinin, which threatens malaria control and elimination strategies. Oxidative stress and protein damage responses have emerged as key players in the generation of artemisinin resistance. In this study, we show that PfGCN5, a histone acetyltransferase, binds to the stress-responsive genes in a poised state and regulates their expression under stress conditions. Furthermore, we show that upon artemisinin exposure, genome-wide binding sites for PfGCN5 are increased and it is directly associated with the genes implicated in artemisinin resistance generation like BiP and TRiC chaperone. Interestingly, expression of genes bound by PfGCN5 was found to be upregulated during stress conditions. Moreover, inhibition of PfGCN5 in artemisinin-resistant parasites increases the sensitivity of the parasites to artemisinin treatment indicating its role in drug resistance generation. Together, these findings elucidate the role of PfGCN5 as a global chromatin regulator of stress-responses with a potential role in modulating artemisinin drug resistance and identify PfGCN5 as an important target against artemisinin-resistant parasites.