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.

Epigenetically regulated RNA-binding proteins signify malaria hypnozoite dormancy.

Dormancy enables relapsing malaria parasites, such as Plasmodium vivax and cynomolgi, to survive unfavorable conditions. It is enabled by hypnozoites, parasites remaining quiescent inside hepatocytes before reactivating and establishing blood-stage infection. We integrate omics approaches to explore gene-regulatory mechanisms underlying hypnozoite dormancy. Genome-wide profiling of activating and repressing histone marks identifies a few genes that get silenced by heterochromatin during hepatic infection of relapsing parasites. By combining single-cell transcriptomics, chromatin accessibility profiling, and fluorescent in situ RNA hybridization, we show that these genes are expressed in hypnozoites and that their silencing precedes parasite development. Intriguingly, these hypnozoite-specific genes mainly encode proteins with RNA-binding domains. We hence hypothesize that these likely repressive RNA-binding proteins keep hypnozoites in a developmentally competent but dormant state and that heterochromatin-mediated silencing of the corresponding genes aids reactivation. Exploring the regulation and exact function of these proteins hence could provide clues for targeted reactivation and killing of these latent pathogens.

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.

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.

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.

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.

Ru-Catalyzed dehydrogenative synthesis of antimalarial arylidene oxindoles.

Ru(ii)-NHC catalyzes α-olefination of 2-oxindoles using diaryl methanols in the absence of an acceptor. A wide array of symmetrical and unsymmetrical diaryl methanols undergoes dehydrogenative coupling with 2-oxindole selectively to generate various substituted 3-(diphenylmethylene)indolin-2-one derivatives in good yields and produces environmentally benign by-products, H2 and H2O. This methodology was successfully applied for the synthesis of a bioactive drug i.e. TAS-301. The biological activities of the synthesized 3-(diphenylmethylene)indolin-2-one derivatives were screened against the Plasmodium falciparum parasite and found to exhibit a significant activity with IC50 = 2.24 µM.

Genome-wide survey and phylogenetic analysis of histone acetyltransferases and histone deacetylases of Plasmodium falciparum.

Malaria parasites can readily sense and adapt to environmental changes, thus making the control and eradication of this disease difficult. Molecular studies have unraveled a very tightly coordinated transcriptional machinery governed by complex regulatory mechanisms including chromatin modification and spatiotemporal compartmentalization. Histone modifying enzymes play key roles in the regulation of chromatin modification and gene expression, which are associated with cell cycle progression, antigenic variation and immune evasion. Here, we present a comprehensive review of the key regulators of the Plasmodium falciparum histone acetylome; histone acetyltransferases (HATs); and histone deacetylases (HDACs). We describe the genome-wide occurrence of HATs and HDACs in the P. falciparum genome and identify novel, as well as previously unclassified HATs. We re-confirm the presence of five known HDACs and identify, a novel putative HDAC. Interestingly, we identify several HATs and HDACs with unique and noncanonical domain combinations indicating their involvement in other associated functions. Moreover, the phylogenetic analyses of HATs and HDACs suggest that many of them are close to the prokaryotic systems and thus potential candidates for drug development. Our review deciphers the phylogeny of HATs and HDACs of the malaria parasite, investigates their role in drug-resistance generation, and highlights their potential as therapeutic targets.