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There is a pressing need to improve the efficiency of drug development, and nowhere is that need more clear than in the case of neglected diseases like malaria. The peculiarities of pyrimidine metabolism in Plasmodium species make inhibition of dihydroorotate dehydrogenase (DHODH) an attractive target for antimalarial drug design. By applying a pair of complementary quantitative structure–activity relationships derived for inhibition of a truncated, soluble form of the enzyme from Plasmodium falciparum (s-PfDHODH) to data from a large-scale phenotypic screen against cultured parasites, we were able to identify a class of antimalarial leads that inhibit the enzyme and abolish parasite growth in blood culture. Novel analogs extending that class were designed and synthesized with a goal of improving potency as well as the general pharmacokinetic and toxicological profiles. Their synthesis also represented an opportunity to prospectively validate our in silico property predictions. The seven analogs synthesized exhibited physicochemical properties in good agreement with prediction, and five of them were more active against P. falciparum growing in blood culture than any of the compounds in the published lead series. The particular analogs prepared did not inhibit s-PfDHODH in vitro, but advanced biological assays indicated that other examples from the class did inhibit intact PfDHODH bound to the mitochondrial membrane. The new analogs, however, killed the parasites by acting through some other, unidentified mechanism 24–48 h before PfDHODH inhibition would be expected to do so.

The RAP (RNA-binding domain abundant in Apicomplexans) protein family has been identified in various organisms. Despite expansion of this protein family in apicomplexan parasites, their main biological functions remain unknown. In this study, we use inducible knockdown studies in the human malaria parasite, Plasmodium falciparum, to show that two RAP proteins, PF3D7_0105200 (PfRAP01) and PF3D7_1470600 (PfRAP21), are essential for parasite survival and localize to the mitochondrion. Using transcriptomics, metabolomics, and proteomics profiling experiments, we further demonstrate that these RAP proteins are involved in mitochondrial RNA metabolism. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (eCLIP-seq), we validate that PfRAP01 and PfRAP21 are true RNA-binding proteins and interact specifically with mitochondrial rRNAs. Finally, mitochondrial enrichment experiments followed by deep sequencing of small RNAs demonstrate that PfRAP21 controls mitochondrial rRNA expression. Collectively, our results establish the role of these RAP proteins in mitoribosome activity and contribute to further understanding this protein family in malaria parasites.

Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary to infect the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. However, the genomes of malaria parasites have been noted for their limited number of transcriptional and chromatin regulators, and the molecular mediators of these changes remain largely unknown. We recently identified homeodomain protein 1 (HDP1) as a DNA-binding protein, first expressed in gametocytes, that enhances the expression of key genes critical for early sexual differentiation. The discovery of HDP1 marks a new class of transcriptional regulator in malaria parasites outside of the better-characterized ApiAP2 family. Here, using molecular biology, biochemistry and microscopy techniques, we show that HDP1 is essential for gametocyte maturation, facilitating the necessary upregulation of inner membrane complex components during early gametocytogenesis that gives P. falciparum gametocytes their characteristic shape.

Plasmodium falciparum erythrocyte membrane and merozoite antigen 1 (PfEMMA1) is a newly characterized malaria protein expressed on the surface of parasitized erythrocytes and merozoites that induces protective antibodies in functional assays, a human epidemiological study, and a mouse immunization model., We previously identified a Plasmodium falciparum (Pf) protein of unknown function encoded by a single-copy gene, PF3D7_1134300, as a target of antibodies in plasma of Tanzanian children in a whole-proteome differential screen. Here we characterize this protein as a blood-stage antigen that localizes to the surface membranes of both parasitized erythrocytes and merozoites, hence its designation as Pf erythrocyte membrane and merozoite antigen 1 (PfEMMA1). Mouse anti-PfEMMA1 antisera and affinity-purified human anti-PfEMMA1 antibodies inhibited growth of P. falciparum strains by up to 68% in growth inhibition assays. Following challenge with uniformly fatal Plasmodium berghei (Pb) ANKA, up to 40% of mice immunized with recombinant PbEMMA1 self-cured, and median survival of lethally infected mice was up to 2.6-fold longer than controls (21 vs. 8 d, P = 0.005). Furthermore, high levels of naturally acquired human anti-PfEMMA1 antibodies were associated with a 46% decrease in parasitemia over 2.5 yr of follow-up of Tanzanian children. Together, these findings suggest that antibodies to PfEMMA1 mediate protection against malaria.

Over the last decades, novel methods have been developed to study the role of chromosome positioning within the nucleus may play in gene regulation. Established proximity ligation-based chromosome conformation capture (3C) techniques such as Hi-C have revealed the existence of chromosome territories, functional nuclear landmarks, and topologically associating domains (TAPs) in most eukaryotic organisms. Adaptation of these methods in apicomplexan parasites has recently uncovered new aspects of 3D genome biology in virulence factors. Here, we describe the Hi-C protocol in the human malaria parasite, Plasmodium falciparum. This method can determine the genome organization in malaria parasites and its role in gene regulation and virulence.

Over the last decades, identification of RNA-proteins complexes and their binding sites was challenging. Recently, techniques based on crosslinking, immunoprecipitation, and high-throughput sequencing have been developed. An optimized method, called eCLIP-seq, enables to identify precisely the targeted RNAs as well as the transcriptome-wide binding sites at nucleotide resolution. Here we describe the eCLIP-seq protocol in asexual stages of the human malaria parasite, Plasmodium falciparum. This method could facilitate the characterization of RNA-binding proteins in this organism for which few data are currently available.

Over the last decades, novel methods have been developed to study the role of chromosome positioning within the nucleus may play in gene regulation. Established proximity ligation-based chromosome conformation capture (3C) techniques such as Hi-C have revealed the existence of chromosome territories, functional nuclear landmarks, and topologically associating domains (TAPs) in most eukaryotic organisms. Adaptation of these methods in apicomplexan parasites has recently uncovered new aspects of 3D genome biology in virulence factors. Here, we describe the Hi-C protocol in the human malaria parasite, Plasmodium falciparum. This method can determine the genome organization in malaria parasites and its role in gene regulation and virulence.

Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary to infect the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. However, the genomes of malaria parasites have been noted for their limited number of transcriptional and chromatin regulators, and the molecular mediators of these changes remain largely unknown. We recently identified homeodomain protein 1 (HDP1) as a DNA-binding protein, first expressed in gametocytes, that enhances the expression of key genes critical for early sexual differentiation. The discovery of HDP1 marks a new class of transcriptional regulator in malaria parasites outside of the better-characterized ApiAP2 family. Here, using molecular biology, biochemistry and microscopy techniques, we show that HDP1 is essential for gametocyte maturation, facilitating the necessary upregulation of inner membrane complex components during early gametocytogenesis that gives P. falciparum gametocytes their characteristic shape.

Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary for transmission to the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. However, the genomes of malaria parasites have been noted for their dearth of transcriptional and chromatin regulators and the molecular mediators of these changes remain largely unknown. We recently identified HomeoDomain Protein 1 (HDP1) as a DNA-binding protein that enhances the expression of key genes that are critical for early sexual differentiation. The discovery of a homeodomain-like DNA-binding protein marks a new class of transcriptional regulator in malaria parasites outside of the better-characterized ApiAP2 family. In this study, we show that HDP1 facilitates the necessary upregulation of inner membrane complex components during early gametocytogenesis that gives P. falciparum gametocytes they characteristic shape and is required for gametocyte maturation and parasite transmission.

Multiple hosts and various life cycle stages prompt the human malaria parasite, Plasmodium falciparum, to acquire sophisticated molecular mechanisms to ensure its survival, spread, and transmission to its next host. To face these environmental challenges, increasing evidence suggests that the parasite has developed complex and complementary layers of regulatory mechanisms controlling gene expression. Here, we discuss the recent developments in the discovery of molecular components that contribute to cell replication and differentiation and highlight the major contributions of epigenetics, transcription factors, and nuclear architecture in controlling gene regulation and life cycle progression in Plasmodium spp.

The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human malaria parasite Plasmodium falciparum , the clustering of a family of virulence genes correlates with their coordinated silencing and has a strong influence on the overall organization of the genome. To identify conserved and species-specific principles of genome organization, we performed Hi-C experiments and generated 3D genome models for five Plasmodium species and two related apicomplexan parasites. Plasmodium species mainly showed clustering of centromeres, telomeres, and virulence genes. In P. falciparum , the heterochromatic virulence gene cluster had a strong repressive effect on the surrounding nuclear space, while this was less pronounced in Plasmodium vivax and Plasmodium berghei , and absent in Plasmodium yoelii . In Plasmodium knowlesi , telomeres and virulence genes were more dispersed throughout the nucleus, but its 3D genome showed a strong correlation with gene expression. The Babesia microti genome showed a classical Rabl organization with colocalization of subtelomeric virulence genes, while the Toxoplasma gondii genome was dominated by clustering of the centromeres and lacked virulence gene clustering. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes. P. falciparum and P. knowlesi , the only two Plasmodium species with gene families involved in antigenic variation, are unique in the effect of these genes on chromosome folding, indicating a potential link between genome organization and gene expression in more virulent pathogens.

Chromatin conformation assays such as Hi-C cannot directly measure differences in 3D architecture between cell types or cell states. For this purpose, two or more Hi-C experiments must be carried out, but direct comparison of the resulting Hi-C matrices is confounded by several features of Hi-C data. Most notably, the genomic distance effect, whereby contacts between pairs of genomic loci that are proximal along the chromosome exhibit many more Hi-C contacts that distal pairs of loci, dominates every Hi-C matrix. Furthermore, the form that this distance effect takes often varies between different Hi-C experiments, even between replicate experiments. Thus, a statistical confidence measure designed to identify differential Hi-C contacts must accurately account for the genomic distance effect or risk being misled by large-scale but artifactual differences. ACCOST (Altered Chromatin Conformation STatistics) accomplishes this goal by extending the statistical model employed by DEseq, re-purposing the “size factors,” which were originally developed to account for differences in read depth between samples, to instead model the genomic distance effect. We show via analysis of simulated and real data that ACCOST provides unbiased statistical confidence estimates that compare favorably with competing methods such as diffHiC, FIND, and HiCcompare. ACCOST is freely available with an Apache license at https://bitbucket.org/noblelab/accost.

Gene expression in Plasmodium falciparum is tightly regulated to ensure successful propagation of the parasite throughout its complex life cycle. The earliest transcriptomics studies in P. falciparum suggested a cascade of transcriptional activity over the course of the 48-hour intraerythrocytic developmental cycle (IDC); however, the just-in-time transcriptional model has recently been challenged by findings that show the importance of post-transcriptional regulation. To further explore the role of transcriptional regulation, we performed the first genome-wide nascent RNA profiling in P. falciparum. Our findings indicate that the majority of genes are transcribed simultaneously during the trophozoite stage of the IDC and that only a small subset of genes is subject to differential transcriptional timing. RNA polymerase II is engaged with promoter regions prior to this transcriptional burst, suggesting that Pol II pausing plays a dominant role in gene regulation. In addition, we found that the overall transcriptional program during gametocyte differentiation is surprisingly similar to the IDC, with the exception of relatively small subsets of genes. Results from this study suggest that further characterization of the molecular players that regulate stage-specific gene expression and Pol II pausing will contribute to our continuous search for novel antimalarial drug targets.

Proteins interacting with DNA are fundamental for mediating processes such as gene expression, DNA replication and maintenance of genome integrity. Accumulating evidence suggests that the chromatin of apicomplexan parasites, such as Plasmodium falciparum, is highly organized, and this structure provides an epigenetic mechanism for transcriptional regulation. To investigate how parasite chromatin structure is being regulated, we undertook comparative genomics analysis using 12 distinct eukaryotic genomes. We identified conserved and parasite-specific chromatin-associated domains (CADs) and proteins (CAPs). We then used the chromatin enrichment for proteomics (ChEP) approach to experimentally capture CAPs in P. falciparum. A topological scoring analysis of the proteomics dataset revealed stage-specific enrichments of CADs and CAPs. Finally, we characterized, two candidate CAPs: a conserved homologue of the structural maintenance of chromosome 3 protein and a homologue of the crowded-like nuclei protein, a plant-like protein functionally analogous to animal nuclear lamina proteins. Collectively, our results provide a comprehensive overview of CAPs in apicomplexans, and contribute to our understanding of the complex molecular components regulating chromatin structure and genome architecture in these deadly parasites.

The chromosomes within the eukaryotic cell nucleus are highly dynamic and adopt complex hierarchical structures. Understanding how this three-dimensional (3D) nuclear architectureaffects gene regulation, cell cycle progression and disease pathogenesis are important biological questions in development and disease. Recently, many genome-wide technologies including chromosome conformation capture (3C) and 3C-based methodologies (4C, 5C, and Hi-C) have been developed to investigate 3D chromatin structure. In this review, we introduce 3D genome methodologies, with a focus on their application for understanding the nuclear architecture of the human malaria parasite, Plasmodium falciparum. An increasing amount of evidence now suggests that gene regulation in the parasite is largely regulated by epigenetic mechanisms and nuclear reorganization. Here, we explore the 3D genome architecture of P. falciparum, including local and global chromatin structure. In addition, molecular components important for maintaining 3D chromatin organization including architectural proteins and long non-coding RNAs are discussed. Collectively, these studies contribute to our understanding of how the plasticity of 3D genome architecture regulates gene expression and cell cycle progression in this deadly parasite.

Due to the unique selective pressures and extreme changes faced by the human malaria parasite Plasmodium falciparum throughout its life cycle, the parasite has evolved distinct features to alter its gene expression patterns. Along with classical gene regulation by transcription factors (TFs), of which only one family, the AP2 TFs, has been described in the parasite genome, a large body of evidence points toward chromatin structure and epigenetic factors mediating the changes in gene expression associated with parasite life cycle stages. These attributes may be critically important for immune evasion, host cell invasion and development of the parasite in its two hosts, the human and the Anopheles vector. Thus, the factors involved in the maintenance and regulation of chromatin and epigenetic features represent potential targets for antimalarial drugs. In this review, we discuss the mechanisms in P. falciparum that regulate chromatin structure, nucleosome landscape, the 3-dimensional structure of the genome and additional distinctive features created by parasite-specific genes and gene families. We review conserved traits of chromatin in eukaryotes in order to highlight what is unique in the parasite.

The development of malaria parasites throughout their various life cycle stages is coordinated by changes in gene expression. We previously showed that the three-dimensional organization of the Plasmodium falciparum genome is strongly associated with gene expression during its replication cycle inside red blood cells. Here, we analyze genome organization in the P. falciparum and P. vivax transmission stages. Major changes occur in the localization and interactions of genes involved in pathogenesis and immune evasion, host cell invasion, sexual differentiation, and master regulation of gene expression. Furthermore, we observe reorganization of subtelomeric heterochromatin around genes involved in host cell remodeling. Depletion of heterochromatin protein 1 (PfHP1) resulted in loss of interactions between virulence genes, confirming that PfHP1 is essential for maintenance of the repressive center. Our results suggest that the three-dimensional genome structure of human malaria parasites is strongly connected with transcriptional activity of specific gene families throughout the life cycle., The development of malaria parasites is controlled by coordinated changes in gene expression. Here, the authors show that the three-dimensional genome structure of human malaria parasites is strongly connected with transcriptional activity of specific gene families throughout the life cycles of Plasmodium falciparum and Plasmodium vivax parasites.

Malaria is caused by Plasmodium parasites that proliferate in the bloodstream. During each replication cycle some parasites differentiate into gametocytes, the only forms able to infect the mosquito vector and transmit malaria. Sexual commitment is triggered by activation of AP2-G, the master transcriptional regulator of gametocytogenesis. Heterochromatin protein 1 (HP1)-dependent silencing of ap2-g prevents sexual conversion in proliferating parasites. Here, we identified Plasmodium falciparum gametocyte development 1 (GDV1) as an upstream activator of sexual commitment. We found that GDV1 targeted heterochromatin and triggered HP1 eviction thus de-repressing ap2-g. Expression of GDV1 was responsive to environmental triggers of sexual conversion and controlled via a gdv1 antisense RNA. Hence, GDV1 appears to act as an effector protein that induces sexual differentiation by antagonizing HP1-dependent gene silencing.

A mixture-based combinatorial library of five Ugi adducts (4-8) incorporating known antitubercular and antimalarial pharmacophores was successfully synthesized, starting from the naturally occurring diisocyanide 3, via parallel Ugi four-center three-component reactions (U-4C-3CR). The novel α-acylamino amides obtained were evaluated for their antiinfective potential against laboratory strains of Mycobacterium tuberculosis H37Rv and chloroquine-susceptible 3D7 Plasmodium falciparum. Interestingly, compounds 4-8 displayed potent in vitro antiparasitic activity with higher cytotoxicity in comparison to their diisocyanide precursor 3, with the best compound exhibiting an IC50 value of 3.6 nM. Additionally, these natural product inspired hybrids potently inhibited in vitro thromboxane B2 (TXB2) and superoxide anion (O2(-)) generation from Escherichia coli lipopolysaccharide (LPS)-activated rat neonatal microglia, with concomitant low short-term toxicity.

© 2015 American Chemical Society. Of the 50+ kalihinane diterpenoids reported to date, only five had been tested for antimalarial activity, in spite of the fact that kalihinol A is the most potent among the members of the larger family of antimalarial isocyanoterpenes. We have validated a strategy designed to access many of the kalihinanes with a 12-step enantioselective synthesis of kalihinol B, the tetrahydrofuran isomer of kalihinol A (a tetrahydropyran). Kalihinol B shows similarly high potency against chloroquine-resistant Plasmodium falciparum.

Bioassay-guided fractionation of the Caribbean sponge Svenzea flava collected near Mona Island, off the west coast of Puerto Rico, led to the isolation of two isocyanide amphilectane-type diterpenes named monamphilectines B and C (2 and 3). Attached to the backbone of each of these compounds is the first α-substituted monocyclic β-lactam ring to be isolated from a marine organism. The molecular structures of 2 and 3 were established by spectroscopic methods and then confirmed unequivocally by chemical correlation and comparison of physical and chemical data with the natural products. The new β-lactams were successfully synthesized in one step, starting from the known diisocyanide 4, via parallel Ugi four-center three-component reactions (U-4C-3CR) that also established their absolute stereostructures. Interestingly, compounds 2 and 3 exhibited activities in the low nanomolar range against the human malaria parasite Plasmodium falciparum.

The human malaria parasite, Plasmodium falciparum, depends on a coordinated regulation of gene expression for development and propagation within the human host. Recent developments suggest that gene regulation in the parasite is largely controlled by epigenetic mechanisms. Here, we discuss recent advancements contributing to our understanding of the mechanisms controlling gene regulation in the parasite, including nucleosome landscape, histone modifications, and nuclear architecture. In addition, various processes involved in regulation of parasite-specific genes and gene families are examined. Finally, we address the use of epigenetic processes as targets for novel antimalarial therapies. Collectively, these topics highlight the unique biology of P. falciparum, and contribute to our understanding of mechanisms regulating gene expression in this deadly parasite.

Plasmodium falciparum is the most deadly human malarial parasite, responsible for an estimated 207 million cases of disease and 627,000 deaths in 2012. Recent studies reveal that the parasite actively regulates a large fraction of its genes throughout its replicative cycle inside human red blood cells and that epigenetics plays an important role in this precise gene regulation. Here, we discuss recent advances in our understanding of three aspects of epigenetic regulation in P. falciparum: changes in histone modifications, nucleosome occupancy and the three-dimensional genome structure. We compare these three aspects of the P. falciparum epigenome to those of other eukaryotes, and show that large-scale compartmentalization is particularly important in determining histone decomposition and gene regulation in P. falciparum. We conclude by presenting a gene regulation model for P. falciparum that combines the described epigenetic factors, and by discussing the implications of this model for the future of malaria research.

Empirical evidence suggests that the malaria parasite Plasmodium falciparum employs a broad range of mechanisms to regulate gene transcription throughout the organism’s complex life cycle. To better understand this regulatory machinery, we assembled a rich collection of genomic and epigenomic data sets, including information about transcription factor (TF) binding motifs, patterns of covalent histone modifications, nucleosome occupancy, GC content, and global 3D genome architecture. We used these data to train machine learning models to discriminate between high-expression and low-expression genes, focusing on three distinct stages of the red blood cell phase of the Plasmodium life cycle. Our results highlight the importance of histone modifications and 3D chromatin architecture in Plasmodium transcriptional regulation and suggest that AP2 transcription factors may play a limited regulatory role, perhaps operating in conjunction with epigenetic factors., Author summary The parasite responsible for the most lethal form of malaria, Plasmodium falciparum, employs a variety of mechanisms to modify the expression of its genes throughout its complex life cycle. In this work, we gather a rich collection of data describing various aspects of the gene regulatory apparatus in P. falciparum, and we use a machine learning approach to help understand the relative importance of each potential regulatory mechanism. Our results highlight the importance of two particular mechanisms: patterns of biochemical modifications on the histone proteins that form the primary scaffold for DNA in the cell and the three-dimensional conformation of DNA in the nucleus.

A suite of pharmacokinetic and pharmacological studies show that bromophycolide A (1), an inhibitor of drug-sensitive and drug-resistant Plasmodium falciparum, displays a typical small molecule profile with low toxicity and good bioavailability. Despite susceptibility to liver metabolism and a short in vivo half-life, 1 significantly decreased parasitemia in a malaria mouse model. Combining these data with prior structure–activity relationship analyses, we demonstrate the potential for future development of 1 and its bioactive ester analogues.

Farnesides A and B (1, 2), linear sesquiterpenoids connected by ether links to a ribose dihydrouracil nucleoside, were isolated from a marine-derived Streptomyces sp., strain CNT-372, grown in saline liquid culture. The structures of the new compounds were assigned by comprehensive spectroscopic analysis primarily involving 1D and 2D NMR analysis and by comparison of spectroscopic data to the recently reported ribose nucleoside JBIR-68 (3). The farnesides are only the second example of this exceedingly rare class of microbial terpenoid nucleoside metabolites. Farneside A (1) was found to have modest antimalarial activity against the parasite Plasmodium falciparum.

Background Gene expression is controlled at multiple levels, including transcription, stability, translation, and degradation. Over the years, it has become apparent that Plasmodium falciparum exerts limited transcriptional control of gene expression, while at least part of Plasmodium’s genome is controlled by post-transcriptional mechanisms. To generate insights into the mechanisms that regulate gene expression at the post-transcriptional level, we undertook complementary computational, comparative genomics, and experimental approaches to identify and characterize mRNA-binding proteins (mRBPs) in P. falciparum. Results Close to 1000 RNA-binding proteins are identified by hidden Markov model searches, of which mRBPs encompass a relatively large proportion of the parasite proteome as compared to other eukaryotes. Several abundant mRNA-binding domains are enriched in apicomplexan parasites, while strong depletion of mRNA-binding domains involved in RNA degradation is observed. Next, we experimentally capture 199 proteins that interact with mRNA during the blood stages, 64 of which with high confidence. These captured mRBPs show a significant overlap with the in silico identified candidate RBPs (p

International audience; Almost a decade after the publication of the complete sequence of the genome of the human malaria parasite Plasmodium falciparum, the mechanisms involved in gene regulation remain poorly understood. Like other eukaryotic organisms, P. falciparum's genomic DNA organizes into nucleosomes. Nucleosomes are the basic structural units of eukaryotic chromatin and their regulation is known to play a key role in regulation of gene expression. Despite its importance, the relationship between nucleosome positioning and gene regulation in the malaria parasite has only been investigated recently. Using two independent and complementary techniques followed by next-generation high-throughput sequencing, our laboratory recently generated a dynamic atlas of nucleosome-bound and nucleosome-free regions (NFRs) at single-nucleotide resolution throughout the parasite erythrocytic cycle. We have found evidences that genome-wide changes in nucleosome occupancy play a critical role in controlling the rigorous parasite replication in infected red blood cells. However, the role of nucleosome positioning at remarkable locations such as transcriptional start sites (TSS) was not investigated. Here we show that a study of NFR in experimentally determined TSS and in silico-predicted promoters can provide deeper insights of how a transcriptionally permissive organization of chromatin can control the parasite's progression through its life cycle. We find that NFRs found at TSS and core promoters are strongly associated with high levels of gene expression in asexual erythrocytic stages, whereas nucleosome-bound TSSs and promoters are associated with silent genes preferentially expressed in sexual stages. The implications in terms of regulatory evolution, adaptation of gene expression and their impact in the design of antimalarial strategies are discussed.

Three antimalarial meroditerpenes have been isolated from two Fijian red macroalgae. The absolute stereochemistry of callophycolide A (1), a unique macrolide from Callophycus serratus, was determined using a combination of Mosher's ester analysis, circular dichroism analysis with a dimolybdenum tetraacetate complex, and conformational analysis using NOEs. In addition, two known tocopherols, β-tocopherylhydroquinone (4) and δ-tocopherylhydroquinone (5), were isolated from Amphiroa crassa. By oxidizing 5 to the corresponding δ-tocopherylquinone (6), antimalarial activity against the human malaria parasite Plasmodium falciparum was increased by more than 20-fold.

Four new bromophycolides, R–U (1–4), were isolated from the Fijian red alga Callophycus serratus and were identified by 1D and 2D NMR and mass spectroscopic analyses. These compounds expand the known structural variety of diterpene-benzoate macrolides and exhibited modest cytotoxicity toward selected human cancer cell lines. Bromophycolide S (2) also showed submicromolar activity against the human malaria parasite Plasmodium falciparum.

Bromophycolides J-Q (1-8) were isolated from extracts of the Fijian red alga Callophycus serratus and identified with 1D and 2D NMR spectroscopy and mass spectral analyses. These diterpene-benzoate macrolides represent two novel carbon skeletons and add to the ten previously reported bromophycolides (9-18) from this alga. Among these 18 bromophycolides, several exhibited activities in the low micromolar range against the human malaria parasite Plasmodium falciparum.

Phytochemical analysis of Fijian populations of the green alga Tydemania expeditionis led to the isolation of two unsaturated fatty acids, 3(zeta)-hydroxy-octadeca-4(E),6(Z),15(Z)-trienoic acid (1) and 3(zeta)-hydroxy-hexadeca-4(E),6(Z)-dienoic acid (2), along with the known 3(zeta)-hydroxy-octadeca-4(E),6(Z)-dienoic acid (4). Investigations of the red alga Hydrolithon reinboldii led to identification of a glycolipid, lithonoside (3), and five known compounds, 15-tricosenoic acid, hexacosa-5,9-dienoic methyl ester, beta-sitosterol, 10(S)-hydroxypheophytin A, and 10(R)-hydroxypheophytin A. The structures of 1-3 were elucidated by spectroscopic methods (1D and 2D NMR spectroscopy and ESI-MS). Compounds 1, 2, and 4, containing conjugated double bonds, demonstrated moderate inhibitory activity against a panel of tumor cell lines (including breast, colon, lung, prostate and ovarian cells) with IC(50) values ranging from 1.3 to 14.4 microM. The similar cell selectivity patterns of these three compounds suggest that they might act by a common, but unknown, mechanism of action.

The marine natural product (−)-8,15-diisocyano-11(20)-amphilectene (1), isolated from the Caribbean sponge Svenzea flava, was used as scaffold to synthetize five new products, all of which were tested against laboratory strains of Plasmodium falciparum and Mycobacterium tuberculosis H37Rv. The scaffold contains two isocyanide units that are amenable to chemical manipulation, enabling them to be elaborated into a small library of sulfur and selenium compounds. Although most of the analogs prepared were less potent than the parent compound, 5 was nearly equipotent showing IC50 values of 0.0066 µM and 0.0025 µM, respectively, against two strains (Dd2 and 3D7) of the malaria parasite. On the other hand, when assayed against the tuberculosis bacterium, analogs 5 and 6 were found to be more potent than 1.

International audience; A major obstacle in understanding the complex biology of the malaria parasite remains to discover how gene transcription is controlled during its life cycle. Accumulating evidence indicates that the parasite’s epigenetic state plays a fundamental role in gene expression and virulence. Using a comprehensive and quantitative mass spectrometry approach, we determined the global and dynamic abundance of histones and their covalent post-transcriptional modifications throughout the intraerythrocytic developmental cycle of Plasmodium falciparum. We detected a total of 232 distinct modifications, of which 160 had never been detected in Plasmodium and 88 had never been identified in any other species. We further validated over 10% of the detected modifications and their expression patterns by multiple reaction monitoring assays. In addition, we uncovered an unusual chromatin organization with parasite-specific histone modifications and combinatorial dynamics that may be directly related to transcriptional activity, DNA replication, and cell cycle progression. Overall, our data suggest that the malaria parasite has a unique histone modification signature that correlates with parasite virulence.

Control of gene expression is poorly understood in the Plasmodium system, where relatively few homologues to known eukaryotic transcription factors have been uncovered. Recent evidence suggests that the parasite may utilize a combinatorial mode of gene regulation, with multiple cis-acting sequences contributing to overall activity at individual promoters [1]. To further probe this mechanism of control, we first searched for over-represented sequence motifs among gene clusters sharing similar expression profiles in Plasmodium falciparum. More specifically, we applied bioinformatic tools to a previously characterized micro-array data set from drug-treated asexual stage cultures (Gunasekera et al., submitted). Cluster analysis of 600 drug responsive genes identified only a single 5' motif, GAGAGAA. Two additional 5' motifs, ACTATAAAGA and TGCAC, were also shared among loci displaying patterns of coordinate expression across varying asexual growth stages. Secondly and most importantly, the functional relevance of each motif was tested in two independent assays-transient transfection and gel-retardation experiments. The GAGAGAA and TGCAC motifs were both active in the former. The GAGAGAA and ACTATAAAGA elements formed specific RNA-protein, but not DNA-protein complexes in gel shift assays, suggesting a key level of control at the RNA level. This is the first report of functionally characterized motifs in P. falciparum that were uncovered following clustering analysis of its asexual stage transcriptome. Together, both the bioinformatic and functional data reported here imply that multiple forms of gene regulation, including post-transcriptional control, may be important in the malarial system.

Background Plasmodium falciparum, the deadliest malaria-causing parasite, has an extremely AT-rich (80.7 %) genome. Because of high AT-content, sequence-based annotation of genes and functional elements remains challenging. In order to better understand the regulatory network controlling gene expression in the parasite, a more complete genome annotation as well as analysis tools adapted for AT-rich genomes are needed. Recent studies on genome-wide nucleosome positioning in eukaryotes have shown that nucleosome landscapes exhibit regular characteristic patterns at the 5’- and 3’-end of protein and non-protein coding genes. In addition, nucleosome depleted regions can be found near transcription start sites. These unique nucleosome landscape patterns may be exploited for the identification of novel genes. In this paper, we propose a computational approach to discover novel putative genes based exclusively on nucleosome positioning data in the AT-rich genome of P. falciparum. Results Using binary classifiers trained on nucleosome landscapes at the gene boundaries from two independent nucleosome positioning data sets, we were able to detect a total of 231 regions containing putative genes in the genome of Plasmodium falciparum, of which 67 highly confident genes were found in both data sets. Eighty-eight of these 231 newly predicted genes exhibited transcription signal in RNA-Seq data, indicative of active transcription. In addition, 20 out of 21 selected gene candidates were further validated by RT-PCR, and 28 out of the 231 genes showed significant matches using BLASTN against an expressed sequence tag (EST) database. Furthermore, 108 (47 %) out of the 231 putative novel genes overlapped with previously identified but unannotated long non-coding RNAs. Collectively, these results provide experimental validation for 163 predicted genes (70.6 %). Finally, 73 out of 231 genes were found to be potentially translated based on their signal in polysome-associated RNA-Seq representing transcripts that are actively being translated. Conclusion Our results clearly indicate that nucleosome positioning data contains sufficient information for novel gene discovery. As distinct nucleosome landscapes around genes are found in many other eukaryotic organisms, this methodology could be used to characterize the transcriptome of any organism, especially when coupled with other DNA-based gene finding and experimental methods (e.g., RNA-Seq). Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2214-9) contains supplementary material, which is available to authorized users.

During the asexual replication cycle of the malaria parasite Plasmodium falciparum, the RNA-binding protein PfAlba1 binds and stabilizes a subset of transcripts for translation at a later time point.Please see related Research article: http://www.genomebiology.com/2015/16/1/212.

The sexual stages of malarial parasites are essential for the mosquito transmission of the disease and therefore are the focus of transmissionblocking drug and vaccine development. In order to better understand genes important to the sexual development process, the transcriptomes of high-purity stage I–V Plasmodium falciparumgametocytes were comprehensively profiled using a full-genome high-density oligonucleotide microarray. The interpretation of this transcriptional data was aided by applying a novel knowledge-based data-mining algorithm termed ontology-based pattern identification (OPI) using current information regarding known sexual stage genes as a guide. This analysis resulted in the identification of a sexual development cluster containing 246 genes, of which ∼75% were hypothetical, exhibiting highly-correlated, gametocyte-specific expression patterns. Inspection of the upstream promoter regions of these 246 genes revealed putative cis-regulatory elements for sexual development transcriptional control mechanisms. Furthermore, OPI analysis was extended using current annotations provided by the Gene Ontology Consortium to identify 380 statistically significant clusters containing genes with expression patterns characteristic of various biological processes, cellular components, and molecular functions. Collectively, these results, available as part of a web-accessible OPI database (http://carrier.gnf.org/publications/Gametocyte ), shed light on the components of molecular mechanisms underlying parasite sexual development and other areas of malarial parasite biology. © 2005 Elsevier B.V. All rights reserved.

Infections with the human parasite Plasmodium falciparum continue to present a great challenge to global health. Fundamental questions regarding the molecular basis of virulence and immune evasion in P. falciparum have been only partially answered. Because of the parasite's intracellular location and complex life cycle, standard genetic approaches to the study of the pathogenesis of malaria have been limited. The present study presents a novel approach to the identification of the biological processes involved in host-pathogen interactions, one that is based on the analysis of in vivo P. falciparum transcripts. We demonstrate that a sufficient quantity of P. falciparum RNA transcripts can be derived from a small blood sample from infected patients for whole-genome microarray analysis. Overall, excellent correlation was observed between the transcriptomes derived from in vivo samples and in vitro samples with ring-stage P. falciparum 3D7 reference strain. However, gene families that encode surface proteins are overexpressed in vivo. Moreover, this analysis has identified a new family of hypothetical genes that may encode surface variant antigens. Comparative studies of the transcriptomes derived from in vivo samples and in vitro 3D7 samples may identify important strategies used by the pathogen for survival in the human host and highlight, for vaccine development, new candidate antigens that were not previously identified through the use of in vitro cultures.

To investigate the role of post-transcriptional controls in the regulation of protein expression for the malaria parasite, Plasmodium falciparum, we have compared mRNA transcript and protein abundance levels for seven different stages of the parasite life cycle. A moderately high positive relationship between mRNA and protein abundance was observed for these stages; the most common discrepancy was a delay between mRNA and protein accumulation. Potentially post-transcriptionally regulated genes are identified, and families of functionally related genes were observed to share similar patterns of mRNA and protein accumulation.

Upon invasion of the erythrocyte cell, the malaria parasite remodels its environment; in particular, it establishes a complex membrane network, which connects the parasitophorous vacuole to the host plasma membrane and is involved in protein transport and trafficking. We have identified a novel subtelomeric gene family in Plasmodium falciparum that encodes 11 transmembrane proteins localized to the Maurer's clefts. Using coimmunoprecipitation and shotgun proteomics, we were able to enrich specifically for these proteins and detect distinct peptides, allowing us to conclude that four to 10 products were present at a given time. Nearly all of the Pfmc-2tm genes are transcribed during the trophozoite stage; this narrow time frame of transcription overlaps with the specific stevor and rif genes that are differentially expressed during the erythrocyte cycle. The description of the structural properties of the proteins led us to manually reannotate published sequences, and to detect potentially homologous gene families in both P. falciparum and Plasmodium yoelii yoelii, where no orthologs were predicted uniquely based on sequence similarity. These basic proteins with two transmembrane domains belong to a larger superfamily, which includes STEVORs and RIFINs.

Metrodorea stipularis stem extracts were studied in the search for possible antichagastic, antimalarial, and antitumoral compounds using cruzain from Trypanosoma cruzi, Plasmodium falciparum, and cathepsins B and L, as molecular targets, respectively. Dihydrochalcones 1, 2, 3, and 4 showed significant inhibitory activity against all the targets. Compounds 1–4 displayed IC(50) values ranging from 7.7 to 21.6 μM against cruzain; dihydrochalcones 2 and 4 inhibited the growth of three different strains of P. falciparum in low micromolar concentrations; and against cathepsins B and L these compounds presented good inhibitory activity with IC(50) values ranging from 1.0 to 14.9 μM. The dihydrochalcones showed good selectivity in their inhibitory activities against the cysteine proteases.

The development of the human malaria parasite Plasmodium falciparum is controlled by coordinated changes in gene expression throughout its complex life cycle, but the corresponding regulatory mechanisms are incompletely understood. To study the relationship between genome architecture and gene regulation in Plasmodium, we assayed the genome architecture of P. falciparum at three time points during its erythrocytic (asexual) cycle. Using chromosome conformation capture coupled with next-generation sequencing technology (Hi-C), we obtained high-resolution chromosomal contact maps, which we then used to construct a consensus three-dimensional genome structure for each time point. We observed strong clustering of centromeres, telomeres, ribosomal DNA, and virulence genes, resulting in a complex architecture that cannot be explained by a simple volume exclusion model. Internal virulence gene clusters exhibit domain-like structures in contact maps, suggesting that they play an important role in the genome architecture. Midway during the erythrocytic cycle, at the highly transcriptionally active trophozoite stage, the genome adopts a more open chromatin structure with increased chromosomal intermingling. In addition, we observed reduced expression of genes located in spatial proximity to the repressive subtelomeric center, and colocalization of distinct groups of parasite-specific genes with coordinated expression profiles. Overall, our results are indicative of a strong association between the P. falciparum spatial genome organization and gene expression. Understanding the molecular processes involved in genome conformation dynamics could contribute to the discovery of novel antimalarial strategies.

Background In eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite. Results Here, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite’s erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes. Conclusions The connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-347) contains supplementary material, which is available to authorized users.

This chapter explains how such data, when combined, can give a comprehensive overview of the transcriptome of the malaria parasite throughout its life cycle. These data are expected to lead to the identification of key Plasmodium-specific regulatory targets, a fundamental step for the development of new antimalarials. Gene expression throughout the cell cycle is usually regulated by complex interactions between DNA regulatory motifs and transcription factors that bind to these motifs, as well as regulation by distinct chromatin structures. Parasite isolates possess a high degree of diversity within their var genes because of duplications, deletions, or recombination events that provide the parasite with a huge repertoire of antigenic determinants. Before the complete release of the Plasmodium falciparum genome sequence, several genomic efforts were accomplished to investigate the transcriptome of the malaria parasite. The first large-scale expression study of P. falciparum was accomplished with a DNA microarray platform constructed using a nuclease-generated genomic library. With the completion of the P. falciparum genome, two transcriptional analyses covering the entire genome were published. Both techniques allow effective and complete genome design. The 70-mer oligonucleotide array study was confined to a high-resolution analysis of the erythrocytic cell cycle. Gene expression in parasitic protozoa has shown itself to be unique when it comes to mechanisms of regulation. For instance, Leishmania and other members of the Trypanosomatidae have polycistronic transcription with maturation of their mRNA by transplicing events.

The regulatory mechanisms of most cyclin dependent protein kinases (CDKs) are well understood and are highly conserved in eukaryotes. CDKs from the malaria parasite, Plasmodium falciparum, appear to be regulated in a similar manner with regard to cyclin binding and phosphorylation. In order to further understand their regulatory mechanisms, we examined two classes of cyclin dependent kinase inhibitors (CDIs) to inhibit a panel of plasmodial CDKs. We find that Pfmrk and PfPK5 are inhibited by heterologous p21(CIP1) with varying degrees of inhibition. In contrast, PfPK6, a kinase with sequence features characteristic of both a CDK and MAP kinase, is unaffected by this CDI. Furthermore, the CDK4/6 specific CDI, p16(INK4), fails to inhibit these plasmodial CDKs. Taken together, these results suggest that plasmodial CDKs may be regulated by the binding of inhibitory proteins in vivo.

Several Plasmodium falciparum genes encoding cdc2-related protein kinases have been identified, but the modalities of their regulation remains largely unexplored. In the present study, we investigated the regulation in vitro of PfPK5, a putative homologue of Cdk1 (cdc2) in P. falciparum. We show that (i) PfPK5 is efficiently activated by heterologous (human) cyclin H and p25, a cyclin-like molecule that specifically activates human Cdk5; (ii) the activated enzyme can be inhibited by chemical Cdk inhibitors; (iii) Pfmrk, a putative P. falciparum homologue of the Cdk-activating kinase, does neither activate nor phosphorylate PfPK5; and (iv) PfPK5 is able to autophosphorylate in the presence of a cyclin. Taken together, these results suggest that the regulation of Plasmodium Cdks may differ in important aspects from that of their human counterparts. Furthermore, we cloned an open reading frame encoding a novel P. falciparum protein possessing maximal homology to cyclin H from various organisms, and we show that this protein, called Pfcyc-1, is able to activate recombinant PfPK5 in vitro with an efficiency similar to that of human cyclin H and p25. This work opens the way to the development of screening procedures aimed at identifying compounds that specifically target the parasite Cdks.

International audience; Cytosine DNA methylation is an epigenetic mark in most eukaryotic cells that regulates numerous processes, including gene expression and stress responses. We performed a genome-wide analysis of DNA methylation in the human malaria parasite Plasmodium falciparum. We mapped the positions of methylated cytosines and identified a single functional DNA methyltransferase (Plasmodium falciparum DNA methyltransferase; PfDNMT) that may mediate these genomic modifications. These analyses revealed that the malaria genome is asymmetrically methylated and shares common features with undifferentiated plant andmammalian cells. Notably, core promoters are hypomethylated, and transcript levels correlate with intraexonic methylation. Additionally, there are sharp methylation transitions at nucleosome and exon-intron boundaries. These data suggest that DNA methylation could regulate virulence gene expression and transcription elongation. Furthermore, the broad range of action of DNA methylation and the uniqueness of PfDNMT suggest that the methylation pathway is a potential target for antimalarial strategies.

International audience; Despite continuous prevention and treatment efforts, malaria remains the deadliest infectious disease in the world. Nonetheless, hope for the future was recently brought by the rapid development of genomics, whose applications have immense potential to create powerful tools to fight malaria and its causing parasite. The affordability of these tools renders possible their use in every part of the world, including developing countries where malaria is endemic. The march toward optimized treatments against malaria has started.