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Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis. We thank R. Gao for her contribution to the initial eforts to sequence the B. duncani genome. C.B.M.’s research was supported by grants from the National Institutes of Health (AI097218, GM110506, AI123321 and R43AI136118), the Steven and Alexandra Cohen Foundation (Lyme 62 2020), and the Global Lyme Alliance. S.L.’s research was supported by grants by the US National Science Foundation (IIS 1814359) and the National Institutes of Health (1R01AI169543-01). K.G.L.R.’s research was supported by the National Institutes of Allergy and Infectious Diseases (R01 AI136511, R01 AI142743-01 and R21 AI142506-01), the University of California, Riverside (NIFA-Hatch-225935) and the Health Institute Carlos III (PI20CIII/00037). Sí

We describe a total synthesis of the rare isocyanoterpene natural product isoneoamphilectane and two of its unnatural diastereomers. The significantly strained ring system of the reported natural product─along with a hypothesis about a biosynthetic relationship to related family members─inspired us to consider a potential misassignment in the structure's relative configuration. As a result, we initially targeted two less strained, more accessible, stereoisomers of the reported natural product. When these compounds failed to exhibit spectroscopic data that matched those of isoneoamphilectane, we embarked on a synthesis of the originally proposed strained structure via an approach that hinged on a challenging cis-to-trans decalone epimerization. Ultimately, we implemented a novel cyclic sulfite pinacol-type rearrangement to generate the strained ring system. Additional features of this work include the application of a stereocontrolled Mukaiyama-Michael addition of an acyclic silylketene acetal, an unusual intramolecular alkoxide-mediated regioselective elimination, and an HAT-mediated alkene hydroazidation to forge the C-N bond of the tertiary isonitrile. Throughout this work, our synthetic planning was heavily guided by computational analyses to inform on key issues of stereochemical control.

Malaria pathogenicity results from the parasite’s ability to invade, multiply within and then egress from the host red blood cell (RBC). Infected RBCs are remodeled, expressing antigenic variant proteins (such as PfEMP1, coded by thevargene family) for immune evasion and survival. These processes require the concerted actions of many proteins, but the molecular regulation is poorly understood. We have characterized an essentialPlasmodiumspecific Apicomplexan AP2 (ApiAP2) transcription factor inPlasmodium falciparum(PfAP2-MRP; Master Regulator of Pathogenesis) during the intraerythrocytic developmental cycle (IDC). An inducible gene knockout approach showed that PfAP2-MRP is essential for development during the trophozoite stage, and critical forvargene regulation, merozoite development and parasite egress. ChIP-seq experiments performed at 16 hour post invasion (h.p.i.) and 40 h.p.i. matching the two peaks of PfAP2-MRP expression, demonstrate binding of PfAP2-MRP to the promoters of genes controlling trophozoite development and host cell remodeling at 16 h.p.i. and antigenic variation and pathogenicity at 40 h.p.i. Using single-cell RNA-seq and fluorescence-activated cell sorting, we show de-repression of mostvargenes inΔpfap2-mrpparasites that express multiple PfEMP1 proteins on the surface of infected RBCs. In addition, theΔpfap2-mrpparasites overexpress several early gametocyte marker genes at both 16 and 40 h.p.i., indicating a regulatory role in the sexual stage conversion. Using the Chromosomes Conformation Capture experiment (Hi-C), we demonstrate that deletion of PfAP2-MRP results in significant reduction of both intra-chromosomal and inter-chromosomal interactions in heterochromatin clusters. We conclude that PfAP2-MRP is a vital upstream transcriptional regulator controlling essential processes in two distinct developmental stages during the IDC that include parasite growth, chromatin structure andvargene expression.

Pathogenic protists are a group of organisms responsible for causing a variety of human diseases including malaria, sleeping sickness, Chagas disease, leishmaniasis, and toxoplasmosis, among others. These diseases, which affect more than one billion people globally, mainly the poorest populations, are characterized by severe chronic stages and the lack of effective antiparasitic treatment. Parasitic protists display complex life-cycles and go through different cellular transformations in order to adapt to the different hosts they live in. Autophagy, a highly conserved cellular degradation process, has emerged as a key mechanism required for these differentiation processes, as well as other functions that are crucial to parasite fitness. In contrast to yeasts and mammals, protist autophagy is characterized by a modest number of conserved autophagy-related proteins (ATGs) that, even though, can drive the autophagosome formation and degradation. In addition, during their intracellular cycle, the interaction of these pathogens with the host autophagy system plays a crucial role resulting in a beneficial or harmful effect that is important for the outcome of the infection. In this review, we summarize the current state of knowledge on autophagy and other related mechanisms in pathogenic protists and their hosts. We sought to emphasize when, how, and why this process takes place, and the effects it may have on the parasitic cycle. A better understanding of the significance of autophagy for the protist life-cycle will potentially be helpful to design novel anti-parasitic strategies.

Mechanisms of cell division are remarkably diverse, suggesting the underlying molecular networks among eukaryotes differ extensively. The Aurora family of kinases orchestrates the process of chromosome segregation and cytokinesis during cell division through precise spatiotemporal regulation of their catalytic activities by distinct scaffolds.Plasmodiumspp., the causative agents of malaria, are unicellular eukaryotes that have three divergent aurora-related kinases (ARKs) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. This includes a rapid threefold genome replication from 1N to 8N with successive cycles of closed mitosis, spindle formation and chromosome segregation within eight minutes (termed male gametogony). Kinome studies had previously suggested likely essential functions for all threePlasmodiumARKs during asexual mitotic cycles; however, little is known about their location, function, or their scaffolding molecules during unconventional sexual proliferative stages. Using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging, we set out to investigate the role of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria modelPlasmodium berghei. We find that ARK2 primarily localises to the spindle apparatus in the vicinity of kinetochores during both mitosis and meiosis. Interactomics and co-localisation studies reveal a unique ARK2 scaffold at the spindle including the microtubule plus end-binding protein EB1, lacking conserved Aurora scaffold proteins. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora kinase spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasitePlasmodium.

The complex life cycle ofPlasmodium falciparumrequires coordinated gene expression regulation to allow host cell invasion, transmission, and immune evasion. However, this cascade of transcripts is unlikely to be regulated by the limited number of identified parasite-specific transcription factors. Increasing evidence now suggests a major role for epigenetic mechanisms in gene expression in the parasite. In eukaryotes, many lncRNAs have been identified and shown to be pivotal regulators of genome structure and gene expression. To investigate the regulatory roles of lncRNAs inP. falciparumwe explored the intergenic lncRNA distribution in nuclear and cytoplasmic subcellular locations. Using nascent RNA expression profiles, we identified a total of 1,768 lncRNAs, of which 58% were identified as novel lncRNAs inP. falciparum. The subcellular localization and stage-specific expression of several putative lncRNAs were validated using RNA fluorescence in situ hybridization (RNA-FISH). Additionally, the genome-wide occupancy of several candidate nuclear lncRNAs was explored using Chromatin Isolation by RNA Purification (ChIRP). ChIRP-seq of candidate lncRNAs revealed that lncRNA occupancy sites within the parasite genome are focal and sequence-specific with a particular enrichment for several parasite-specific gene families, including those involved in pathogenesis, erythrocyte remodeling, and regulation of sexual differentiation. We further validated the function of one specific lncRNA (lncRNA-ch14) using the CRISPR-Cas9 genome editing tool. Genomic and phenotypic analysis of the △lncRNA-ch14 line demonstrated the importance of this lncRNA in sexual differentiation and sexual reproduction. Our findings bring a new level of insight into the role of lncRNAs in pathogenicity, gene regulation and sexual differentiation. These findings also open new avenues for targeted approaches towards therapeutic strategies against the deadly malaria parasite.

Babesia species are tick-transmitted apicomplexan pathogens and the causative agents of babesiosis, a malaria-like disease of major medical and veterinary importance. Of the different species of Babesia reported so far, Babesia duncani causes severe to lethal infection in patients. Despite the highly virulent nature of this parasite and the risk it may pose as an emerging pathogen, little is known about its biology, metabolic requirements, and pathogenesis. B. duncani is unique among apicomplexan parasites that infect red blood cells in that it can be continuously cultured in vitro in human erythrocytes but can also infect mice leading to fulminant babesiosis infection and death. Here we have taken advantage of the recent advances in the propagation of this parasite in vitro and in vivo to conduct detailed molecular, genomic and transcriptomic analyses and to gain insights into its biology. We report the assembly, 3D structure, and annotation of the nuclear genome of this parasite as well as its transcriptomic profile and an atlas of its metabolism during its intraerythrocytic life cycle. Detailed examination of the B. duncani genome and comparative genomic analyses identified new classes of candidate virulence factors, suitable antigens for diagnosis of active infection, and several attractive drug targets. Translational analyses and efficacy studies identified highly potent inhibitors of B. duncani thus enriching the pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.

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.

Summary Condensin is a multi-subunit protein complex regulating chromosome condensation and segregation during cell division. In Plasmodium spp., the causative agent of malaria, cell division is atypical and the role of condensin is unclear. Here we examine the role of SMC2 and SMC4, the core subunits of condensin, during endomitosis in schizogony and endoreduplication in male gametogenesis. During early schizogony, SMC2/SMC4 localize to a distinct focus, identified as the centromeres by NDC80 fluorescence and chromatin immunoprecipitation sequencing (ChIP-seq) analyses, but do not form condensin I or II complexes. In mature schizonts and during male gametogenesis, there is a diffuse SMC2/SMC4 distribution on chromosomes and in the nucleus, and both condensin I and condensin II complexes form at these stages. Knockdown of smc2 and smc4 gene expression reveals essential roles in parasite proliferation and transmission. The condensin core subunits (SMC2/SMC4) form different complexes and may have distinct functions at various stages of the parasite life cycle., Graphical Abstract, Highlights • SMC2/SMC4 and condensin complex I and II proteins are present in Plasmodium • SMC2/SMC4GFP has discrete centromeric localization in nuclei during early schizogony • SMC2/SMC4GFP are dispersed in the nucleus in male gametogenesis and late schizogony • SMC2/SMC4 gene knockdown (KD) impairs oocyst development and parasite transmission, Pandey et al. use live cell imaging, proteomics, and genetic approaches to show that Plasmodium condensin core subunits (SMC2/SMC4) have differential cellular location and condensin complex formation during parasite development and have essential roles in parasite proliferation and transmission.

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.

COVID-19 is a devastating respiratory and inflammatory illness caused by a new coronavirus that is rapidly spreading throughout the human population. Over the past 12 months, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has already infected over 160 million (&gt, 20% located in United States) and killed more than 3.3 million people around the world (&gt, 20% deaths in USA). As we face one of the most challenging times in our recent history, there is an urgent need to identify drug candidates that can attack SARS-CoV-2 on multiple fronts. We have therefore initiated a computational dynamics drug pipeline using molecular modeling, structure simulation, docking and machine learning models to predict the inhibitory activity of several million compounds against two essential SARS-CoV-2 viral proteins and their host protein interactors—S/Ace2, Tmprss2, Cathepsins L and K, and Mpro—to prevent binding, membrane fusion and replication of the virus, respectively. All together, we generated an ensemble of structural conformations that increase high-quality docking outcomes to screen over &gt, 6 million compounds including all FDA-approved drugs, drugs under clinical trial (&gt, 3000) and an additional &gt, 30 million selected chemotypes from fragment libraries. Our results yielded an initial set of 350 high-value compounds from both new and FDA-approved compounds that can now be tested experimentally in appropriate biological model systems. We anticipate that our results will initiate screening campaigns and accelerate the discovery of COVID-19 treatments.

The flagship member of the antiplasmodial isocyanoterpenes, 7,20-diisocyanoadociane (DICA), was synthesized from dehydrocryptone in 10 steps, and in 13 steps from commercially available material. Our previous formal synthesis was reengineered, leveraging only productive transformations to deliver DICA in fewer than half the number of steps of our original effort. Important contributions, in addition to the particularly concise strategy, include a solution to the problem of axial nucleophilic methylation of a late-stage cyclohexanone, and the first selective synthesis and antiplasmodial evaluation of the DICA stereoisomer with both isonitriles equatorial.

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.

The NLR-receptor RPP7 mediates race-specific immunity in Arabidopsis. Previous screens for enhanced downy mildew (edm) mutants identified the co-chaperone SGT1b (EDM1) and the PHD-finger protein EDM2 as critical regulators of RPP7. Here, we describe a third edm mutant compromised in RPP7 immunity, edm3. EDM3 encodes a nuclear-localized protein featuring an RNA-recognition motif. Like EDM2, EDM3 promotes histone H3 lysine 9 dimethylation (H3K9me2) at RPP7. Global profiling of H3K9me2 showed EDM3 to affect this silencing mark at a large set of loci. Importantly, both EDM3 and EDM2 co-associate in vivo with H3K9me2-marked chromatin and transcripts at a critical proximal polyadenylation site of RPP7, where they suppress proximal transcript polyadeylation/termination. Our results highlight the complexity of plant NLR gene regulation, and establish a functional and physical link between a histone mark and NLR-transcript processing.

The RNA binding domain abundant in apicomplexans (RAP) is a protein domain identified in a diverse group of proteins, called RAP proteins, many of which have been shown to be involved in RNA binding. To understand the expansion and potential function of the RAP proteins, we conducted a hidden Markov model based screen among the proteomes of 54 eukaryotes, 17 bacteria and 12 archaea. We demonstrated that the domain is present in closely and distantly related organisms with particular expansions in Alveolata and Chlorophyta, and are not unique to Apicomplexa as previously believed. All RAP proteins identified can be decomposed into two parts. In the N-terminal region, the presence of variable helical repeats seems to participate in the specific targeting of diverse RNAs, while the RAP domain is mostly identified in the C-terminal region and is highly conserved across the different phylogenetic groups studied. Several conserved residues defining the signature motif could be crucial to ensure the function(s) of the RAP proteins. Modelling of RAP domains in apicomplexan parasites confirmed an ⍺/β structure of a restriction endonuclease-like fold. The phylogenetic trees generated from multiple alignment of RAP domains and full-length proteins from various distantly related eukaryotes indicated a complex evolutionary history of this family. We further discuss these results to assess the potential function of this protein family in apicomplexan parasites.

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.

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.

We are presenting our on going studies with inhibitory research on Tmprss2, S-protein:Ace2, and 3CLpro using compound screening coupled with X-ray crystallography, molecular modeling, live virus screening using host human cells (BSL3 facility at UC Center for Infectious Disease and Vector Research, and organ-on-a-chip at Harvard Medical School for safety profiling before proceeding to animal models with InVivo BioSystems for ADMET.We have derived a useful chemical toolkit of 350 compounds for the community to study with biochemical assays and other biophysical-chemical studies that will prove useful in searching for optimal inhibitors of these targets to find suitable pharmacophores for blocking each of these enzyme's activities -- that would be beneficial for human health. Our past successes with these methodologies have resulted in over 28 patents, 11 technologies and two startup companies.

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.

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.

Toxoplasma gondii is an obligate intracellular parasite that has a significant impact on human health, especially in the immunocompromised. This parasite is also a useful genetic model for intracellular parasitism given its ease of culture in the laboratory and relevant animal models. However, as for many other eukaryotes, the T. gondii genome is incomplete, containing hundreds of sequence gaps due to the presence of repetitive and/or uncloneable sequences that prevent complete telomere-to-telomere de novo chromosome assembly. Here, we report the first use of single molecule DNA sequencing to generate near complete de novo genome assemblies for T. gondii and its near relative, N. caninum. Using the Oxford Nanopore Minion platform, we dramatically improved the contiguity of the T. gondii genome (N50 of ∼6.6Mb) and increased overall assembled sequence compared to current reference sequences by ∼2 Mb. Multiple complete chromosomes were fully assembled as evidenced by clear telomeric repeats on the end of each contig. Interestingly, for all of the Toxoplasma gondii strains that we sequenced (RH, CTG, II×III F1 progeny clones CL13, S27, S21, and S26), the largest contig ranged in size between 11.9 and 12.1 Mb in size, which is larger than any previously reported T. gondii chromosome. This was due to a repeatable and consistent fusion of chromosomes VIIb and VIII. These data were further validated by mapping existing T. gondii ME49 Hi-C data to our assembly, providing parallel lines of evidence that the T. gondii karyotype consists of 13, rather than 14, chromosomes. In addition revising the molecular karyotype we were also able to resolve hundreds of repeats derived from both coding and non-coding tandem sequence expansions. For well-known host-targeting effector loci like rhoptry protein 5 (ROP5) and ROP38, we were also able to accurately determine the precise gene count, order and orientation using established assembly approaches and the most likely primary sequence of each using our own assembly correction scripts tailored to correcting homopolymeric run errors in tandem sequence arrays. Finally, when we compared the T. gondii and N. caninum assemblies we found that while the 13 chromosome karyotype was conserved, we determined that previously unidentified large scale translocation events occurred in T. gondii and N. caninum since their most recent common ancestry.

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.

Eukaryotic cell proliferation requires chromosome replication and precise segregation to ensure daughter cells have identical genomic copies. The genus Plasmodium, the causative agent of malaria, displays remarkable aspects of nuclear division throughout its lifecycle to meet some peculiar and unique challenges of DNA replication and chromosome segregation. The parasite undergoes atypical endomitosis and endoreduplication with an intact nuclear membrane and intranuclear mitotic spindle. To understand these diverse modes of Plasmodium cell division, we have studied the behaviour and composition of the outer kinetochore NDC80 complex, a key part of the mitotic apparatus that attaches the centromere of chromosomes to microtubules of the mitotic spindle. Using NDC80-GFP live-cell imaging in Plasmodium berghei we observe dynamic spatiotemporal changes during proliferation, including highly unusual kinetochore arrangements during sexual stages. We identify a very divergent candidate for the SPC24 subunit of the NDC80 complex, previously thought to be missing in Plasmodium, which completes a canonical, albeit unusual, NDC80 complex structure. Altogether, our studies reveal the kinetochore as an ideal tool to investigate the non-canonical modes of chromosome segregation and cell division in Plasmodium.

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.