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Abstract Programmed-cell death is an antimicrobial defense mechanism that promotes clearance of intracellular pathogens. Toxoplasma counteracts host immune defenses by secreting effector proteins into host cells; however, how the parasite evades lytic cell death and the effectors involved remain poorly characterized. We identified ROP55, a rhoptry protein that promotes parasite survival by preventing lytic cell death in absence of IFN-γ stimulation. RNA-Seq analysis revealed that ROP55 acts as a repressor of host pro-inflammatory responses. In THP-1 monocytes ΔROP55 infection increased NF-κB p65 nuclear translocation, IL-1β production, and GSDMD cleavage compared to wild type or complemented parasites. ΔROP55 infection also induced RIPK3-dependent necroptosis in human and mouse primary macrophages. Moreover, ΔROP55 parasites were significantly impaired in virulence in female mice and prevented NF-κB activation and parasite clearance in mBMDM. These findings place ROP55 as a major virulence factor, dampening lytic cell death and enabling Toxoplasma to evade clearance from infected cells.

The environmental challenges the human malaria parasite, Plasmodium falciparum, faces during its progression into its various lifecycle stages warrant the use of effective and highly regulated access to chromatin for transcriptional regulation. Microrchidia (MORC) proteins have been implicated in DNA compaction and gene silencing across plant and animal kingdoms. Accumulating evidence has shed light on the role MORC protein plays as a transcriptional switch in apicomplexan parasites. In this study, using the CRISPR/Cas9 genome editing tool along with complementary molecular and genomics approaches, we demonstrate that PfMORC not only modulates chromatin structure and heterochromatin formation throughout the parasite erythrocytic cycle, but is also essential to the parasite survival. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments suggests that PfMORC binds to not only sub-telomeric regions and genes involved in antigenic variation but may also play a role in modulating stage transition. Protein knockdown experiments followed by chromatin conformation capture (Hi-C) studies indicate that downregulation of PfMORC impairs key histone marks and induces the collapse of the parasite heterochromatin structure leading to its death. All together these findings confirm that PfMORC plays a crucial role in chromatin structure and gene regulation, validating this factor as a strong candidate for novel antimalarial strategies.

Babesiosis, caused by protozoan parasites of the genus Babesia, is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of new Babesia species underscores the ongoing risk of zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental changes. One such pathogen, Babesia MO1, previously implicated in severe cases of human babesiosis in the United States, was initially considered a subspecies of B. divergens, the predominant agent of human babesiosis in Europe. Here we report comparative multiomics analyses of B. divergens and B. MO1 that offer insight into their biology and evolution. Our analysis shows that despite their highly similar genomic sequences, substantial genetic and genomic divergence occurred throughout their evolution resulting in major differences in gene functions, expression and regulation, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens, and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.

Abstract Ribonucleoprotein complexes are composed of RNA, RNA-dependent proteins (RDPs) and RNA-binding proteins (RBPs), and play fundamental roles in RNA regulation. However, in the human malaria parasite, Plasmodium falciparum, identification and characterization of these proteins are particularly limited. In this study, we use an unbiased proteome-wide approach, called R-DeeP, a method based on sucrose density gradient ultracentrifugation, to identify RDPs. Quantitative analysis by mass spectrometry identifies 898 RDPs, including 545 proteins not yet associated with RNA. Results are further validated using a combination of computational and molecular approaches. Overall, this method provides the first snapshot of the Plasmodium protein-protein interaction network in the presence and absence of RNA. R-DeeP also helps to reconstruct Plasmodium multiprotein complexes based on co-segregation and deciphers their RNA-dependence. One RDP candidate, PF3D7_0823200, is functionally characterized and validated as a true RBP. Using enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq), we demonstrate that this protein interacts with various Plasmodium non-coding transcripts, including the var genes and ap2 transcription factors.

Abstract The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei, using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite.

Abstract The complex life cycle of Plasmodium falciparum requires coordinated gene expression regulation to allow host cell invasion, transmission, and immune evasion. Increasing evidence now suggests a major role for epigenetic mechanisms in gene expression in the parasite. In eukaryotes, many lncRNAs have been identified to be pivotal regulators of genome structure and gene expression. To investigate the regulatory roles of lncRNAs in P. falciparum we explore the intergenic lncRNA distribution in nuclear and cytoplasmic subcellular locations. Using nascent RNA expression profiles, we identify a total of 1768 lncRNAs, of which 718 (~41%) are novels in P. falciparum. The subcellular localization and stage-specific expression of several putative lncRNAs are validated using RNA-FISH. Additionally, the genome-wide occupancy of several candidate nuclear lncRNAs is explored using ChIRP. The results reveal that lncRNA occupancy sites are focal and sequence-specific with a particular enrichment for several parasite-specific gene families, including those involved in pathogenesis and sexual differentiation. Genomic and phenotypic analysis of one specific lncRNA demonstrate its importance in sexual differentiation and reproduction. Our findings bring a new level of insight into the role of lncRNAs in pathogenicity, gene regulation and sexual differentiation, opening new avenues for targeted therapeutic strategies against the deadly malaria parasite.

Abstract Mosquito-borne disease remains a significant burden on global health. In the United States, the major threat posed by mosquitoes is transmission of arboviruses, including West Nile virus by mosquitoes of the Culex genus. Virus metagenomic analysis of mosquito small RNA using deep sequencing and advanced bioinformatic tools enables the rapid detection of viruses and other infecting organisms, both pathogenic and non-pathogenic to humans, without any precedent knowledge. In this study, we sequenced small RNA samples from over 60 pools of Culex mosquitoes from two major areas of Southern California from 2017 to 2019 to elucidate the virome and immune responses of Culex. Our results demonstrated that small RNAs not only allowed the detection of viruses but also revealed distinct patterns of viral infection based on location, Culex species, and time. We also identified miRNAs that are most likely involved in Culex immune responses to viruses and Wolbachia bacteria, and show the utility of using small RNA to detect antiviral immune pathways including piRNAs against some pathogens. Collectively, these findings show that deep sequencing of small RNA can be used for virus discovery and surveillance. One could also conceive that such work could be accomplished in various locations across the world and over time to better understand patterns of mosquito infection and immune response to many vector-borne diseases in field samples.

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. Abbreviations: AAs: amino acids; ATGs: autophagy-related proteins; ADCD; autophagy-dependent cell death; AMPK: 5’ adenosine monophosphate-activated protein kinase; CD40: Cluster of differentiation 40; gHBSS: Hanks’ Balanced Salt Solution; GO: gene ontology; IFN-γ: IFN-gamma; LC3: mammalian microtubule-associated protein light chain 3; LAP; LC3-associated phagocytosis; LECA: last eukaryotic common ancestor; 3-MA: 3-methyladenine; MTOR; Mechanistic target of rapamycin kinase; MDC: monodansylcadaverine; NDP52: nuclear dot protein 52; PAAR: Plasmodium-Associated Autophagy-Related response; PE: phosphatidylethanolamine: PCD: programmed cell death; PND: programmed nuclear death; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; PV: parasitophorous vacuole; PVM: parasitophorous vacuole membrane; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment receptor; SQSTM1/p62: sequestosome-1; TEM: transmission electron microscopy; TNF-α: tumor necrosis factor-alpha; TVN: tubovesicular network; Ub: ubiquitin; UPS: ubiquitin-proteasome system; Vps: vacuolar protein sorting.

The function of RNA-binding domain abundant in Apicomplexans (RAP) protein family members is largely unknown. Here, using high-throughput functional genomics, including metabolomics, Hollin et al. characterize two RAP proteins that are essential for Plasmodium falciparum survival and control mitochondrial rRNAs.

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria.

The recent Coronavirus Disease 2019 pandemic has once again reminded us the importance of understanding infectious diseases. One important but understudied area in infectious disease research is the role of nuclear architecture or the physical arrangement of the genome in the nucleus in controlling gene regulation and pathogenicity. Recent advances in research methods, such as Genome-wide chromosome conformation capture using high-throughput sequencing (Hi-C), have allowed for easier analysis of nuclear architecture and chromosomal reorganization in both the infectious disease agents themselves as well as in their host cells. This review will discuss broadly on what is known about nuclear architecture in infectious disease, with an emphasis on chromosomal reorganization, and briefly discuss what steps are required next in the field.

Over the past decade, we have witnessed significant progresses in understanding gene regulation in Apicomplexa including the human malaria parasite, Plasmodium falciparum. This parasite possesses the ability to convert in multiple stages in various hosts, cell types, and environments. Recent findings indicate that P. falciparum is talented at using efficient and complementary molecular mechanisms to ensure a tight control of gene expression at each stage of its life cycle. Here, we review the current understanding on the contribution of the epigenome, atypical transcription factors, and chromatin organization to regulate stage conversion in P. falciparum. The adjustment of these regulatory mechanisms occurring during the progression of the life cycle will be extensively discussed.

Plant NLR-type receptors serve as sensitive triggers of host immunity. Their expression has to be well-balanced, due to their interference with various cellular processes and dose-dependency of their defense-inducing activity. A genetic "arms race" with fast-evolving pathogenic microbes requires plants to constantly innovate their NLR repertoires. We previously showed that insertion of the COPIA-R7 retrotransposon into RPP7 co-opted the epigenetic transposon silencing signal H3K9me2 to a new function promoting expression of this Arabidopsis thaliana NLR gene. Recruitment of the histone binding protein EDM2 to COPIA-R7-associated H3K9me2 is required for optimal expression of RPP7. By profiling of genome-wide effects of EDM2, we now uncovered additional examples illustrating effects of transposons on NLR gene expression, strongly suggesting that these mobile elements can play critical roles in the rapid evolution of plant NLR genes by providing the "raw material" for gene expression mechanisms. We further found EDM2 to have a global role in NLR expression control. Besides serving as a positive regulator of RPP7 and a small number of other NLR genes, EDM2 acts as a suppressor of a multitude of additional NLR genes. We speculate that the dual functionality of EDM2 in NLR expression control arose from the need to compensate for fitness penalties caused by high expression of some NLR genes by suppression of others. Moreover, we are providing new insights into functional relationships of EDM2 with its interaction partner, the RNA binding protein EDM3/AIPP1, and its target gene IBM1, encoding an H3K9-demethylase.

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.

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. : 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. Keywords: condensin, SMC4, atypical mitosis, Plasmodium, mosquito, transmision, chromosome, protein complex, kleisin, oocyst

Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the plus ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics in the different proliferative stages are not well understood. Genome-wide shared orthology analysis of Plasmodium spp. revealed the presence of two kinesin-8 motor proteins, kinesin-8X and kinesin-8B. Here we studied the biochemical properties of kinesin-8X and its role in parasite proliferation. In vitro, kinesin-8X has motility and depolymerization activities like other kinesin-8 motors. To understand the role of Plasmodium kinesin-8X in cell division, we used fluorescence-tagging and live cell imaging to define its location, and gene targeting to analyse its function, during all proliferative stages of the rodent malaria parasite P. berghei life cycle. The results revealed a spatio-temporal involvement of kinesin-8X in spindle dynamics and an association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the kinesin-8X gene revealed a defect in oocyst development, confirmed by ultrastructural studies, suggesting that this protein is required for oocyst development and sporogony. Transcriptome analysis of Δkinesin-8X gametocytes revealed modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression, supporting a role for kinesin-8X in cell division. Kinesin-8X is thus required for parasite proliferation within the mosquito and for transmission to the vertebrate host.

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.

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 (>20% located in United States) and killed more than 3.3 million people around the world (>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 >6 million compounds including all FDA-approved drugs, drugs under clinical trial (>3000) and an additional >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.

Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.

Ubiquitin-dependent protein degradation within malarial parasites is a burgeoning field of interest due to several encouraging reports of proteasome inhibitors that were able to confer antimalarial activity. Despite the growing interest in the Plasmodium proteasome system, relatively little investigation has been done to actually characterize the parasite degradation machinery. In this report, we provide an initial biological investigation of the ubiquitylating components of the endoplasmic reticulum-associated degradation (ERAD) system, which is a major pathway in targeting misfolded proteins from the ER to the cytosol for proteasome degradation. We are able to show that the ERAD system is essential for parasite survival and that the putative Plasmodium HRD1 (E3 ubiquitin ligase), UBC (E2 ubiquitin conjugating enzyme) and UBA1 (E1 ubiquitin activating enzyme) are able to mediate in vitro ubiquitylation. Furthermore, by using immunofluorescence, we report that Plasmodium HRD1 localizes to the ER membranes, while the Plasmodium UBC and UBA1 localize to the cytosol. In addition, our gene disruption experiments indicate that the Plasmodium HRD1 is likely essential. We have conducted an initial characterization of the ubiquitylating components of the Plasmodium ERAD system, a major pathway for protein degradation and parasite maintenance. In conjunction with promising proteasome inhibitor studies, we explore the possibility of targeting the Plasmodium ERAD system for future bottom-up drug development approaches.

BACKGROUND: Reversible modification of proteins through the attachment of ubiquitin or ubiquitin-like modifiers is an essential post-translational regulatory mechanism in eukaryotes. The conjugation of ubiquitin or ubiquitin-like proteins has been demonstrated to play roles in growth, adaptation and homeostasis in all eukaryotes, with perturbation of ubiquitin-mediated systems associated with the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the use of an HMM search of functional Pfam domains found in the key components of the ubiquitin-mediated pathway necessary to activate and reversibly modify target proteins in eight apicomplexan parasitic protozoa for which complete or late-stage genome projects exist. In parallel, the same search was conducted on five model organisms, single-celled and metazoans, to generate data to validate both the search parameters employed and aid paralog classification in Apicomplexa. For each of the 13 species investigated, a set of proteins predicted to be involved in the ubiquitylation pathway has been identified and demonstrates increasing component members of the ubiquitylation pathway correlating with organism and genome complexity. Sequence homology and domain architecture analyses facilitated prediction of apicomplexan-specific protein function, particularly those involved in regulating cell division during these parasite's complex life cycles. CONCLUSIONS/SIGNIFICANCE: This study provides a comprehensive analysis of proteins predicted to be involved in the apicomplexan ubiquitin-mediated pathway. Given the importance of such pathway in a wide variety of cellular processes, our data is a key step in elucidating the biological networks that, in part, direct the pathogenicity of these parasites resulting in a massive impact on global health. Moreover, apicomplexan-specific adaptations of the ubiquitylation pathway may represent new therapeutic targets for much needed drugs against apicomplexan parasites.