Acute response to pathogens in the early human placenta at single-cell resolution.

The placenta is a selective maternal-fetal barrier that provides nourishment and protection from infections. However, certain pathogens can attach to and even cross the placenta, causing pregnancy complications with potential lifelong impacts on the child's health. Here, we profiled at the single-cell level the placental responses to three pathogens associated with intrauterine complications-Plasmodium falciparum, Listeria monocytogenes, and Toxoplasma gondii. We found that upon exposure to the pathogens, all placental lineages trigger inflammatory responses that may compromise placental function. Additionally, we characterized the responses of fetal macrophages known as Hofbauer cells (HBCs) to each pathogen and propose that they are the probable niche for T. gondii. Finally, we revealed how P. falciparum adapts to the placental microenvironment by modulating protein export into the host erythrocyte and nutrient uptake pathways. Altogether, we have defined the cellular networks and signaling pathways mediating acute placental inflammatory responses that could contribute to pregnancy complications.

Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma.

Apicomplexans are ubiquitous intracellular parasites of animals. These parasites use a programmed sequence of secretory events to find, invade, and then re-engineer their host cells to enable parasite growth and proliferation. The secretory organelles micronemes and rhoptries mediate the first steps of invasion. Both secrete their contents through the apical complex which provides an apical opening in the parasite's elaborate inner membrane complex (IMC) - an extensive subpellicular system of flattened membrane cisternae and proteinaceous meshwork that otherwise limits access of the cytoplasm to the plasma membrane for material exchange with the cell exterior. After invasion, a second secretion programme drives host cell remodelling and occurs from dense granules. The site(s) of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii, small subapical annular structures that are embedded in the IMC have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown. Here, we determined that integral membrane proteins of the plasma membrane occur specifically at these apical annular sites, that these proteins include SNARE proteins, and that the apical annuli are sites of vesicle fusion and exocytosis. Specifically, we show that dense granules require these structures for the secretion of their cargo proteins. When secretion is perturbed at the apical annuli, parasite growth is strongly impaired. The apical annuli, therefore, represent a second type of IMC-embedded structure to the apical complex that is specialised for protein secretion, and reveal that in Toxoplasma there is a physical separation of the processes of pre- and post-invasion secretion that mediate host-parasite interactions.

Mapping diversity in African trypanosomes using high resolution spatial proteomics.

African trypanosomes are dixenous eukaryotic parasites that impose a significant human and veterinary disease burden on sub-Saharan Africa. Diversity between species and life-cycle stages is concomitant with distinct host and tissue tropisms within this group. Here, the spatial proteomes of two African trypanosome species, Trypanosoma brucei and Trypanosoma congolense, are mapped across two life-stages. The four resulting datasets provide evidence of expression of approximately 5500 proteins per cell-type. Over 2500 proteins per cell-type are classified to specific subcellular compartments, providing four comprehensive spatial proteomes. Comparative analysis reveals key routes of parasitic adaptation to different biological niches and provides insight into the molecular basis for diversity within and between these pathogen species.

Stable endocytic structures navigate the complex pellicle of apicomplexan parasites.

Apicomplexan parasites have immense impacts on humanity, but their basic cellular processes are often poorly understood. Where endocytosis occurs in these cells, how conserved this process is with other eukaryotes, and what the functions of endocytosis are across this phylum are major unanswered questions. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. We identify that these endocytic structures correspond to the 'micropore' that has been observed throughout the Apicomplexa. Moreover, conserved molecular adaptation of this structure is seen in apicomplexans including the kelch-domain protein K13 that is central to malarial drug-resistance. We determine that a dominant function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.

Cryptosporidium uses multiple distinct secretory organelles to interact with and modify its host cell.

Cryptosporidium is a leading cause of diarrheal disease in children and an important contributor to early childhood mortality. The parasite invades and extensively remodels intestinal epithelial cells, building an elaborate interface structure. How this occurs at the molecular level and the contributing parasite factors are largely unknown. Here, we generated a whole-cell spatial proteome of the Cryptosporidium sporozoite and used genetic and cell biological experimentation to discover the Cryptosporidium-secreted effector proteome. These findings reveal multiple organelles, including an original secretory organelle, and generate numerous compartment markers by tagging native gene loci. We show that secreted proteins are delivered to the parasite-host interface, where they assemble into different structures including a ring that anchors the parasite into its unique epicellular niche. Cryptosporidium thus uses a complex set of secretion systems during and following invasion that act in concert to subjugate its host cell.

Mitochondrial Genomes in Perkinsus Decode Conserved Frameshifts in All Genes.

Mitochondrial genomes of apicomplexans, dinoflagellates, and chrompodellids that collectively make up the Myzozoa, encode only three proteins (Cytochrome b [COB], Cytochrome c oxidase subunit 1 [COX1], Cytochrome c oxidase subunit 3 [COX3]), contain fragmented ribosomal RNAs, and display extensive recombination, RNA trans-splicing, and RNA-editing. The early-diverging Perkinsozoa is the final major myzozoan lineage whose mitochondrial genomes remained poorly characterized. Previous reports of Perkinsus genes indicated independent acquisition of non-canonical features, namely the occurrence of multiple frameshifts. To determine both ancestral myzozoan and novel perkinsozoan mitochondrial genome features, we sequenced and assembled mitochondrial genomes of four Perkinsus species. These data show a simple ancestral genome with the common reduced coding capacity but disposition for rearrangement. We identified 75 frameshifts across the four species that occur as distinct types and that are highly conserved in gene location. A decoding mechanism apparently employs unused codons at the frameshift sites that advance translation either +1 or +2 frames to the next used codon. The locations of frameshifts are seemingly positioned to regulate protein folding of the nascent protein as it emerges from the ribosome. The cox3 gene is distinct in containing only one frameshift and showing strong selection against residues that are otherwise frequently encoded at the frameshift positions in cox1 and cob. All genes lack cysteine codons implying a reduction to 19 amino acids in these genomes. Furthermore, mitochondrion-encoded rRNA fragment complements are incomplete in Perkinsus spp. but some are found in the nuclear DNA suggesting import into the organelle. Perkinsus demonstrates further remarkable trajectories of organelle genome evolution including pervasive integration of frameshift translation into genome expression.

The convoluted history of haem biosynthesis.

The capacity of haem to transfer electrons, bind diatomic gases, and catalyse various biochemical reactions makes it one of the essential biomolecules on Earth and one that was likely used by the earliest forms of cellular life. Since the description of haem biosynthesis, our understanding of this multi-step pathway has been almost exclusively derived from a handful of model organisms from narrow taxonomic contexts. Recent advances in genome sequencing and functional studies of diverse and previously neglected groups have led to discoveries of alternative routes of haem biosynthesis that deviate from the 'classical' pathway. In this review, we take an evolutionarily broad approach to illuminate the remarkable diversity and adaptability of haem synthesis, from prokaryotes to eukaryotes, showing the range of strategies that organisms employ to obtain and utilise haem. In particular, the complex evolutionary histories of eukaryotes that involve multiple endosymbioses and horizontal gene transfers are reflected in the mosaic origin of numerous metabolic pathways with haem biosynthesis being a striking case. We show how different evolutionary trajectories and distinct life strategies resulted in pronounced tensions and differences in the spatial organisation of the haem biosynthesis pathway, in some cases leading to a complete loss of a haem-synthesis capacity and, rarely, even loss of a requirement for haem altogether.

A Prioritized and Validated Resource of Mitochondrial Proteins in Plasmodium Identifies Unique Biology.

Plasmodium species have a single mitochondrion that is essential for their survival and has been successfully targeted by antimalarial drugs. Most mitochondrial proteins are imported into this organelle, and our picture of the Plasmodium mitochondrial proteome remains incomplete. Many data sources contain information about mitochondrial localization, including proteome and gene expression profiles, orthology to mitochondrial proteins from other species, coevolutionary relationships, and amino acid sequences, each with different coverage and reliability. To obtain a comprehensive, prioritized list of Plasmodium falciparum mitochondrial proteins, we rigorously analyzed and integrated eight data sets using Bayesian statistics into a predictive score per protein for mitochondrial localization. At a corrected false discovery rate of 25%, we identified 445 proteins with a sensitivity of 87% and a specificity of 97%. They include proteins that have not been identified as mitochondrial in other eukaryotes but have characterized homologs in bacteria that are involved in metabolism or translation. Mitochondrial localization of seven Plasmodium berghei orthologs was confirmed by epitope labeling and colocalization with a mitochondrial marker protein. One of these belongs to a newly identified apicomplexan mitochondrial protein family that in P. falciparum has four members. With the experimentally validated mitochondrial proteins and the complete ranked P. falciparum proteome, which we have named PlasmoMitoCarta, we present a resource to study unique proteins of Plasmodium mitochondria. IMPORTANCE The unique biology and medical relevance of the mitochondrion of the malaria parasite Plasmodium falciparum have made it the subject of many studies. However, we actually do not have a comprehensive assessment of which proteins reside in this organelle. Many omics data are available that are predictive of mitochondrial localization, such as proteomics data and expression data. Individual data sets are, however, rarely complete and can provide conflicting evidence. We integrated a wide variety of available omics data in a manner that exploits the relative strengths of the data sets. Our analysis gave a predictive score for the mitochondrial localization to each nuclear encoded P. falciparum protein and identified 445 likely mitochondrial proteins. We experimentally validated the mitochondrial localization of seven of the new mitochondrial proteins, confirming the quality of the complete list. These include proteins that have not been observed mitochondria before, adding unique mitochondrial functions to P. falciparum.

Development of the Myzozoan Aquatic Parasite Perkinsus marinus as A Versatile Experimental Genetic Model Organism.

The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation was previously achieved for Perkinsus spp. but with few tools for transgene expression and limited selection efficacy. We sought to expand the power of experimental genetic tools for Perkinsus using P. marinus as a model. We constructed a modular plasmid assembly system for expression of multiple genes simultaneously. We developed efficient selection systems for three drugs, puromycin, bleomycin and blasticidin, that are effective in as little as three weeks. We developed eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination but with transgene fragmentation including deletion of some elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide great new capacity to genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.

Repurposing of synaptonemal complex proteins for kinetochores in Kinetoplastida.

Chromosome segregation in eukaryotes is driven by the kinetochore, a macromolecular complex that connects centromeric DNA to microtubules of the spindle apparatus. Kinetochores in well-studied model eukaryotes consist of a core set of proteins that are broadly conserved among distant eukaryotic phyla. By contrast, unicellular flagellates of the class Kinetoplastida have a unique set of 36 kinetochore components. The evolutionary origin and history of these kinetochores remain unknown. Here, we report evidence of homology between axial element components of the synaptonemal complex and three kinetoplastid kinetochore proteins KKT16-18. The synaptonemal complex is a zipper-like structure that assembles between homologous chromosomes during meiosis to promote recombination. By using sensitive homology detection protocols, we identify divergent orthologues of KKT16-18 in most eukaryotic supergroups, including experimentally established chromosomal axis components, such as Red1 and Rec10 in budding and fission yeast, ASY3-4 in plants and SYCP2-3 in vertebrates. Furthermore, we found 12 recurrent duplications within this ancient eukaryotic SYCP2-3 gene family, providing opportunities for new functional complexes to arise, including KKT16-18 in the kinetoplastid parasite Trypanosoma brucei. We propose the kinetoplastid kinetochore system evolved by repurposing meiotic components of the chromosome synapsis and homologous recombination machinery that were already present in early eukaryotes.

Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species.

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

A Comprehensive Subcellular Atlas of the Toxoplasma Proteome via hyperLOPIT Provides Spatial Context for Protein Functions.

Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to highly specialized cell compartments and structures. These adaptations drive their recognition, nondestructive penetration, and elaborate reengineering of the host's cells to promote their growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty. Consequently, half of apicomplexan proteins are unique and uncharacterized. Here, we determine the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii. This provides unprecedented comprehensive molecular definition of these unicellular eukaryotes and their specialized compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.

Integrated omics unveil the secondary metabolic landscape of a basal dinoflagellate.

BACKGROUND: Some dinoflagellates cause harmful algal blooms, releasing toxic secondary metabolites, to the detriment of marine ecosystems and human health. Our understanding of dinoflagellate toxin biosynthesis has been hampered by their unusually large genomes. To overcome this challenge, for the first time, we sequenced the genome, microRNAs, and mRNA isoforms of a basal dinoflagellate, Amphidinium gibbosum, and employed an integrated omics approach to understand its secondary metabolite biosynthesis. RESULTS: We assembled the ~ 6.4-Gb A. gibbosum genome, and by probing decoded dinoflagellate genomes and transcriptomes, we identified the non-ribosomal peptide synthetase adenylation domain as essential for generation of specialized metabolites. Upon starving the cells of phosphate and nitrogen, we observed pronounced shifts in metabolite biosynthesis, suggestive of post-transcriptional regulation by microRNAs. Using Iso-Seq and RNA-seq data, we found that alternative splicing and polycistronic expression generate different transcripts for secondary metabolism. CONCLUSIONS: Our genomic findings suggest intricate integration of various metabolic enzymes that function iteratively to synthesize metabolites, providing mechanistic insights into how dinoflagellates synthesize secondary metabolites, depending upon nutrient availability. This study provides insights into toxin production associated with dinoflagellate blooms. The genome of this basal dinoflagellate provides important clues about dinoflagellate evolution and overcomes the large genome size, which has been a challenge previously.

Real-time dynamics of Plasmodium NDC80 reveals unusual modes of chromosome segregation during parasite proliferation.

Eukaryotic cell proliferation requires chromosome replication and precise segregation to ensure daughter cells have identical genomic copies. Species of the genus Plasmodium, the causative agents of malaria, display remarkable aspects of nuclear division throughout their life cycle to meet some peculiar and unique challenges to 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 to be an ideal tool to investigate the non-canonical modes of chromosome segregation and cell division in Plasmodium.

Evolution, Composition, Assembly, and Function of the Conoid in Apicomplexa.

The phylum Apicomplexa has been defined by the presence of the apical complex, a structure composed of secretory organelles and specific cytoskeletal elements. A conspicuous feature of the apical complex in many apicomplexans is the conoid, a hollow tapered barrel structure composed of tubulin fibers. In Toxoplasma gondii, the apical complex is a central site of convergence for calcium-related and lipid-mediated signaling pathways that coordinate conoid protrusion, microneme secretion, and actin polymerization, to initiate gliding motility. Through cutting-edge technologies, great progress has recently been made in discovering the structural subcomponents and proteins implicated in the biogenesis and stability of the apical complex and, in turn, these discoveries have shed new light on the function and evolution of this definitive structure.

The Biochemistry and Evolution of the Dinoflagellate Nucleus.

Dinoflagellates are known to possess a highly aberrant nucleus-the so-called dinokaryon-that exhibits a multitude of exceptional biological features. These include: (1) Permanently condensed chromosomes; (2) DNA in a cholesteric liquid crystalline state, (3) extremely large DNA content (up to 200 pg); and, perhaps most strikingly, (4) a deficit of histones-the canonical building blocks of all eukaryotic chromatin. Dinoflagellates belong to the Alveolata clade (dinoflagellates, apicomplexans, and ciliates) and, therefore, the biological oddities observed in dinoflagellate nuclei are derived character states. Understanding the sequence of changes that led to the dinokaryon has been difficult in the past with poor resolution of dinoflagellate phylogeny. Moreover, lack of knowledge of their molecular composition has constrained our understanding of the molecular properties of these derived nuclei. However, recent advances in the resolution of the phylogeny of dinoflagellates, particularly of the early branching taxa; the realization that divergent histone genes are present; and the discovery of dinoflagellate-specific nuclear proteins that were acquired early in dinoflagellate evolution have all thrown new light nature and evolution of the dinokaryon.

A new marine prasinophyte genus alternates between a flagellate and a dominant benthic stage with microrhizoids for adhesion.

Prasinophytes (Chlorophyta) are a diverse, paraphyletic group of planktonic microalgae for which benthic species are largely unknown. Here, we report a sand-dwelling, marine prasinophyte with several novel features observed in clonal cultures established from numerous locations around Australia. The new genus and species, which we name Microrhizoidea pickettheapsiorum (Mamiellophyceae), alternates between a benthic palmelloid colony, where cell division occurs, and a planktonic flagellate. Flagellates are short lived, settle and quickly resorb their flagella, the basal bodies then nucleate novel tubular appendages, termed "microrhizoids", that lack an axoneme and function to anchor benthic cells to the substratum. To our knowledge, microrhizoids have not been observed in any other green alga or protist, are slightly smaller in diameter than flagella, generally contain nine microtubules, are long (3-5 times the length of flagella) and are not encased in scales. Following settlement, cell divisions result in a loose, palmelloid colony, each cell connected to the substratum by two microrhizoids. Flagellates are round to bean-shaped with two long, slightly uneven flagella. Both benthic cells and flagellates, along with their flagella, are encased in thin scales. Phylogenies based on the complete chloroplast genome of Microrhizoidea show that it is clearly a member of the Mamiellophyceae, most closely related to Dolichomastix tenuilepsis. More taxon-rich phylogenetic analyses of the 18S rRNA gene, including metabarcodes from the Tara Oceans and Ocean Sampling Day projects, confidently show the distinctive nature of Microrhizoidea, and that the described biodiversity of the Mamiellophyceae is a fraction of its real biodiversity. The discovery of a largely benthic prasinophyte changes our perspective on this group of algae and, along with the observation of other potential benthic lineages in environmental sequences, illustrates that benthic habitats can be a rich ground for algal biodiscovery.

An essential pentatricopeptide repeat protein in the apicomplexan remnant chloroplast.

The malaria parasite Plasmodium and other apicomplexans such as Toxoplasma evolved from photosynthetic organisms and contain an essential, remnant plastid termed the apicoplast. Transcription of the apicoplast genome is polycistronic with extensive RNA processing. Yet little is known about the mechanism of apicoplast RNA processing. In plants, chloroplast RNA processing is controlled by multiple pentatricopeptide repeat (PPR) proteins. Here, we identify the single apicoplast PPR protein, PPR1. We show that the protein is essential and that it binds to RNA motifs corresponding with previously characterized processing sites. Additionally, PPR1 shields RNA transcripts from ribonuclease degradation. This is the first characterization of a PPR protein from a nonphotosynthetic plastid.

Systematic analysis of Plasmodium myosins reveals differential expression, localisation, and function in invasive and proliferative parasite stages.

The myosin superfamily comprises of actin-dependent eukaryotic molecular motors important in a variety of cellular functions. Although well studied in many systems, knowledge of their functions in Plasmodium, the causative agent of malaria, is restricted. Previously, six myosins were identified in this genus, including three Class XIV myosins found only in Apicomplexa and some Ciliates. The well characterized MyoA is a Class XIV myosin essential for gliding motility and invasion. Here, we characterize all other Plasmodium myosins throughout the parasite life cycle and show that they have very diverse patterns of expression and cellular location. MyoB and MyoE, the other two Class XIV myosins, are expressed in all invasive stages, with apical and basal locations, respectively. Gene deletion revealed that MyoE is involved in sporozoite traversal, MyoF and MyoK are likely essential in the asexual blood stages, and MyoJ and MyoB are not essential. Both MyoB and its essential light chain (MCL-B) are localised at the apical end of ookinetes but expressed at completely different time points. This work provides a better understanding of the role of actomyosin motors in Apicomplexan parasites, particularly in the motile and invasive stages of Plasmodium during sexual and asexual development within the mosquito.