Plasmodium LCCL domain-containing modular proteins have their origins in the ancestral alveolate.

Plasmodium species encode a unique set of six modular proteins named LCCL lectin domain adhesive-like proteins (LAPs) that operate as a complex and that are essential for malaria parasite transmission from mosquito to vertebrate. LAPs possess complex architectures obtained through unique assemblies of conserved domains associated with lipid, protein and carbohydrate interactions, including the name-defining LCCL domain. Here, we assessed the prevalence of Plasmodium LAP orthologues across eukaryotic life. Our findings show orthologous conservation in all apicomplexans, with lineage-specific repertoires acquired through differential lap gene loss and duplication. Besides Apicomplexa, LAPs are found in their closest relatives: the photosynthetic chromerids, which encode the broadest repertoire including a novel membrane-bound LCCL protein. LAPs are notably absent from other alveolate lineages (dinoflagellates, perkinsids and ciliates), but are encoded by predatory colponemids, a sister group to the alveolates. These results reveal that the LAPs are much older than previously thought and pre-date not only the Apicomplexa but the Alveolata altogether.

Proteomic approaches for protein kinase substrate identification in Apicomplexa.

Apicomplexa is a phylum of protist parasites, notable for causing life-threatening diseases including malaria, toxoplasmosis, cryptosporidiosis, and babesiosis. Apicomplexan pathogenesis is generally a function of lytic replication, dissemination, persistence, host cell modification, and immune subversion. Decades of research have revealed essential roles for apicomplexan protein kinases in establishing infections and promoting pathogenesis. Protein kinases modify their substrates by phosphorylating serine, threonine, tyrosine, or other residues, resulting in rapid functional changes in the target protein. Post-translational modification by phosphorylation can activate or inhibit a substrate, alter its localization, or promote interactions with other proteins or ligands. Deciphering direct kinase substrates is crucial to understand mechanisms of kinase signaling, yet can be challenging due to the transient nature of kinase phosphorylation and potential for downstream indirect phosphorylation events. However, with recent advances in proteomic approaches, our understanding of kinase function in Apicomplexa has improved dramatically. Here, we discuss methods that have been used to identify kinase substrates in apicomplexan parasites, classifying them into three main categories: i) kinase interactome, ii) indirect phosphoproteomics and iii) direct labeling. We briefly discuss each approach, including their advantages and limitations, and highlight representative examples from the Apicomplexa literature. Finally, we conclude each main category by introducing prospective approaches from other fields that would benefit kinase substrate identification in Apicomplexa.

Time to switch gears: how long noncoding RNAs function as epigenetic regulators in Apicomplexan parasites.

Long noncoding RNAs (lncRNA) are emerging as important regulators of gene expression in eukaryotes. In recent years, a large repertoire of lncRNA were discovered in Apicomplexan parasites and were implicated in several mechanisms of gene expression, including marking genes for activation, contributing to the formation of subnuclear compartments and organization, regulating the deposition of epigenetic modifications, influencing chromatin and chromosomal structure and manipulating host gene expression. Here, we aim to update recent knowledge on the role of lncRNAs as regulators in Apicomplexan parasites and highlight the possible molecular mechanisms by which they function. We hope that some of the hypotheses raised here will contribute to further investigation and lead to new mechanistic insight and better understanding of the role of lncRNA in parasite's biology.

Histone code: a common language and multiple dialects to meet the different developmental requirements of apicomplexan parasites.

Apicomplexan parasites have complex life cycles, often requiring transmission between two different hosts, facing periods of dormancy within the host or in the environment to maximize chances of transmission. To support survival in these different conditions, tightly regulated and correctly timed gene expression is critical. The modification of histones and nucleosome composition makes a significant contribution to this regulation, and as eukaryotes, the fundamental mechanisms underlying this process in apicomplexans are similar to those in model eukaryotic organisms. However, single-celled intracellular parasites face unique challenges, and regulation of gene expression at the epigenetic level provides tight control for responses that must often be rapid and robust. Here, we discuss the recent advances in understanding the dynamics of histone modifications across Apicomplexan life cycles and the molecular mechanisms that underlie epigenetic regulation of gene expression to promote parasite life cycle progression, dormancy, and transmission.

Transcript tinkering: RNA modifications in protozoan parasites.

Apicomplexan and trypanosomatid parasites have evolved a wide range of post-transcriptional processes that allow them to replicate, differentiate, and transmit within and among multiple different tissue, host, and vector environments. In this review, we highlight the recent advances that point toward the regulatory potential of RNA modifications in mediating these processes on the coding and noncoding transcriptome throughout the life cycle of protozoan parasites. We discuss the recent technical advancements enabling the study of the 'epitranscriptome' and how parasites evolved RNA modification-mediated mechanisms adapted to their unique lifestyles.

Examination of Secondary Metabolite Biosynthesis in Apicomplexa.

Natural product discovery has traditionally relied on the isolation of small molecules from producing species, but genome-sequencing technology and advances in molecular biology techniques have expanded efforts to a wider array of organisms. Protists represent an underexplored kingdom for specialized metabolite searches despite bioinformatic analysis that suggests they harbor distinct biologically active small molecules. Specifically, pathogenic apicomplexan parasites, responsible for billions of global infections, have been found to possess multiple biosynthetic gene clusters, which hints at their capacity to produce polyketide metabolites. Biochemical studies have revealed unique features of apicomplexan polyketide synthases, but to date, the identity and function of the polyketides synthesized by these megaenzymes remains unknown. Herein, we discuss the potential for specialized metabolite production in protists and the possible evolution of polyketide biosynthetic gene clusters in apicomplexan parasites. We then focus on a polyketide synthase from the apicomplexan Toxoplasma gondii to discuss the unique domain architecture and properties of these proteins when compared to previously characterized systems, and further speculate on the possible functions for polyketides in these pathogenic parasites.

Development and validation of a machine learning algorithm prediction for dense granule proteins in Apicomplexa.

BACKGROUND: Apicomplexa consist of numerous pathogenic parasitic protistan genera that invade host cells and reside and replicate within the parasitophorous vacuole (PV). Through this interface, the parasite exchanges nutrients and affects transport and immune modulation. During the intracellular life-cycle, the specialized secretory organelles of the parasite secrete an array of proteins, among which dense granule proteins (GRAs) play a major role in the modification of the PV. Despite this important role of GRAs, a large number of potential GRAs remain unidentified in Apicomplexa. METHODS: A multi-view attention graph convolutional network (MVA-GCN) prediction model with multiple features was constructed using a combination of machine learning and genomic datasets, and the prediction was performed on selected Neospora caninum protein data. The candidate GRAs were verified by a CRISPR/Cas9 gene editing system, and the complete NcGRA64(a,b) gene knockout strain was constructed and the phenotypes of the mutant were analyzed. RESULTS: The MVA-GCN prediction model was used to screen N. caninum candidate GRAs, and two novel GRAs (NcGRA64a and NcGRA64b) were verified by gene endogenous tagging. Knockout of complete genes of NcGRA64(a,b) in N. caninum did not affect the parasite's growth and replication in vitro and virulence in vivo. CONCLUSIONS: Our study showcases the utility of the MVA-GCN deep learning model for mining Apicomplexa GRAs in genomic datasets, and the prediction model also has certain potential in mining other functional proteins of apicomplexan parasites.

The mystery of massive mitochondrial complexes: the apicomplexan respiratory chain.

The mitochondrial respiratory chain is an essential pathway in most studied eukaryotes due to its roles in respiration and other pathways that depend on mitochondrial membrane potential. Apicomplexans are unicellular eukaryotes whose members have an impact on global health. The respiratory chain is a drug target for some members of this group, notably the malaria-causing Plasmodium spp. This has motivated studies of the respiratory chain in apicomplexan parasites, primarily Toxoplasma gondii and Plasmodium spp. for which experimental tools are most advanced. Studies of the respiratory complexes in these organisms revealed numerous novel features, including expansion of complex size. The divergence of apicomplexan mitochondria from commonly studied models highlights the diversity of mitochondrial form and function across eukaryotic life.

The ferredoxin redox system - an essential electron distributing hub in the apicoplast of Apicomplexa.

The apicoplast, a relict plastid found in most species of the phylum Apicomplexa, harbors the ferredoxin redox system which supplies electrons to enzymes of various metabolic pathways in this organelle. Recent reports in Toxoplasma gondii and Plasmodium falciparum have shown that the iron-sulfur cluster (FeS)-containing ferredoxin is essential in tachyzoite and blood-stage parasites, respectively. Here we review ferredoxin's crucial contribution to isoprenoid and lipoate biosynthesis as well as tRNA modification in the apicoplast, highlighting similarities and differences between the two species. We also discuss ferredoxin's potential role in the initial reductive steps required for FeS synthesis as well as recent evidence that offers an explanation for how NADPH required by the redox system might be generated in Plasmodium spp.

The flexibility of Apicomplexa parasites in lipid metabolism.

Apicomplexa are obligate intracellular parasites responsible for major human infectious diseases such as toxoplasmosis and malaria, which pose social and economic burdens around the world. To survive and propagate, these parasites need to acquire a significant number of essential biomolecules from their hosts. Among these biomolecules, lipids are a key metabolite required for parasite membrane biogenesis, signaling events, and energy storage. Parasites can either scavenge lipids from their host or synthesize them de novo in a relict plastid, the apicoplast. During their complex life cycle (sexual/asexual/dormant), Apicomplexa infect a large variety of cells and their metabolic flexibility allows them to adapt to different host environments such as low/high fat content or low/high sugar levels. In this review, we discuss the role of lipids in Apicomplexa parasites and summarize recent findings on the metabolic mechanisms in host nutrient adaptation.

Eimeria proteins: order amidst disorder.

Apicomplexans are important pathogens that cause severe infections in humans and animals. The biology and pathogeneses of these parasites have shown that proteins are intrinsically modulated during developmental transitions, physiological processes and disease progression. Also, proteins are integral components of parasite structural elements and organelles. Among apicomplexan parasites, Eimeria species are an important disease aetiology for economically important animals wherein identification and characterisation of proteins have been long-winded. Nonetheless, this review seeks to give a comprehensive overview of constitutively expressed Eimeria proteins. These molecules are discussed across developmental stages, organelles and sub-cellular components vis-à-vis their biological functions. In addition, hindsight and suggestions are offered with intention to summarise the existing trend of eimerian protein characterisation and to provide a baseline for future studies.

Pantothenate and CoA biosynthesis in Apicomplexa and their promise as antiparasitic drug targets.

The Apicomplexa phylum comprises thousands of distinct intracellular parasite species, including coccidians, haemosporidians, piroplasms, and cryptosporidia. These parasites are characterized by complex and divergent life cycles occupying a variety of host niches. Consequently, they exhibit distinct adaptations to the differences in nutritional availabilities, either relying on biosynthetic pathways or by salvaging metabolites from their host. Pantothenate (Pan, vitamin B5) is the precursor for the synthesis of an essential cofactor, coenzyme A (CoA), but among the apicomplexans, only the coccidian subgroup has the ability to synthesize Pan. While the pathway to synthesize CoA from Pan is largely conserved across all branches of life, there are differences in the redundancy of enzymes and possible alternative pathways to generate CoA from Pan. Impeding the scavenge of Pan and synthesis of Pan and CoA have been long recognized as potential targets for antimicrobial drug development, but in order to fully exploit these critical pathways, it is important to understand such differences. Recently, a potent class of pantothenamides (PanAms), Pan analogs, which target CoA-utilizing enzymes, has entered antimalarial preclinical development. The potential of PanAms to target multiple downstream pathways make them a promising compound class as broad antiparasitic drugs against other apicomplexans. In this review, we summarize the recent advances in understanding the Pan and CoA biosynthesis pathways, and the suitability of these pathways as drug targets in Apicomplexa, with a particular focus on the cyst-forming coccidian, Toxoplasma gondii, and the haemosporidian, Plasmodium falciparum.

Using Diatom and Apicomplexan Models to Study the Heme Pathway of Chromera velia.

Heme biosynthesis is essential for almost all living organisms. Despite its conserved function, the pathway's enzymes can be located in a remarkable diversity of cellular compartments in different organisms. This location does not always reflect their evolutionary origins, as might be expected from the history of their acquisition through endosymbiosis. Instead, the final subcellular localization of the enzyme reflects multiple factors, including evolutionary origin, demand for the product, availability of the substrate, and mechanism of pathway regulation. The biosynthesis of heme in the apicomonad Chromera velia follows a chimeric pathway combining heme elements from the ancient algal symbiont and the host. Computational analyses using different algorithms predict complex targeting patterns, placing enzymes in the mitochondrion, plastid, endoplasmic reticulum, or the cytoplasm. We employed heterologous reporter gene expression in the apicomplexan parasite Toxoplasma gondii and the diatom Phaeodactylum tricornutum to experimentally test these predictions. 5-aminolevulinate synthase was located in the mitochondria in both transfection systems. In T. gondii, the two 5-aminolevulinate dehydratases were located in the cytosol, uroporphyrinogen synthase in the mitochondrion, and the two ferrochelatases in the plastid. In P. tricornutum, all remaining enzymes, from ALA-dehydratase to ferrochelatase, were placed either in the endoplasmic reticulum or in the periplastidial space.

Signs of the plastid: Enzymes involved in plastid-localized metabolic pathways in a eugregarine species.

Apicomplexa mainly comprises parasitic species and some of them, which infect and cause severe diseases to humans and livestock, have been extensively studied due to the clinical and industrial importance. Besides, apicomplexans are a popular subject of the studies focusing on the evolution initiated by a secondary loss of photosynthesis. By interpreting the position in the tree of eukaryotes and lifestyles of the phylogenetic relatives parsimoniously, the extant apicomplexans are predicted to be the descendants of a parasite bearing a non-photosynthetic (cryptic) plastid. The plastid-bearing characteristic for the ancestral apicomplexan is further strengthened by non-photosynthetic plastids found in the extant apicomplexans. The research on apicomplexan members infecting invertebrates is much less advanced than that on the pathogens to humans and livestock. Gregarines are apicomplexans that infect diverse invertebrates and recent studies based on transcriptome data revealed the presence of cryptic plastids in a subset of the species investigated. In this study, we isolated gregarine-like organisms (GLOs) from three arthropod species and conducted transcriptome analyses on the isolated cells. A transcriptome-based, multi-gene phylogenetic analysis clearly indicated that all of the three GLOs are eugregarines. Significantly, the transcriptome data from the GLO in a centipede appeared to contain the transcripts encoding enzymes involved in the non-mevalonate pathway for isopentenyl diphosphate biosynthesis and C5 pathway for heme biosynthesis. The enzymes involved in the two plastid-localized metabolic pathways circumstantially but strongly suggest that the particular GLO possesses a cryptic plastid. The evolution of cryptic plastids in eugregarines is revised by incorporating the new data obtained from the three GLOs in this study.

Identification and phylogenetic analysis of RNA binding domain abundant in apicomplexans or RAP proteins.

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 ⍺/ß 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.

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.

Theileria secretes proteins to subvert its host leukocyte.

Theileria parasites are classified in the phylum Apicomplexa that includes several genera of medical and veterinary importance such as Plasmodium, Babesia, Toxoplasma and Cryptosporidium. These protozoans have evolved subtle ways to reshape their intracellular niche for their own benefit and Theileria is no exception. This tick transmitted microorganism is unique among all eukaryotes in that its intracellular schizont stage is able to transform its mammalian host leukocytes into an immortalised highly disseminating cell that phenocopies tumour cells. Here, we describe what is known about secreted Theileria-encoded host cell manipulators.

Structural role of essential light chains in the apicomplexan glideosome.

Gliding, a type of motility based on an actin-myosin motor, is specific to apicomplexan parasites. Myosin A binds two light chains which further interact with glideosome associated proteins and assemble into the glideosome. The role of individual glideosome proteins is unclear due to the lack of structures of larger glideosome assemblies. Here, we investigate the role of essential light chains (ELCs) in Toxoplasma gondii and Plasmodium falciparum and present their crystal structures as part of trimeric sub-complexes. We show that although ELCs bind a conserved MyoA sequence, P. falciparum ELC adopts a distinct structure in the free and MyoA-bound state. We suggest that ELCs enhance MyoA performance by inducing secondary structure in MyoA and thus stiffen its lever arm. Structural and biophysical analysis reveals that calcium binding has no influence on the structure of ELCs. Our work represents a further step towards understanding the mechanism of gliding in Apicomplexa.

Photoparasitism as an Intermediate State in the Evolution of Apicomplexan Parasites.

Despite the benefits of phototrophy, many algae have lost photosynthesis and have converted back to heterotrophy. Parasitism is a heterotrophic strategy, with apicomplexans being among the most devastating parasites for humans. The presence of a nonphotosynthetic plastid in apicomplexan parasites suggests their phototrophic ancestry. The discovery of related phototrophic chromerids has unlocked the possibility to study the transition between phototrophy and parasitism in the Apicomplexa. The chromerid Chromera velia can live as an intracellular parasite in coral larvae as well as a free-living phototroph, combining phototrophy and parasitism in what I call photoparasitism. Since early-branching apicomplexans live extracellularly, their evolution from an intracellular symbiont is unlikely. In this opinion article I discuss possible evolutionary trajectories from an extracellular photoparasite to an obligatory apicomplexan parasite.

Fatty Acid Biosynthesis in Chromerids.

Fatty acids are essential components of biological membranes, important for the maintenance of cellular structures, especially in organisms with complex life cycles like protozoan parasites. Apicomplexans are obligate parasites responsible for various deadly diseases of humans and livestock. We analyzed the fatty acids produced by the closest phototrophic relatives of parasitic apicomplexans, the chromerids Chromera velia and Vitrella brassicaformis, and investigated the genes coding for enzymes involved in fatty acids biosynthesis in chromerids, in comparison to their parasitic relatives. Based on evidence from genomic and metabolomic data, we propose a model of fatty acid synthesis in chromerids: the plastid-localized FAS-II pathway is responsible for the de novo synthesis of fatty acids reaching the maximum length of 18 carbon units. Short saturated fatty acids (C14:0-C18:0) originate from the plastid are then elongated and desaturated in the cytosol and the endoplasmic reticulum. We identified giant FAS I-like multi-modular enzymes in both chromerids, which seem to be involved in polyketide synthesis and fatty acid elongation. This full-scale description of the biosynthesis of fatty acids and their derivatives provides important insights into the reductive evolutionary transition of a phototropic algal ancestor to obligate parasites.