Apicomplexan phosphodiesterases in cyclic nucleotide turnover: conservation, function, and therapeutic potential.

Apicomplexa encompasses a large number of intracellular parasites infecting a wide range of animals. Cyclic nucleotide signaling is crucial for a variety of apicomplexan life stages and cellular processes. The cyclases and kinases that synthesize and respond to cyclic nucleotides (i.e., 3',5'-cyclic guanosine monophosphate and 3',5'-cyclic adenosine monophosphate) are highly conserved and essential throughout the parasite phylum. Growing evidence indicates that phosphodiesterases (PDEs) are also critical for regulating cyclic nucleotide signaling via cyclic nucleotide hydrolysis. Here, we discuss recent advances in apicomplexan PDE biology and opportunities for therapeutic interventions, with special emphasis on the major human apicomplexan parasite genera Plasmodium, Toxoplasma, Cryptosporidium, and Babesia. In particular, we show a highly flexible repertoire of apicomplexan PDEs associated with a wide range of cellular requirements across parasites and lifecycle stages. Despite this phylogenetic diversity, cellular requirements of apicomplexan PDEs for motility, host cell egress, or invasion are conserved. However, the molecular wiring of associated PDEs is extremely malleable suggesting that PDE diversity and redundancy are key for the optimization of cyclic nucleotide turnover to respond to the various environments encountered by each parasite and life stage. Understanding how apicomplexan PDEs are regulated and integrating multiple signaling systems into a unified response represent an untapped avenue for future exploration.

Plasmodium ARK2 and EB1 drive unconventional spindle dynamics, during chromosome segregation in sexual transmission stages.

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.

A Plasmodium membrane receptor platform integrates cues for egress and invasion in blood forms and activation of transmission stages.

Critical events in the life cycle of malaria-causing parasites depend on cyclic guanosine monophosphate homeostasis by guanylyl cyclases (GCs) and phosphodiesterases, including merozoite egress or invasion of erythrocytes and gametocyte activation. These processes rely on a single GCα, but in the absence of known signaling receptors, how this pathway integrates distinct triggers is unknown. We show that temperature-dependent epistatic interactions between phosphodiesterases counterbalance GCα basal activity preventing gametocyte activation before mosquito blood feed. GCα interacts with two multipass membrane cofactors in schizonts and gametocytes: UGO (unique GC organizer) and SLF (signaling linking factor). While SLF regulates GCα basal activity, UGO is essential for GCα up-regulation in response to natural signals inducing merozoite egress and gametocyte activation. This work identifies a GC membrane receptor platform that senses signals triggering processes specific to an intracellular parasitic lifestyle, including host cell egress and invasion to ensure intraerythrocytic amplification and transmission to mosquitoes.

An Sfi1-like centrin-interacting centriolar plaque protein affects nuclear microtubule homeostasis.

Malaria-causing parasites achieve rapid proliferation in human blood through multiple rounds of asynchronous nuclear division followed by daughter cell formation. Nuclear divisions critically depend on the centriolar plaque, which organizes intranuclear spindle microtubules. The centriolar plaque consists of an extranuclear compartment, which is connected via a nuclear pore-like structure to a chromatin-free intranuclear compartment. Composition and function of this non-canonical centrosome remain largely elusive. Centrins, which reside in the extranuclear part, are among the very few centrosomal proteins conserved in Plasmodium falciparum. Here we identify a novel centrin-interacting centriolar plaque protein. Conditional knock down of this Sfi1-like protein (PfSlp) caused a growth delay in blood stages, which correlated with a reduced number of daughter cells. Surprisingly, intranuclear tubulin abundance was significantly increased, which raises the hypothesis that the centriolar plaque might be implicated in regulating tubulin levels. Disruption of tubulin homeostasis caused excess microtubules and aberrant mitotic spindles. Time-lapse microscopy revealed that this prevented or delayed mitotic spindle extension but did not significantly interfere with DNA replication. Our study thereby identifies a novel extranuclear centriolar plaque factor and establishes a functional link to the intranuclear compartment of this divergent eukaryotic centrosome.

The Skp1-Cullin1-FBXO1 complex is a pleiotropic regulator required for the formation of gametes and motile forms in Plasmodium berghei.

Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational modifications. While ubiquitination by multi-component E3 ligases is key to regulate a wide range of cellular processes in eukaryotes, little is known about its role in Plasmodium. Here we show that Plasmodium berghei expresses a conserved SKP1/Cullin1/FBXO1 (SCFFBXO1) complex showing tightly regulated expression and localisation across multiple developmental stages. It is key to cell division for nuclear segregation during schizogony and centrosome partitioning during microgametogenesis. It is additionally required for parasite-specific processes including gamete egress from the host erythrocyte, as well as integrity of the apical and the inner membrane complexes (IMC) in merozoite and ookinete, two structures essential for the dissemination of these motile stages. Ubiquitinomic surveys reveal a large set of proteins ubiquitinated in a FBXO1-dependent manner including proteins important for egress and IMC organisation. We additionally demonstrate an interplay between FBXO1-dependent ubiquitination and phosphorylation via calcium-dependent protein kinase 1. Altogether we show that Plasmodium SCFFBXO1 plays conserved roles in cell division and is also important for parasite-specific processes in the mammalian and mosquito hosts.

Plasmodium ARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation and chromosome segregation of sexual transmission stages.

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. Plasmodium spp., 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 three Plasmodium ARKs 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 model Plasmodium 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 parasite Plasmodium .

Plasmodium ARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation and chromosome segregation of sexual transmission stages.

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. Plasmodium spp., 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 three Plasmodium ARKs 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, omics and live-cell fluorescence imaging, we set out to investigate the contribution of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria model Plasmodium berghei. We find that ARK2 primarily localises to the spindle apparatus associated with 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 and lacking some other conserved molecules. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium.

Conoid extrusion regulates glideosome assembly to control motility and invasion in Apicomplexa.

Members of Apicomplexa are defined by apical cytoskeletal structures and secretory organelles, tailored for motility, invasion and egress. Gliding is powered by actomyosin-dependent rearward translocation of apically secreted transmembrane adhesins. In the human parasite Toxoplasma gondii, the conoid, composed of tubulin fibres and preconoidal rings (PCRs), is a dynamic organelle of undefined function. Here, using ultrastructure expansion microscopy, we established that PCRs serve as a hub for glideosome components including Formin1. We also identified components of the PCRs conserved in Apicomplexa, Pcr4 and Pcr5, that contain B-box zinc-finger domains, assemble in heterodimer and are essential for the formation of the structure. The fitness conferring Pcr6 tethers the PCRs to the cone of tubulin fibres. F-actin produced by Formin1 is used by Myosin H to generate the force for conoid extrusion which directs the flux of F-actin to the pellicular space, serving as gatekeeper to control parasite motility.

Composition and organization of kinetochores show plasticity in apicomplexan chromosome segregation.

Kinetochores are multiprotein assemblies directing mitotic spindle attachment and chromosome segregation. In apicomplexan parasites, most known kinetochore components and associated regulators are apparently missing, suggesting a minimal structure with limited control over chromosome segregation. In this study, we use interactomics combined with deep homology searches to identify 13 previously unknown components of kinetochores in Apicomplexa. Apicomplexan kinetochores are highly divergent in sequence and composition from animal and fungal models. The nanoscale organization includes at least four discrete compartments, each displaying different biochemical interactions, subkinetochore localizations and evolutionary rates across the phylum. We reveal alignment of kinetochores at the metaphase plate in both Plasmodium berghei and Toxoplasma gondii, suggestive of a conserved "hold signal" that prevents precocious entry into anaphase. Finally, we show unexpected plasticity in kinetochore composition and segregation between apicomplexan lifecycle stages, suggestive of diverse requirements to maintain fidelity of chromosome segregation across parasite modes of division.

Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle.

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.

N-acetylation of secreted proteins in Apicomplexa is widespread and is independent of the ER acetyl-CoA transporter AT1.

Acetyl-CoA participates in post-translational modification of proteins and in central carbon and lipid metabolism in several cell compartments. In mammals, acetyl-CoA transporter 1 (AT1, also known as SLC33A1) facilitates the flux of cytosolic acetyl-CoA into the endoplasmic reticulum (ER), enabling the acetylation of proteins of the secretory pathway, in concert with the activity of dedicated acetyltransferases such as NAT8. However, the involvement of the ER acetyl-CoA pool in acetylation of ER-transiting proteins in Apicomplexa is unknown. Here, we identified homologs of AT1 and NAT8 in Toxoplasma gondii and Plasmodium berghei parasites. Proteome-wide analyses revealed widespread N-terminal acetylation of secreted proteins in both species. Such extensive acetylation of N-terminally processed proteins has not been observed previously in any other organism. Deletion of AT1 homologs in both T. gondii and P. berghei resulted in considerable reductions in parasite fitness. In P. berghei, AT1 was found to be important for growth of asexual blood stages, production of female gametocytes and male gametocytogenesis, implying its requirement for parasite transmission. In the absence of AT1, lysine acetylation and N-terminal acetylation in T. gondii remained globally unaltered, suggesting an uncoupling between the role of AT1 in development and active acetylation occurring along the secretory pathway.

Expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a bipartite microtubule organisation centre coordinating mitosis with axoneme assembly.

Transmission of malaria-causing parasites to mosquitoes relies on the production of gametocyte stages and their development into gametes. These stages display various microtubule cytoskeletons and the architecture of the corresponding microtubule organisation centres (MTOC) remains elusive. Combining ultrastructure expansion microscopy (U-ExM) with bulk proteome labelling, we first reconstructed in 3D the subpellicular microtubule network which confers cell rigidity to Plasmodium falciparum gametocytes. Upon activation, as the microgametocyte undergoes three rounds of endomitosis, it also assembles axonemes to form eight flagellated microgametes. U-ExM combined with Pan-ExM further revealed the molecular architecture of the bipartite MTOC coordinating mitosis with axoneme formation. This MTOC spans the nuclear membrane linking cytoplasmic basal bodies to intranuclear bodies by proteinaceous filaments. In P. berghei, the eight basal bodies are concomitantly de novo assembled in a SAS6- and SAS4-dependent manner from a deuterosome-like structure, where centrin, γ-tubulin, SAS4 and SAS6 form distinct subdomains. Basal bodies display a fusion of the proximal and central cores where centrin and SAS6 are surrounded by a SAS4-toroid in the lumen of the microtubule wall. Sequential nucleation of axonemes and mitotic spindles is associated with a dynamic movement of γ-tubulin from the basal bodies to the intranuclear bodies. This dynamic architecture relies on two non-canonical regulators, the calcium-dependent protein kinase 4 and the serine/arginine-protein kinase 1. Altogether, these results provide insights into the molecular organisation of a bipartite MTOC that may reflect a functional transition of a basal body to coordinate axoneme assembly with mitosis.

Correction: Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid.

[This corrects the article DOI: 10.1371/journal.pbio.3001020.].

Protein phosphatase 1 regulates atypical mitotic and meiotic division in Plasmodium sexual stages.

PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the malaria parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time live-cell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut, suggesting that small molecule inhibitors of PP1 should be explored for blocking parasite transmission.

Ca2+ signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG.

Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in Plasmodium is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages. Phosphoproteomic analyses reveal multiple ICM1 phosphorylation events dependent on PKG activity. Stage-specific depletion of Plasmodium berghei ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization, while conditional loss of Plasmodium falciparum ICM1 is detrimental for the parasite resulting in severely reduced calcium mobilization, defective egress, and lack of invasion. Our findings suggest that ICM1 is a key missing link in transducing PKG-dependent signals and provide previously unknown insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.

Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid.

Malaria is caused by unicellular Plasmodium parasites. Plasmodium relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication, and dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy (EM). Here, we demonstrate that the nanoscale cytoskeleton organisation is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows monitoring the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the diversity across Plasmodium stages and species of the subpellicular microtubule arrays that confer cell rigidity. In ookinetes, we additionally identify an apical tubulin ring (ATR) that colocalises with markers of the conoid in related apicomplexan parasites. This tubulin-containing structure was presumed to be lost in Plasmodium despite its crucial role in motility and invasion in other apicomplexans. Here, U-ExM reveals that a divergent and considerably reduced form of the conoid is actually conserved in Plasmodium species.

cGMP homeostasis in malaria parasites-The key to perceiving and integrating environmental changes during transmission to the mosquito.

Malaria-causing parasites are transmitted from humans to mosquitoes when developmentally arrested gametocytes are taken up by a female Anopheles during a blood meal. The changes in environment from human to mosquito activate gametogenesis, including a drop in temperature, a rise in pH, and a mosquito-derived molecule, xanthurenic acid. Signaling receptors have not been identified in malaria parasites but mounting evidence indicates that cGMP homeostasis is key to sensing extracellular cues in gametocytes. Low levels of cGMP maintained by phosphodiesterases prevent precocious activation of gametocytes in the human blood. Upon ingestion, initiation of gametogenesis depends on the activation of a hybrid guanylyl cyclase/P4-ATPase. Elevated cGMP levels lead to the rapid mobilization of intracellular calcium that relies upon the activation of both cGMP-dependent protein kinase and phosphoinositide phospholipase C. Once calcium is released, a cascade of phosphorylation events mediated by calcium-dependent protein kinases and phosphatases regulates the cellular processes required for gamete formation. cGMP signaling also triggers timely egress from the host cell at other life cycle stages of malaria parasites and in Toxoplasma gondii, a related apicomplexan parasite. This suggests that cGMP signaling is a versatile platform transducing external cues into calcium signals at important decision points in the life cycle of apicomplexan parasites.

PfMAP-2 is essential for male gametogenesis in the malaria parasite Plasmodium falciparum.

In malaria parasites, male gametogenesis is a proliferative stage essential for parasite transmission to the mosquito vector. It is a rapid process involving three rounds of genome replication alternating with closed endomitoses, and assembly of axonemes to produce eight flagellated motile microgametes. Studies in Plasmodium berghei have highlighted tight regulation of gametogenesis by a network of kinases. The P. berghei MAPK homologue PbMAP-2 is dispensable for asexual development but important at the induction of axoneme motility. However, in P. falciparum, causing the most severe form of human malaria, PfMAP-2 was suggested to be essential for asexual proliferation indicating distinct functions for MAP-2 in these two Plasmodium species. We here show that PfMAP-2 is dispensable for asexual growth but important for male gametogenesis in vitro. Similar to PbMAP-2, PfMAP-2 is required for initiating axonemal beating but not for prior DNA replication or axoneme formation. In addition, single and double null mutants of PfMAP-2 and the second P. falciparum MAPK homologue PfMAP-1 show no defect in asexual proliferation, sexual commitment or gametocytogenesis. Our results suggest that MAPK activity plays no major role in the biology of both asexual and sexual blood stage parasites up until the point of male gametogenesis.

A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of a malaria parasite during gametogony and transmission.

Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.