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.

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.

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.

Trypanosome KKIP1 Dynamically Links the Inner Kinetochore to a Kinetoplastid Outer Kinetochore Complex.

Kinetochores perform an essential role in eukaryotes, coupling chromosomes to the mitotic spindle. In model organisms they are composed of a centromere-proximal inner kinetochore and an outer kinetochore network that binds to microtubules. In spite of universal function, the composition of kinetochores in extant eukaryotes differs greatly. In trypanosomes and other Kinetoplastida, kinetochores are extremely divergent, with most components showing no detectable similarity to proteins in other systems. They may also be very different functionally, potentially binding to the spindle directly via an inner-kinetochore protein. However, we do not know the extent of the trypanosome kinetochore, and proteins interacting with a highly divergent Ndc80/Nuf2-like protein (KKIP1) suggest the existence of more centromere-distal complexes. Here we use quantitative proteomics from multiple start-points to define a stable 9-protein kinetoplastid outer kinetochore (KOK) complex. This complex incorporates proteins recruited from other nuclear processes, exemplifying the role of moonlighting proteins in kinetochore evolution. The outer kinetochore complex is physically distinct from inner-kinetochore proteins, but nanometer-scale label separation shows that KKIP1 bridges the two plates in the same orientation as Ndc80. Moreover, KKIP1 exhibits substantial elongation at metaphase, altering kinetochore structure in a manner consistent with pulling at the outer plate. Together, these data suggest that the KKIP1/KOK likely constitute the extent of the trypanosome outer kinetochore and that this assembly binds to the spindle with sufficient strength to stretch the kinetochore, showing design parallels may exist in organisms with very different kinetochore composition.

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.

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.

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.

Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.