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.

Apicomplexan cytoskeleton and motors: key regulators in morphogenesis, cell division, transport and motility.

Protozoan parasites of the phylum Apicomplexa undergo a lytic cycle whereby a single zoite produced by the previous cycle has to encounter a host cell, invade it, multiply to differentiate into a new zoite generation and escape to resume a new cycle. At every step of this lytic cycle, the cytoskeleton and/or the gliding motility apparatus play a crucial role and recent results have elucidated aspects of these processes, especially in terms of the molecular characterization and interaction of the increasing number of partners involved, and the signalling mechanisms implicated. The present review aims to summarize the most recent findings in the field.

New insights into myosin evolution and classification.

Myosins are eukaryotic actin-dependent molecular motors important for a broad range of functions like muscle contraction, vision, hearing, cell motility, and host cell invasion of apicomplexan parasites. Myosin heavy chains consist of distinct head, neck, and tail domains and have previously been categorized into 18 different classes based on phylogenetic analysis of their conserved heads. Here we describe a comprehensive phylogenetic examination of many previously unclassified myosins, with particular emphasis on sequences from apicomplexan and other chromalveolate protists including the model organism Toxoplasma, the malaria parasite Plasmodium, and the ciliate Tetrahymena. Using different phylogenetic inference methods and taking protein domain architectures, specific amino acid polymorphisms, and organismal distribution into account, we demonstrate a hitherto unrecognized common origin for ciliate and apicomplexan class XIV myosins. Our data also suggest common origins for some apicomplexan myosins and class VI, for classes II and XVIII, for classes XII and XV, and for some microsporidian myosins and class V, thereby reconciling evolutionary history and myosin structure in several cases and corroborating the common coevolution of myosin head, neck, and tail domains. Six novel myosin classes are established to accommodate sequences from chordate metazoans (class XIX), insects (class XX), kinetoplastids (class XXI), and apicomplexans and diatom algae (classes XXII, XXIII, and XXIV). These myosin (sub)classes include sequences with protein domains (FYVE, WW, UBA, ATS1-like, and WD40) previously unknown to be associated with myosin motors. Regarding the apicomplexan "myosome," we significantly update class XIV classification, propose a systematic naming convention, and discuss possible functions in these parasites.

Functional and biophysical analyses of the class XIV Toxoplasma gondii myosin D.

The obligate intracellular parasite Toxoplasma gondii uses gliding motility to migrate across the biological barriers of the host and to invade cells. This unique form of locomotion requires an intact actin cytoskeleton and involves at least one motor protein (TgMyoA) that belongs to the class XIV of the myosin superfamily. TgMyoA is anchored in the inner membrane complex and is essential for the gliding motion, host cell invasion and egress of T. gondii tachyzoites. TgMyoD is the smallest T. gondii myosin and is structurally very closely related to TgMyoA. We show here that TgMyoD exhibits similar transient kinetic properties as the fast single-headed TgMyoA. To determine if TgMyoD also contributes to parasite gliding motility, the TgMyoD gene was disrupted by double homologous recombination. In contrast to TgMyoA, TgMyoD gene is dispensable for tachyzoite propagation and motility. Parasites lacking TgMyoD glide normally and their virulence is not compromised in mice. The fact that TgMyoD is predominantly expressed in bradyzoites explains the absence of a phenotype observed with myodko in tachyzoites and does not exclude a role of this motor in gliding that would be restricted to the cyst forming but nevertheless motile stage of the parasite.

The transcription machinery and the molecular toolbox to control gene expression in Toxoplasma gondii and other protozoan parasites.

The phylum of Apicomplexa groups a large variety of obligate intracellular protozoan parasites that exhibit complicated life cycles, involving transmission and differentiation within and between different hosts. Little is known about the level of regulation and the nature of the factors controlling gene expression throughout their life stages. Unravelling the mechanisms that govern gene regulation is critical for the development of adequate tools to manipulate these parasites and modulate gene expression, in order to study their function in molecular terms in vivo. A comparative analysis of the transcriptional machinery of several apicomplexan genomes and other protozoan parasites has revealed the existence of a primitive eukaryotic transcription apparatus consisting only of a subset of the general transcription factors found in higher eukaryotes. These findings have some direct implications on development of tools.

Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion.

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.

Rhomboid-like proteins in Apicomplexa: phylogeny and nomenclature.

Rhomboids form a family of polytopic intramembrane serine proteases. In Toxoplasma gondii, an essential activity called microneme protein protease 1 (MPP1) cleaves secreted adhesive proteins within their transmembrane domains, at a site conserved in similar proteins of other Apicomplexa. Current evidence suggests that MPP1 is ubiquitous in the phylum and is encoded by a rhomboid gene. In this article, we present the current repertoire of rhomboid-like proteins in Apicomplexa using a nomenclature based on phylogenetic analyses.

Apicomplexan rhomboids have a potential role in microneme protein cleavage during host cell invasion.

Apicomplexan parasites secrete transmembrane (TM) adhesive proteins as part of the process leading to host cell attachment and invasion. These microneme proteins are cleaved in their TM domains by an unidentified protease termed microneme protein protease 1 (MPP1). The cleavage site sequence (IA downward arrowGG), mapped in the Toxoplasma gondii microneme proteins TgMIC2 and TgMIC6, is conserved in microneme proteins of other apicomplexans including Plasmodium species. We report here the characterisation of novel T. gondii proteins belonging to the rhomboid family of intramembrane-cleaving serine proteases. T. gondii possesses six genes encoding rhomboid-like proteins. Four are localised along the secretory pathway and therefore constitute possible candidates for MPP1 activity. Toxoplasma rhomboids TgROM1, TgROM2 and TgROM5 cleave the TM domain of Drosophila Spitz, an established substrate for rhomboids from several species, demonstrating that they are active proteases. In addition, TgROM2 cleaves chimeric proteins that contain the TM domains of TgMIC2 and TgMIC12.

Tetracycline analogue-regulated transgene expression in Plasmodium falciparum blood stages using Toxoplasma gondii transactivators.

Genetic manipulation has revolutionized research in the Apicomplexan parasite Plasmodium falciparum, the most important causative agent of malaria. However, to date no techniques have been established that allow modifications that are deleterious to blood-stage growth, such as the disruption of essential genes or the expression of dominant-negative transgenes. The recent establishment of a screen for functional transactivators in the related parasite Toxoplasma gondii prompted us to identify transactivators in T. gondii and to examine their functionality in P. falciparum. Tetracycline-responsive minimal promoters were generated based on the characterized P. falciparum calmodulin promoter and used to assess transactivators in P. falciparum. We demonstrate that artificial tetracycline-regulated transactivators isolated in T. gondii are also functional in P. falciparum. By using the tetracycline analogue anhydrotetracycline, efficient, stage-specific gene regulation was achieved in P. falciparum. This regulatable expression technology has clear potential for the study of essential gene function in P. falciparum blood stages. On the other hand, the identified transactivators are not functional in mammalian cells, consistent with the fundamental differences in the mechanism of gene regulation between Apicomplexan parasites and their human hosts.

Molecular and functional aspects of parasite invasion.

Apicomplexan parasites have evolved an efficient mechanism to gain entry into non-phagocytic cells, hence challenging their hosts by the establishment of infection in immuno-privileged tissues. Gliding motility is a prerequisite for the invasive stage of most apicomplexans, allowing them to migrate across tissues, and actively invade and egress host cells. In the late 1960s, detailed morphological studies revealed that motile apicomplexans share an elaborate architecture comprising a subpellicular cytoskeleton and apical organelles. Since 1993, the development of technologies for transient and stable transfection have provided powerful tools with which to identify gene products associated with these structures and organelles, as well as to understand their functions. In combination with access to several parasite genomes, it is now possible to compare and contrast the strategies and molecular machines that have been selectively designed by distinct life stages within a species, or by different apicomplexan species, to optimize infection.

Host cell invasion by the apicomplexans: the significance of microneme protein proteolysis.

Intracellular life-style has been adopted by many pathogens as a successful immune evasion mechanism. To gain entry to a large variety of host cells and to establish an intracellular niche, Toxoplasma gondii and other apicomplexans rely on an active process distinct from phagocytosis. Calcium-regulated secretion of microneme proteins and parasite actin polymerization together with the action of at least one myosin motor act in concert to generate the gliding motility necessary to propel the parasite into host cells. During this active penetration, host cell transmembrane proteins are excluded from the forming parasitophorous vacuole hence conferring the resistance to acidification and degradative fusion. Apicomplexans possess a large repertoire of microneme proteins that contribute to invasion, but their precise role and the level of functional redundancy remain to be evaluated. Remarkably, most microneme proteins are proteolytically cleaved during biogenesis and post-exocytosis. The significance of the processing events and the identification of the proteases implicated are the object of intensive investigations. These proteases may constitute potential drug targets for intervention against malaria and other diseases caused by these parasites.

The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa.

The apicomplexans are obligate intracellular protozoan parasites that rely on gliding motility for their migration across biological barriers and for host-cell invasion and egress. This unusual form of substrate-dependent motility is powered by the "glideosome", a macromolecular complex consisting of adhesive proteins that are released apically and translocated to the posterior pole of the parasite by the action of an actomyosin system anchored in the inner membrane complex of the parasite. Recent studies have revealed new insights into the composition and biogenesis of Toxoplasma gondii myosin-A motor complex and have identified an exciting set of small molecules that can interfere with different aspects of glideosome function.

Trans-genera reconstitution and complementation of an adhesion complex in Toxoplasma gondii.

Eimeria tenella and Toxoplasma gondii are obligate intracellular parasites belonging to the phylum Apicomplexa. In T. gondii, the microneme protein TgMIC2 contains two well-defined adhesive motifs and is thought to be a key participant in the attachment and invasion of host cells. However, several attempts by different laboratories to generate a knockout (KO) of TgMIC2 have failed, implying that TgMIC2 is an essential gene. As Eimeria and Toxoplasma utilize the same mechanisms of invasion and have highly conserved adhesive proteins, we hypothesized that the orthologous molecule in Eimeria, EtMIC1, could functionally substitute in Toxoplasma to allow a knockout of TgMIC2. TgMIC2 is partnered with a protein called TgM2AP, which corresponds to EtMIC2 in Eimeria. Because the activity of TgMIC2 is most likely tightly linked to its association with TgM2AP, it was thought that the activity of EtMIC1 might similarly require its partner EtMIC2. EtMIC1 and EtMIC2 were introduced into T. gondii, and the presence of EtMIC1 allowed the first knockout clone of TgMIC2 to be obtained. The TgMIC2 KO resulted in significantly decreased numbers of invaded parasites compared to the parental clone. In the absence of TgMIC2, TgM2AP was incorrectly processed and mistargeted to the parasitophorous vacuole instead of the micronemes. These findings indicate that the EtMIC1 can compensate for the essential requirement of TgMIC2, but it cannot fully functionally substitute for TgMIC2 in the invasion process or for supporting the correct maturation and targeting of TgM2AP.

Toxoplasma as a novel system for motility.

Motility is a characteristic of most living organisms and often requires specialized structures like cilia or flagella. An alternative is amoeboid movement, where the polymerization/depolymerization of actin leads to the formation of pseudopodia, filopodia and/or lamellipodia that enable the cell to crawl along a surface. Despite their lack of locomotive organelles and in absence of cell deformation, members of the apicomplexan parasites employ a unique form of locomotion called gliding motility to promote their migration across biological barriers and to power host-cell invasion and egress. Detailed studies in Toxoplasma gondii and Plasmodium species have revealed that this unique mode of movement is dependent on a myosin of class XIV and necessitates actin dynamics and the concerted discharge and processing of adhesive proteins. Gliding is essential for the survival and infectivity of these obligate intracellular parasites, which cause severe disease in humans and animals.

The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury.

Superoxide dismutase, catalase, glutathione peroxidase and peroxiredoxins form an antioxidant network protecting cells against reactive oxygen species (ROS). Catalase is a potent H2O2-detoxifying enzyme, which is unexpectedly absent in some members of the Kinetoplastida and Apicomplexa, but present in Toxoplasma gondii. In T. gondii, catalase appears to be cytosolic. In addition, T. gondii also possesses genes coding for other types of peroxidases, including glutathione/thioredoxin-like peroxidases and peroxiredoxins. This study presents a detailed analysis of the role of catalase in the parasite and reports the existence of antioxidant enzymes localized in the cytosol and the mitochondrion of T. gondii. The catalase gene was disrupted and, in addition, T. gondii cell lines overexpressing either catalase or a cytosolic 1-cys peroxiredoxin, TgPrx2, under the control of a strong promoter were created. Analysis of these mutants confirmed that the catalase activity is cytosolic and is encoded by a unique gene in T. gondii. Furthermore, the catalase confers protection against H2O2 exposure and contributes to virulence in mice. The overexpression of Prx2 also increases protection against H2O2 treatment, suggesting that catalase and other peroxidases function as a defence mechanism against endogenously produced reactive oxygen intermediates and the oxidative stress imposed by the host.

The induction and kinetics of antigen-specific CD8 T cells are defined by the stage specificity and compartmentalization of the antigen in murine toxoplasmosis.

Toxoplasma gondii forms different life stages, fast-replicating tachyzoites and slow-growing bradyzoites, in mammalian hosts. CD8 T cells are of crucial importance in toxoplasmosis, but it is unknown which parasite stage is recognized by CD8 T cells. To analyze stage-specific CD8 T cell responses, we generated various recombinant Toxoplasma gondii expressing the heterologous Ag beta-galactosidase (beta-gal) and studied whether 1) secreted or cytoplasmic Ags and 2) tachyzoites or bradyzoites, which persist intracerebrally, induce CD8 T cells. We monitored the frequencies and kinetics of beta-gal-specific CD8 T cells in infected mice by MHC class I tetramer staining. Upon oral infection of B6C (H-2(bxd)) mice, only beta-gal-secreting tachyzoites induced beta-gal-specific CD8 T cells. However, upon secondary infection of mice that had received a primary infection with tachyzoites secreting beta-gal, beta-gal-secreting tachyzoites and bradyzoites transiently increased the frequency of intracerebral beta-gal-specific CD8 T cells. Frequencies of splenic and cerebral beta-gal-specific CD8 T cells peaked at day 23 after infection, thereafter persisting at high levels in the brain but declining in the spleen. Splenic and cerebral beta-gal-specific CD8 T cells produced IFN-gamma and were cytolytic upon specific restimulation. Thus, compartmentalization and stage specificity of an Ag determine the induction of CD8 T cells in toxoplasmosis.

Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion.

Obligate intracellular apicomplexan parasites rely on gliding motion powered by their actomyosin system to disperse throughout tissues and to penetrate host cells. Toxoplasma gondii myosin A has been implicated in this process, but direct proof has been lacking. We designed a genetic screen to generate a tetracycline-inducible transactivator system in T. gondii. The MyoA gene was disrupted in the presence of a second regulatable copy of MyoA. Conditional removal of this myosin caused severe impairment in host cell invasion and parasite spreading in cultured cells, and unambiguously established the pathogenic function of this motor in an animal model.

'The glideosome': a dynamic complex powering gliding motion and host cell invasion by Toxoplasma gondii.

Motion is an intrinsic property of all living organisms, and each cell displays a variety of shapes and modes of locomotion. How structural proteins support cellular movement and how cytoskeletal dynamics and motor proteins are harnessed to generate order and movement are among the fundamental and not fully resolved questions in biology today. Protozoan parasites belonging to the Apicomplexa are of enormous medical and veterinary significance, being responsible for a wide variety of diseases in human and animals, including malaria, toxoplasmosis, coccidiosis and cryptosporidiosis. These obligate intracellular parasites exhibit a unique form of actin-based gliding motility, which is essential for host cell invasion and spreading of parasites throughout the infected hosts. A motor complex composed of a small myosin of class XIV associated with a myosin light chain and a plasma membrane-docking protein is present beneath the parasite's plasma membrane. According to the capping model, this complex is connected directly or indirectly to transmembrane adhesin complexes, which are delivered to the parasite surface upon microneme secretion. Together with F-actin and as yet unknown bridging molecules and proteases, these complexes are among the structural and functional components of the 'glideosome'.

Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor.

Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite. We report here an extensive biochemical characterization of the functional TgMyoA motor complex. TgMyoA is anchored at the plasma membrane and binds a novel type of myosin light chain (TgMLC1). Despite some unusual features, the kinetic and mechanical properties of TgMyoA are unexpectedly similar to those of fast skeletal muscle myosins. Microneedle-laser trap and sliding velocity assays established that TgMyoA moves in unitary steps of 5.3 nm with a velocity of 5.2 microm/s towards the plus end of actin filaments. TgMyoA is the first fast, single-headed myosin and fulfils all the requirements for power parasite gliding.

Intramembrane cleavage of microneme proteins at the surface of the apicomplexan parasite Toxoplasma gondii.

Apicomplexan parasites actively secrete proteins at their apical pole as part of the host cell invasion process. The adhesive micronemal proteins are involved in the recognition of host cell receptors. Redistribution of these receptor-ligand complexes toward the posterior pole of the parasites is powered by the actomyosin system of the parasite and is presumed to drive parasite gliding motility and host cell penetration. The microneme protein protease termed MPP1 is responsible for the removal of the C-terminal domain of TgMIC2 and for shedding of the protein during invasion. In this study, we used site-specific mutagenesis to determine the amino acids essential for this cleavage to occur. Mapping of the cleavage site on TgMIC6 established that this processing occurs within the membrane-spanning domain, at a site that is conserved throughout all apicomplexan microneme proteins. The fusion of the surface antigen SAG1 with these transmembrane domains excluded any significant role for the ectodomain in the cleavage site recognition and provided evidence that MPP1 is constitutively active at the surface of the parasites, ready to sustain invasion at any time.