A role for coccidian cGMP-dependent protein kinase in motility and invasion

The coccidian parasite cGMP-dependent protein kinase is the primary target of a novel coccidiostat, the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine (compound 1), which effectively controls the proliferation of Eimeria tenella and Toxoplasma gondii parasites in animal models. The efficacy of compound 1 in parasite-specific metabolic assays of infected host cell monolayers is critically dependent on the timing of compound addition. Simultaneous addition of compound with extracellular E. tenella sporozoites or T. gondii tachyzoites inhibited [3H]-uracil uptake in a dose-dependent manner, while minimal efficacy was observed if compound addition was delayed, suggesting a block in host cell invasion. Immunofluorescence assays confirmed that compound 1 blocks the attachment of Eimeria sporozoites or Toxoplasma tachyzoites to host cells and inhibits parasite invasion and gliding motility. Compound 1 also inhibits the secretion of micronemal adhesins (E. tenella MIC1, MIC2 and T. gondii MIC2), an activity closely linked to invasion and motility in apicomplexan parasites. The inhibition of T. gondii MIC2 adhesin secretion by compound 1 was not reversed by treatment with calcium ionophores or by ethanol (a microneme secretagogue), suggesting a block downstream of calcium-dependent events commonly associated with the discharge of the microneme organelle in tachyzoites. Transgenic Toxoplasma strains expressing cGMP-dependent protein kinase mutant alleles that are refractory to compound 1 (including cGMP-dependent protein kinase knock-out lines complemented by such mutants) were used as tools to validate the potential role of cGMP-dependent protein kinase in invasion and motility. In these strains, parasite adhesin secretion, gliding motility, host cell attachment and invasion displayed a reduced sensitivity to compound 1. These data clearly demonstrate that cGMP-dependent protein kinase performs an important role in the host-parasite interaction.     

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.     

Proteomics and glycomics analyses of N-glycosylated structures involved in Toxoplasma gondii--host cell interactions

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.     

Toxoplasma gondii microneme secretion involves intracellular Ca(2+) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores

Calcium-mediated microneme secretion in Toxoplasma gondii is stimulated by contact with host cells, resulting in the discharge of adhesins that mediate attachment. The intracellular source of calcium and the signaling pathway(s) triggering release have not been characterized, prompting our search for mediators of calcium signaling and microneme secretion in T. gondii. We identified two stimuli of microneme secretion, ryanodine and caffeine, which enhanced release of calcium from parasite intracellular stores. Ethanol, a previously characterized trigger of microneme secretion, stimulated an increase in parasite inositol 1,4,5-triphosphate, implying that this second messenger may mediate intracellular calcium release. Consistent with this observation, xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, inhibited microneme secretion and blocked parasite attachment and invasion of host cells. Collectively, these results suggest that T. gondii possess an intracellular calcium release channel with properties of the inositol 1,4,5-triphosphate/ryanodine receptor superfamily. Intracellular calcium channels, previously studied almost exclusively in multicellular animals, appear to also be critical to the control of parasite calcium during the initial steps of host cell entry.     

Calmodulin distribution and the actomyosin cytoskeleton in Toxoplasma gondii.

The gliding motility of the protozoan parasite Toxoplasma gondii and its invasion of cells are powered by an actin-myosin motor. We have studied the spatial distribution and relationship between these two cytoskeleton proteins and calmodulin (CaM), the Ca(2+)-dependent protein involved in invasion by T. gondii. A 3D reconstruction using labeling and tomographic studies showed that actin was present as a V-like structure in the conoidal part of the parasite. The myosin distribution overlapped that of actin, and CaM was concentrated at the center of the apical pole. We demonstrated that the actomyosin network, CaM, and myosin light-chain kinases are confined to the apical pole of the T. gondii tachyzoite. MLCK could act as an intermediate molecule between CaM and the cytoskeleton proteins. We have developed a model of the organization of the actomyosin-CaM complex and the steps of a signaling pathway for parasite motility.     

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.     

Galactose recognition by the apicomplexan parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.     

Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response

Apicomplexan parasites exhibit actin-dependent gliding motility that is essential for migration across biological barriers and host cell invasion. Profilins are key contributors to actin polymerization, and the parasite Toxoplasma gondii possesses a profilin-like protein that is recognized by Toll-like receptor TLR11 in the host innate immune system. Here, we show by conditional disruption of the corresponding gene that T.gondii profilin, while not required for intracellular growth, is indispensable for gliding motility, host cell invasion, active egress from host cells, and virulence in mice. Furthermore, parasites lacking profilin are unable to induce TLR11-dependent production in vitro and in vivo of the defensive host cytokine interleukin-12. Thus, profilin is an essential element of two aspects of T. gondii infection. Like bacterial flagellin, profilin plays a role in motility while serving as a microbial ligand recognized by the host innate immune system.     

Unusual N-glycan structures required for trafficking Toxoplasma gondii GAP50 to the inner membrane complex regulate host cell entry through parasite motility

Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.     

Induction and regulation of conoid extrusion in Toxoplasma gondii

Cell invasion by the intracellular parasite Toxoplasma gondii occurs through an active process that involves dynamic events, such as gliding motility and conoid extrusion, followed by a sequential secretion from specialized secretory organelles. Increase of intracellular Ca²⁺ by ionophores induces conoid extrusion, although in an irreversible way, thus limiting the characterization of the regulatory pathways. In this report we studied the effect of different activating conoid conditions to characterize the regulatory mechanisms involved. Exposure of tachyzoites to ethanol, a well-known activator of microneme secretion through the increase of intracellular Ca²⁺, induced conoid extrusion without affecting parasite viability nor its in vitro invasive capability, in a process that could be completely reverted and repeatedly reactivated. A temporal relationship between conoid extrusion and microneme secretion was here studied. Under this condition, signal transduction pathways and the precise role of the parasite cytoskeleton were characterized. Our results indicate that phospholipase C, Ca²⁺ released through channels sensitive to inositol-3-phosphate and ryanodine, as well as myosin together with actin filaments, but not microtubules, all participate in conoid extrusion. Specific inhibitors for serine-threonine kinases blocked conoid extrusion; in contrast, calmodulin inhibitors did not affect the induction. A regulatory model for conoid activation is here proposed.     

Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ～98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer-sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.     

Secretion of micronemal proteins is associated with toxoplasma invasion of host cells

Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of its pervasive cell invasion is not completely understood. Here, we demonstrate, using several independent assays, that Toxoplasma invasion of host cells is tightly coupled to the release of proteins stored within apical secretory granules called micronemes. Both microneme secretion and cell invasion were highly temperature dependent, and partial depletion of microneme resulted in a transient loss of infectivity. Chelation of parasite intracellular calcium strongly inhibited both microneme release and invasion of host cells, and this effect was partially reversed by raising intracellular calcium using the ionophore A23187. We also provide evidence that a staurosporine-sensitive kinase activity regulates microneme discharge and is required for parasite invasion of host cells. Additionally, we demonstrate that, during apical attachment to the host cell, the micronemal protein MIC2 is released at the junction between the parasite and the host cell. During invasion, MIC2 is successively translocated towards the posterior end of the parasite and is shed before entry of the parasite into the vacuole. Furthermore, we show that the full-length cellular form of MIC2, but not the proteolytically modified secreted form of MIC2, binds specifically to host cells. Collectively, these observations strongly imply that micronemal proteins play a role in Toxoplasma invasion of host cells.     

A cleavable propeptide influences Toxoplasma infection by facilitating the trafficking and secretion of the TgMIC2-M2AP invasion complex

Propeptides regulate protein function and trafficking in many eukaryotic systems and have emerged as important features of regulated secretory proteins in parasites of the phylum Apicomplexa. Regulated protein secretion from micronemes and host cell invasion are inextricably linked and essential processes for the apicomplexan parasite Toxoplasma gondii. TgM2AP is a propeptide-containing microneme protein found in a heterohexameric complex with the microneme protein TgMIC2, a protein that has a demonstrated fundamental role in gliding motility and invasion. TgM2AP function is also central to these processes, because disruption of TgM2AP (m2apKO) results in secretory retention of TgMIC2, leading to reduced TgMIC2 secretion from the micronemes and impaired invasion. Because the TgM2AP propeptide is predicted to be processed in an intracellular site near where TgMIC2 is retained in m2apKO parasites, we hypothesized that the propeptide and its proteolytic removal influence trafficking and secretion of the complex. We found that proTgM2AP traffics through endosomal compartments and that deletion of the propeptide leads to defective trafficking of the complex within or near this site, resulting in aberrant processing and decreased secretion of TgMIC2, impaired invasion, and reduced virulence in vivo, mirroring the phenotypes observed in m2apKO parasites. In contrast, mutation of several cleavage site residues resulted in normal localization, but it affected the stability and secretion of the complex from the micronemes. Therefore, the propeptide and its cleavage site influence distinct aspects of TgMIC2-M2AP function, with both impacting the outcome of infection.     

Role of the parafusin orthologue,PRP1, in microneme exocytosis and cell invasion in Toxoplasma gondii

The association of PRP1, a Paramecium parafusin orthologue, with Toxoplasma gondii micronemes, now confirmed by immunoelectron microscopy, has here been studied in relation to exocytosis and cell invasion. PRP1 becomes labelled in vivo by inorganic 32P and is dephosphorylated when ethanol is used to stimulate Ca2+-dependent exocytosis of the micronemes. The ethanol Ca2+-stimulated exocytosis is accompanied by translocation of PRP1 and microneme content protein (MIC3) from the apical end of the parasite. Immunoblotting showed that PRP1 is redistributed inside the parasite, while microneme content is secreted. To study whether similar changes occur during cell invasion, quantitative microscopy was performed during secretion, invasion and exit (egress) from the host cell. Time-course experiments showed that fluorescence intensities of PRP1 and MIC3 immediately after invasion were reduced 10-fold compared to preinvasion levels, indicating that PRP1 translocation and microneme secretion accompanies invasion. MIC3 regained fluorescence intensity and apical distribution after 15 min, while PRP1 recovered after 1 h. Intensity of both proteins then increased throughout the parasite division period until host cell lysis, suggesting the need to secrete microneme proteins to egress. These studies suggest that PRP1 associated with the secretory vesicle scaffold serves an important role in Ca2+-regulated exocytosis and cell invasion.     

Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii

Apicomplexan parasites, including Toxoplasma gondii, apically attach to their host cells before invasion. Recent studies have implicated the contents of micronemes, which are small secretory organelles confined to the apical region of the parasite, in the process of host cell attachment. Here, we demonstrate that microneme discharge is regulated by parasite cytoplasmic free Ca2+ and that the micronemal contents, including the MIC2 adhesin, are released through the extreme apical tip of the parasite. Microneme secretion was triggered by Ca2+ ionophores in both the presence and the absence of external Ca2+, while chelation of intracellular Ca2+ prevented release. Mobilization of intracellular calcium with thapsagargin or NH4Cl also triggered microneme secretion, indicating that intracellular calcium stores are sufficient to stimulate release. Following activation of secretion by the Ca2+ ionophore A23187, MIC2 initially occupied the apical surface of the parasite, but was then rapidly treadmilled to the posterior end and released into the culture supernatant. This capping and release of MIC2 by ionophore-stimulated tachyzoites mimics the redistribution of MIC2 that occurs during attachment and penetration of host cells, and both events are dependent on the actin-myosin cytoskeleton of the parasite. These studies indicate that microneme release is a stimulus-coupled secretion system responsible for releasing adhesins involved in cell attachment.     

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.     

Assembly and export of a Toxoplasma microneme complex in Giardia lamblia

The microneme proteins of Toxoplasma gondii belong to a large family of adhesins of apicomplexan parasites involved in motility and host cell invasion. During secretory transport, soluble micronemes associate with membrane-bound carriers/escorters and become exposed on the parasite surface as complexes with an array of adhesive domains. Previously, we have exploited the intestinal protozoan Giardia lamblia as an expression system to produce correctly folded and unglycosylated monomeric surface proteins of T. gondii. Here, we report assembly and export of a trimeric microneme (MIC1/4/6) adhesin complex from Toxoplasma. Co-expressed, recombinant microneme proteins were used to investigate structural requirements for microneme complex formation. In addition, export of a microneme subunit induced development of novel Golgi-like compartments demonstrating the existence of post endoplasmic reticulum structures involved in constitutive secretion in this 'Golgi-less' cell. Recreation of the trimeric microneme escorter-cargo system in Giardia is a versatile tool to analyse universal requirements for complex assembly, receptor-ligand interactions and Golgi neogenesis in the basal Giardia secretory system.     

The toxoplasma proteins MIC2 and M2AP form a hexameric complex necessary for intracellular survival

Toxoplasma gondii parasites gain entry into host cells through a process that depends on apically stored adhesins that are strategically released during invasion. One of these adhesins, microneme protein 2 (MIC2), is a type one transmembrane protein that binds to an accessory protein known as MIC2-associated protein (M2AP). Together the MIC2 x M2AP complex participates in host cell attachment and invasion. The short cytoplasmic C-domain of MIC2 is implicated in protein trafficking and mediating an association with the parasite cytoskeleton. To define the role of the cytoplasmic domain of MIC2, proteins lacking the C-domain were expressed in transgenic T. gondii. Surprisingly, protein trafficking and secretion were not affected. We hypothesized that mutant mic2 lacking the C-domain might be escorted to the micronemes by association with endogenous wild-type MIC2 possessing functional transmembrane and cytoplasmic domains. To investigate this interaction, native blue gels and gel filtration were employed to identify a stable macromolecular MIC2 x M2AP complex of approximately 450 kDa. Our findings reveal that MIC2 and M2AP proteins form stable hexamers consisting of three alphabeta dimers. Resolution of this complex has implications for how MIC2 x M2AP associates with host cell receptors and the cytoskeleton to facilitate parasite motility and invasion.     

A conserved subtilisin-like protein TgSUB1 in microneme organelles of Toxoplasma gondii

Proteolytic processing plays a significant role in the process of invasion by the obligate intracellular parasite Toxoplasma gondii. We have cloned a gene, TgSUB1, encoding for a subtilisin-type serine protease found in T. gondii tachyzoites. TgSUB1 protein is homologous to other Apicomplexan and bacterial subtilisins and is processed within the secretory pathway of the parasite. Initial cleavage occurs in the endoplasmic reticulum, after which the protein is transported to micronemes, vesicles that secrete early during host cell invasion. Upon stimulation of microneme secretion, TgSUB1 is cleaved into smaller products that are secreted from the parasite. This secondary processing is inhibited by brefeldin A and serine protease inhibitors. TgSUB1 is a candidate processing enzyme for several microneme proteins cleaved within the secretory pathway or during invasion.