Signaling during the invasion of host cells by Toxoplasma gondii

Invasion of host cells is essential for the pathogenicity of Toxoplasma gondii. This review examines the signal transduction pathways that lead to the internalization of T. gondii. We demonstrate that extra- and intracellular Ca(2+) mobilization, Ca(2+)-calmodulin complex and phospholipase A(2) activities are required for T. gondii entry. T. gondii also causes the activation of mitogen-activated protein kinase in infected cells and modifies its ionic environment during its intracellular state. Thus, many of the signaling systems found in other eukaryotes are operative in Toxoplasma invasion.     

Host cell egress and invasion induce marked relocations of glycolytic enzymes in Toxoplasma gondii tachyzoites

Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K(+)] experienced during egress and invasion, a signal that requires changes of [Ca(2+)](c) in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites.     

Molecular partitioning during host cell penetration by Toxoplasma gondii

During invasion by Toxoplasma gondii, host cell transmembrane proteins are excluded from the forming parasitophorous vacuole membrane (PVM) by the tight apposition of host and parasite cellular membranes. Previous studies suggested that the basis for the selective partitioning of membrane constituents may be a preference for membrane microdomains, and this hypothesis was herein tested. The partitioning of a diverse group of molecular reporters for raft and nonraft membrane subdomains was monitored during parasite invasion by time-lapse video or confocal microscopy. Unexpectedly, both raft and nonraft lipid probes, as well as both raft and nonraft cytosolic leaflet proteins, flowed unhindered past the host-parasite junction into the PVM. Moreover, neither a raft-associated type 1 transmembrane protein nor its raft-dissociated counterpart accessed the PVM, while a multispanning membrane raft protein readily did so. Considered together with previous data, these studies demonstrate that selective partitioning at the host-parasite interface is a highly complex process, in which raft association favors, but is neither necessary nor sufficient for, inclusion into the T. gondii PVM.     

Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction

Rhoptries are unique secretory organelles shared by all Apicomplexan invasive stages. They are exocytosed upon host cell invasion and their contents are involved in creating the moving junction that propels the parasite in the cell and in building the parasitophorous vacuole in which the parasite will develop. In addition, some rhoptry proteins are targeted to the host cell nucleus. The array of roles played by these organelles has considerably expanded in the recent years, making them a major clue to the understanding of the early interaction between these parasites and their host. Yet, our knowledge on these organelles is still very poor and much has to be done before we get a clear view of the part they play in Apicomplexan biology.     

Influence of calcium ion on host cell invasion and intracellular replication by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of Ca2+ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and Ca2+ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the Ca2+ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular Ca2+ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.     

Attachment and invasion of host cells by Toxoplasma gondii

Recent studies indicate that Toxoplasma gondii attachment is mediated via a parasite ligand-host cell receptor interaction. Lloyd Kosper and Jose Mineo here survey factors involved in the attachment to and penetration and invasion of host cells by T. gondii.     

Toxoplasma gondii: characterization of monoclonal antibodies that recognize rhoptries

We have previously reported on a series of monoclonal antibodies that recognize the rhoptries of Toxoplasma gondii and that interfere with the action of penetration enhancing factor. The antibodies immunoprecipitate several related antigens from [35S]methionine-labeled parasites that range in size from 60 to 43 kDa. By immunoblot, one of the antibodies reacts with the 60 kDa protein in the presence of protease inhibitors. Trypsin digestion of the antigen destroyed antigenic reactivity indicating that the 60 kDa antigen is a protein. The antigen was stable to periodate oxidation and failed to react with Schiff's reagent, indicating that the antigen contains little or no carbohydrate. Two-dimensional gel electrophoresis followed by immunoblot showed that the antigen recognized by Tg 49 was an acidic protein with an approximate pI of 5.8.     

Characterisation of hexokinase in Toxoplasma gondii tachyzoites

We have cloned the hexokinase [E.C. 2.7.1.1] gene of Toxoplasma gondii tachyzoite and obtained an active recombinant enzyme with a calculated molecular mass of 51,465Da and an isoelectric point of 5.82. Southern blot analysis indicated that the hexokinase gene existed as a single copy in the tachyzoites of T. gondii. The sequence of T. gondii hexokinase exhibited the highest identity (44%) to that of Plasmodium falciparum hexokinase and lower identity of less than 35% to those of hexokinases from other organisms. The specific activity of the homogeneously purified recombinant enzyme was 4.04 micromol/mg protein/min at 37 degrees C under optimal conditions. The enzyme could use glucose, fructose, and mannose as substrates, though it preferred glucose. Adenosine triphosphate was exclusively the most effective phosphorus donor, and pyrophosphate did not serve as a substrate. K(m) values for glucose and adenosine triphosphate were 8.0+/-0.8 microM and 1.05+/-0.25mM, respectively. No allosteric effect of substrates was observed, and the products, glucose 6-phosphate and adenosine diphosphate, had no inhibitory effect on T. gondii hexokinase activity. Other phosphorylated hexoses, fructose 6-phosphate, trehalose 6-phosphate which is an inhibitor of yeast hexokinase, and pyrophosphate, also did not affect T. gondii hexokinase activity. Native hexokinase activity was recovered in both the cytosol and membrane fractions of the whole lysate of T. gondii tachyzoites. This result suggests that T. gondii hexokinase weakly associates with the membrane or particulate fraction of the tachyzoite cell.     

Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum-coated slides. The myosin light-chain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii .     

Toxoplasma gondii: immunocytochemistry of four immunodominant antigens with monoclonal antibodies

Four monoclonal antibodies in which diagnostic usefulness has been observed, concerning congenital, acquired, and reactivated toxoplasmosis, were raised against Toxoplasma gondii tachyzo?tes in order to localize immunodominant antigens. On immunoblots, it appears that McAb IV47, McAB GII9, McAb II38, and McAb IE10 identify families of proteins with estimated molecular weights of 28-30 kDa, 30 kDa, 45-50 kDa, and 66-70 kDa, respectively. By immunogold preembedding techniques one can observe an homogeneous labeling of the outer pellicle of the tachyzo?tes with the McAb GII9 and IV47 and a light labeling with the McAb II38 and IE10. The three-dimensional observation of cell surface antigens is performed by applying a modified metal extraction replica method, i.e., A plasma polymerization method of glow discharge by Tanaka (1979). By immunogold preembedding techniques [with saponin permeabilization (0.1%)], and by immunogold postembedding techniques, a labeling of the rhoptries is observed with McAb GII9 and McAb IV47 but essentially all label is found with McAb II38 and IE10. With McAb GII9 a uniform labeling is observed on the cell surface. By immunoenzymatic techniques (peroxidase) a cell surface labeling is observed with the four McAb. Intracellular Toxoplasma, the outer pellicle, and the vesicles of the network (elaborated by Toxoplasma in parasitophorous vacuole) are also labeled with McAb IE10. These results indicate that McAb GII9 recognizes antigens of the antigen family (P 30) located on the cell surface and in the rhoptries. The antigen recognized by McAb IV47 is essentially located on and beneath the Toxoplasma cell surface membrane, and McAb II38 and IE10 identify preferentially rhoptry proteins.     

The single mitochondrion of tachyzoites of Toxoplasma gondii

The infective tachyzoite form of the protozoan Toxoplasma gondii is able to penetrate into vertebrate host cells and to survive and multiply within a cytoplasmic vacuole known as the parasitophorous vacuole. Previous observations, confirmed in the present study, showed that extracellular, but not intravacuolar, tachyzoites are labeled with rhodamine 123, a dye that specifically binds to functional mitochondria, which present a high transmembrane potential. These observations led to the suggestion that intravacuolar tachyzoites do not possess functional mitochondria. However, our present observations using the new dye CMXRos and observation by confocal laser scanning microscopy (CLSM) showed that the mitochondria of both extracellular and intravacuolar tachyzoites were intensely labeled, indicating that they were functional. In addition, cytochrome c activity could be cytochemically detected in the inner mitochondrial membrane of intravacuolar tachyzoites. Three-dimensional reconstruction of serial optical sections of CMXRos-stained tachyzoites observed by CLSM and of serial thin sections examined by transmission electron microscopy revealed that the protozoan presented only one ramified mitochondrion, reinforcing previous observations by Seeber et al. (1998, Exp. Parasitol. 89, 137-139) Petitprez and Vivier (1972, Protistologica VIII, 199-221).     

Toxoplasma gondii: the role of a 30-kDa surface protein in host cell invasion.

C1E3, a monoclonal antibody recognizing protein P30, a major surface antigen of Toxoplasma gondii tachyzoites, was shown to have a consistent effect on invasion in adult bovine kidney cells. In 10 replicate assays, the overall invasion was reduced to 37% of control values (P less than 0.0001). These results support the role of a functional role for P30 in mediating invasion.     

'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'.     

Developmental differentiation between tachyzoites and bradyzoites of Toxoplasma gondii

An important event in the pathogenesis of toxoplasmosis is the interconversion between the bradyzoite and the tachyzoite stage of Toxoplasma gondii within the intermediate host. The factors that influence either cyst formation (bradyzoites) or reactivation (tachyzoites) are unknown. Uwe Gross, Wolfgang Bohne, Martine Soête and Jean Fran?ois Dubremetz here describe current knowledge about the mechanisms that might lead to the induction of stage differentiation of this protozoan parasite.     

Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii

The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.     

Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites

Since we had previously demonstrated the protective role played by Toxoplasma excreted-secreted antigens, the aim of the present work was to produce monoclonal antibodies directed against these antigens in order to determine if their localization in the parasite is compatible with a mechanism of excretion or secretion. Western immunoblotting analysis revealed three monoclonal antibodies (TG17-179, TG17-43, and TG17-113) raised against excreted-secreted antigens of 28.5, 27, and 21 kDa, respectively. The TG17-179 which reacts with antigens isolated by Concanavalin A affinity chromatography is directed against a glycosylated 28.5-kDa component. Colloidal immunogold labeling showed the ultrastructural localization of the 21-, 27-, and 28.5-kDa antigens in the matrix of the dense granules of tachyzoites and associated with the microvilli network of the parasitophorous vacuole, after host cell invasion. These observations suggest the following mechanism of Toxoplasma secretion: secreted antigens are first stored in tachyzoite-dense granules and are then released inside the parasitophorous vacuole. Among the secretory molecules characterized here, the native 27-kDa antigen recognized by TG17-43 is a calcium-binding protein found to be intermixed with the 21- and 28.5-kDa antigens inside the dense granules and hence could play a role in the packaging of secretory products. In addition, the 21- and 28.5-kDa antigens were also located beneath the parasite plasma-lemma. This particular location could reflect a transient step characteristic of T. gondii secretion.     

Toxoplasma gondii: isolation of tachyzoites rhoptries and incorporation into Iscom

Rhoptries have been isolated from Toxoplasma gondii tachyzoites by subcellular fractionation in isopynic density sucrose gradient. Five bands were observed, and transmission electron microscopy of these indicated that rhoptries were in band 3. This band had a density of 1.17 g/cm(3). Fraction 1 had membrane structures of the parasite. Fraction 2 contained membranes and mitochondria. Fraction 4 had mostly conoid structure and fraction 5 showed ghosts. The electrophoretic and Western blotting analysis of the fractions indicated the presence of a number of proteins. Iscoms were constructed from band 3, which contained the rhoptry structures. Iscom showed a only protein incorporated of 55 kDa. Isolation of the parasite organelles has got in this work is necessary to identification, characterization, and function elucidation of the organelle proteins.     

Toxoplasma gondii RH Ankara: production of evolving tachyzoites using a novel cell culture method

Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of Plasmodium falciparum trophozoites. The product of PFL1555w, a putative cytochrome b₅, localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b₅ reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.     

Toxoplasma gondii: role of the phosphatidylcholine-specific phospholipase C during cell invasion and intracellular development

The effect of D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C, was investigated on cyst development of the Prugniaud strain of Toxoplasma gondii in vitro. Following treatment with the inhibitor 24 h after cell infection, cyst development was affected as assessed by staining with the bradyzoite-specific mAb CC2: the CC2-reactive antigen was shown to be differently located (in the wall versus the matrix under control conditions). This correlated with a decrease in parasite multiplication induced by D609. Pretreatment of the parasites with D609 inhibited their entry into the host cells, whereas pretreatment of the host cells enhanced the intracellular multiplication of the para sites, without any effect on cell invasion or cyst formation. Our results suggest a crucial role for phosphatidylcholine-specific phospholipase C in the pathophysiology of toxoplasmosis.     

Role of calcium during Toxoplasma gondii invasion and egress

Calcium is a ubiquitous signalling molecule involved in a large number of cellular processes in eukaryotic cells. In the obligate intracellular parasite, Toxoplasma gondii, for example, a rise in calcium concentration is associated with significant morphological changes, secretion of proteins involved in host cell invasion and rapid egress from the host cell. Recent findings indicate that calcium released from the parasite's intracellular pools is necessary and sufficient to induce some of the events critical for invasion and egress. In addition, ethanol, a powerful inducer of invasion-related events, is shown here to also induce rapid egress from the host cell, indicating that a common mechanism for calcium release might be involved during both invasion and egress.