Cell cycle-dependent entry of Toxoplasma gondii into synchronized HL-60 cells

The degree of attraction of Toxoplasma gondii to vertebrate cells varies with cell type and cell phase. Human promyelocytic leukemia cells, HL-60, were synchronized by double thymidine block method and co-cultured with Toxoplasma for 1 hr at each cell stage to investigate the cell cycle specific susceptibility of parasites to host cells. For 30 hr the average number of Toxoplasma that invaded was a little changed except at 3 hr from G1/S phase boundary which concurred with the peak point of DNA synthesis. At 3 hr which is a relatively short interval compared to whole S phase, modification of cells by parasitic invasion was most remarkable. The number of Toxoplasma that penetrated was increased to more than six times. The shape of the cells became sludgy and almost indiscernible by strong accessibility of parasites only for an hour of mid-S phase. The same fluctuation was also observed at the second peak of S phase but weakly. This suggests that there be surface molecules concerning with the attachment of Toxoplasma to the host cells, which is expressed at special point of S phase. Further studies on the specific protein or similar molecules related could be carried out using synchronized HL-60 cells.     

Toxoplasma gondii: Ultrastructure study of the entry of tachyzoites into mammalian cells

Toxoplama gondii (Apicomplexa: Coccidia), an obligatory intracellular parasite with a unique capacity to invade virtually all nucleated cell type from warm-blooded vertebrate hosts. Despite the efficiency with which Toxoplasma enters its host cell, it remains unresolved if invasion occurs by direct penetration of the parasite or through phagocytosis. In the present work, electron microscopic study was designed to examine the entry process of Toxoplasma (RH strain) into macrophages and non phagocytic-host cells (Hela cells) and to observe the ultrastructure changes associated with intracellular parasitism. The results showed that both active invasion and phagocytosis were occurred and revealed that invasion is an ordered process that initiates with binding of the parasite at its apical end followed by tight-fitting invagination of the host cell membrane and a prominent constriction in the parasite at the site of penetration. The process ended by the professional parasitophorous vacuole that is distinct at the outset from those formed by phagocytosis in which once Toxoplasma triggered, phagocytic uptake can proceed by capture of the parasite within a loose fitting vacuole formed by localized membrane ruffling. The cytopathic effects of the parasite on macrophages and Hela cells were demonstrated within 5–15 h post-inoculation in the form of degenerative mitochondria, swelling Golgi apparatus and widening of endoplasmic reticulum indicating intracellular oedema. These changes were exaggerated and several cells were found dead after 48–72 h.     

Toxoplasma gondii: penetration into differentiating Friend erythroleukemia cells.

The ability of Toxoplasma gondii tachyzoites to penetrate erythroid-differentiating Friend erythroleukemia cells (FL cells) was examined in vitro. The parasites were incubated for 4 hr with FL cells which had been induced to synthesize spectrin, a major erythrocyte membrane protein, or hemoglobin by culturing the cells in the presence of dimethylsulfoxide (DMSO) or sodium butyrate. The penetration rate, in terms of both the average number of T. gondii per cell and the percentage of cells containing parasities, observed in the DMSO-treated FL cells was depressed as compared with that found in untreated cells. However, this depression was not related to the presence of spectrin or hemoglobin. This conclusion was based on the fact that the penetration rate in the butyrate-treated cells was the same as that in untreated cells. These results suggest that it is not the presence of spectrin and hemoglobin that inhibits penetration by T. gondii. The failure of T. gondii to enter mammalian erythrocytes is discussed in relation to surface changes in FL cells undergoing erythroid differentiation.     

Influenza entry pathways in polarized MDCK cells

In non-polarized cell culture models, influenza virus has been shown to enter host cells via multiple endocytic pathways, including classical clathrin-mediated endocytic routes (CME), clathrin- and caveolae-independent routes and macropinocytosis. However, little is known about the entry route of influenza virus in differentiated epithelia, in vivo site of infection for influenza virus. Here, we show that in polarized Madin–Darby canine kidney type II (MDCK II) cells, influenza virus has a specific utilization of the clathrin-mediated endocytic pathway and requires Eps15 for host cell entry.     

Invasion of vertebrate cells by Toxoplasma gondii

Toxoplasma gondii actively invades its host cell rather than entering by conventional phagocytosis. Invasion is powered by a microfilament-based motility system in the parasite that leads to invagination of the host cell plasma membrane to form a novel intracellular vacuole. This vacuole resists fusion with the endocytic network of the host cell and provides a protective environment for replication of the parasite.     

Effect of extracellular ions on motility and cell entry in Toxoplasma gondii

Toxoplasma gondii tachyzoites were quiescent in mouse peritoneal fluid or in K2SO4 buffer at pH 8.2. They became consistently motile when K+ was replaced by other monovalent or divalent cations at a constant pH (pH = 8.2). They also became motile when Cl- was substituted for SO4(2-). Nitrate or SCN-, can also be substituted for Cl- to a certain extent. Tachyzoites showed independent movement for more than 15 min in KCl, and for about 5 min in the other buffers at pH 8.2 after which they were exhausted and stopped. These tachyzoites could not then be further stimulated to motility by renewal of the suspension buffer. Infection of monolayer cells was demonstrated only with parasites which were motile during inoculation. The highest infectivity was thus obtained either with freshly collected tachyzoites or with those preincubated in K2SO4 buffer for 30 min at 37 degrees C at alkaline pH and thus not yet exhausted for motility. Approximately 34 to 38% of these latter organisms were seen to enter cells when they were inoculated into cultures immediately after being resuspended in MEM for 30 min at 37 degrees C. Conversely, those whose motility had been exhausted by the preincubation in buffers other than K2SO4, pH 8.2 could not enter monolayer cells. Additionally, parasites were unable to enter cells when inoculated into cultures in K2SO4 buffer at alkaline pH; instead they remained quiescent on the surface of the monolayer cells, suggesting that Toxoplasma enters the host cells by active invasion.     

Tight junction formation in cultured epithelial cells (MDCK)

Summary Synthesis and assembly of tight junctions are studied in monolayers of MDCK cells plated at a density sufficient for confluence, allowed to attach for 1 hr, and transferred to fresh media without cells containing or not Ca²⁺, 20 hr later, while monolayers with Ca²⁺ have fully developed junctions that confer an electrical resistance across of 346±51 cm², those without Ca²⁺ have a negligible resistance. If at this time Ca²⁺ is added, junctions assemble and seal with a fast kinetics, that can be followed through the development of electrical resistance, penetration of ruthenium red, and electron microscopy. Drugs that impair synthesis, maturation and transport of proteins (cycloheximide, tunicamycin, monensin) indicate that protein components are synthesized early upon plating, do not seem to require N-glycosylation, and are stored in the Golgi compartment. Upon addition of Ca²⁺ they are transferred to the membrane with the participation of microfilaments but not of microtubules. These components seem to insert directly in the position they occupy in the strands, and the cell circles its perimeter with one strand as early as 15 min, even if in some segments it only consists of a row of particles. New strands develop in association with previous ones, and the pattern completes in 4 to 6 hr. Ca²⁺ is required for the maintenance of the assembly and also for the sealing with neighboring cells. These processes cannot occur below 25°C. Serum is not required. Polarized distribution of intramembrane particles (IMP) in apical and basolateral regions follows the same time course as junction formation, in spite of the fence constituted by those strands that are already assembled. This suggests that IMP do not redistribute by lateral displacements in the plane of the membrane, but by removal and insertion in the apical and basolateral domains.     

Differential development of Toxoplasma gondii in neural cells

In this article, Remi Fagard and colleagues discuss the properties of neurons that lead to their low infection by Toxoplasma gondii, and the role that cytokines such as tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) might play.     

Toxoplasma gondii inhibits granzyme B-mediated apoptosis by the inhibition of granzyme B function in host cells

Host defense to the apicomplexan parasite Toxoplasma gondii is critically dependent on CD8⁺ T cells, whose effector functions include the induction of apoptosis in target cells following the secretion of granzyme proteases. Here we demonstrate that T. gondii induces resistance of host cells to apoptosis induced by recombinant granzyme B. Granzyme B induction of caspase-independent cytochrome c release was blocked in T. gondii-infected cells. Prevention of apoptosis could not be attributed to altered expression of the Bcl-2 family of apoptotic regulatory proteins, but was instead associated with reduced granzyme B-mediated, caspase-independent cleavage of procaspase 3 to the p20 form in T. gondii-infected cells, as well as reduced granzyme B-mediated cleavage of the artificial granzyme B substrate, GranToxiLux. The reduction in granzyme B proteolytic function in T. gondii-infected cells could not be attributed to altered granzyme B uptake or reduced trafficking of granzyme B to the cytosol, implying a T. gondii-mediated inhibition of granzyme B activity. Apoptosis and GranToxiLux cleavage were similarly inhibited in T. gondii-infected cells exposed to the natural killer-like cell line YT-1. The endogenous granzyme B inhibitor PI-9 was not up-regulated in infected cells. We believe these findings represent the first demonstration of granzyme B inhibition by a cellular pathogen and indicate a new modality for host cell protection by T. gondii that may contribute to parasite immune evasion.     

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.     

Penetration of maturating red blood cells by Toxoplasma gondii

Penetration of Toxoplasma gondii tachyzoites was studied in vitro using murine erythroid cells at different stages of development. Toxoplasma gondii penetrated nucleated erythroblasts and macroreticulocytes from foetal mouse liver and the circulating erythrocytes of foetal, neonatal or severely anaemic adult mice. Immature reticulocytes were more susceptible to penetration than mature ones, indicating that some change in their membrane properties occurred during maturation. The present results confirmed our previous finding that the major erythrocyte membrane-specific proteins do not prevent erythrocyte penetration since these proteins are known to be present in the reticulocyte membrane.     

Cytoskeleton of Toxoplasma gondii

The cytoskeleton of Toxoplasma gondii was studied by electron microscopy using whole mounts of detergent-extracted parasites and thin sections of routine preparations, tannic acid-stained organisms, and detergent-extracted parasites. In whole mounts, the spiral arrangement of the 22 pellicular microtubules closely corresponded to the pattern of surface ridges seen previously by scanning electron microscopy and reflected the torsion of the parasite body during locomotion. The microtubules had free posterior ends and were anchored anteriorly in the polar ring, presumed to be a microtubule organizing center (MTOC). The insertions of the microtubules were supported by blunt projections of the polar ring, forming a cogwheel pattern in transverse view. The internal microtubules had 13 protofilaments and were twice the length of the conoid. They extended through the conoid and ended at the anterior preconoidal ring, presumably a second MTOC. The subunits of the conoid were arranged in a counterclockwise spiral when traced from base to tip, as were the pellicular microtubules. We postulate that as the conoid moves, the polar ring complex moves along the spiral pathway of the conoid subunits. Retraction of the conoid would then rotate the polar ring, producing the torsion of the body we observed by SEM.     

Molecular cloning of the 82-kDa heat shock protein (HSP90) of Toxoplasma gondii associated with the entry into and growth in host cells

Among the monoclonal antibodies (mAb) against Toxoplasma gondii, mAb Tg485 specifically reacted with an 82-kDa cytoplasmic protein of tachyzoites. The protein was secreted from extracellular tachyzoites, but was not released into the parasitophorous vacuole after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg485. The full-length cDNA was amplified by the 5(')-RACE method and sequenced. The deduced amino acid sequence of the 82 kDa protein reacting with Tg485 revealed a polypeptide of 708 amino acids showing significant homology to the heat shock protein 90 (HSP90) family of other organisms, especially to those of apicomplexan species. Treatment with geldanamycin, a drug known to interfere with HSP90 function, did not affect the secretion of TgHSP90 from extracellular tachyzoites, but the entry of the tachyzoites into host cells and the intracellular growth of the parasite were significantly disturbed.     

Atomic resolution insight into host cell recognition by Toxoplasma gondii

The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Micronemal proteins (MICs) are released onto the parasite surface just before invasion of host cells and play important roles in host cell recognition, attachment and penetration. Here, we report the atomic structure for a key MIC, TgMIC1, and reveal a novel cell-binding motif called the microneme adhesive repeat (MAR). Using glycoarray analyses, we identified a novel interaction with sialylated oligosaccharides that resolves several prevailing misconceptions concerning TgMIC1. Structural studies of various complexes between TgMIC1 and sialylated oligosaccharides provide high-resolution insights into the recognition of sialylated oligosaccharides by a parasite surface protein. We observe that MAR domains exist in tandem repeats, which provide a highly specialized structure for glycan discrimination. Our work uncovers new features of parasite-receptor interactions at the early stages of host cell invasion, which will assist the design of new therapeutic strategies.     

Regulation of the MDCK Cell Tight Junction

The sodium flux across individual tight junctions (TJ) of low-resistance MDCK cell monolayers grown on glass coverslips was determined as a measure of paracellular permeability. Increases in perfusate glucose concentration from 5 to 25 mm decreased tight junction Na permeability. This permeability decrease was not specific as nonmetabolizable analogues of glucose caused similar diminutions in TJ Na permeability. Stimulation of protein kinase A increased TJ Na permeability, and inhibition of protein kinase A decreased TJ Na permeability. Transepithelial electrical resistance of monolayers grown on permeable supports did not change as predicted from the observed alterations in TJ Na permeability of monolayers grown on glass coverslips. Fluorescent labeling of cell F-actin showed that increased F-actin in the perijunctional ring correlated with higher TJ Na permeability. Although a low dose of cytochalasin D did not change TJ Na permeability, it disrupted the cytoskeleton and blocked the decrease in TJ Na permeability caused by glucose. Cytochalasin D failed to block the effects of protein kinase A stimulation or inhibition on TJ Na permeability. We conclude that tight junction sodium permeability is regulated both by protein kinase A activity and by other processes involving the actin cytoskeleton. Received: 17 June 1997/Revised: 28 August 1997     

Interactions between Toxoplasma gondii and its mammalian host cells

Toxoplasma is a protozoan parasite that is uniquely adapted for invading and surviving within a wide range of host cells. The parasite actively invades the cell, forming a novel vacuole that originates from the host cell plasma membrane. The vacuole membrane is rapidly modified to remove host cell proteins and this compartment subsequently resists fusion with all other host cell endocytic compartments. Shortly after invasion, the parasite secretes a variety of proteins by a process of regulation exocytosis and elaborates an extensive array of membranous tubules that form a network connecting with the vacuolar membrane. Understanding the formation and modification of this unique vacuole may reveal novel mechanisms for subverting host cell endocytic pathways that lead to intracellular survival.     

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.