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.     

Ca2+-Dependence of Conoid Extrusion in Toxoplasma gondii Tachyzoites

The role of Ca²⁺ in conoid extrusion was investigated in isolated Toxoplasma gondii tachyzoites by treatment with Ca²⁺-ionophores, Ca²⁺-chelating agents and an inhibitor of the Ca²⁺-ATPase at the endoplasmic reticulum. The results were evaluated by light phase-contrast microscopy and electron microscopy. lonomycin (0.5-1 μM) caused an immediate and sustained extrusion of the conoid in up to 80% of the tachyzoites, depending on the concentrations of ionophore and Ca²⁺ in the medium. However, over 50% of the tachyzoites extruded the conoid when treated with ionomycin in Ca²⁺-free saline complemented with EGTA. The effect of ionomycin was reversible and could be induced a second time in about half of the responsive population. Similar results were obtained with A23187. Conoid extrusion induced by ionomycin in Ca²⁺-free medium was almost completely abolished when the tachyzoites were previously loaded with a permeable compound known to chelate intracellular Ca²⁺ (BAPTA/AM; 25μM). On the other hand, exposure of tachyzoites to the Ca²⁺-ATPase inhibitor thapsigargin (0.5-1μM) produced significant extrusion of the conoid. Tachyzoites loaded with BAPTA/AM as well as those treated with ionomycin, i.e. with conoids paralyzed in opposite positions, had a diminished capacity to invade cultured epithelial cells. A substantial reduction in the response to stimulation by ionomycin was found also in parasites treated with cytochalasin-D, a drug that depolymerizes actin-filaments. The results suggest that Ca²⁺-release from internal stores may act as a key signal to activate a mechanism of conoid extrusion probably mediated, at least in part, by actin-filaments.     

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: 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.     

Spontaneous cystogenesis in vitro of a Brazilian strain of Toxoplasma gondii

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation in the life cycle of the parasite have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK₂ epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK₂ cells after 4 days of infection, when 72.3 ± 15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the fully conversion of parasites presenting the typical characteristics of bradyzoites as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also detected. Besides, the scanning microscopy revealed that the intracyst matrix tubules were shorter than those from the parasitophorous vacuole intravacuolar network and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle.     

Scanning Electron Microscopy of Toxoplasma gondii: Parasite Torsion and Host-Cell Responses during Invasion1

Scanning electron microscopy confirmed our previous finding that toxoplasmas actively invade mouse peritoneal cells that are inhibited from phagocytosis. The parasites entered cells with the conoid end first and sometimes showed a counter-clockwise torsion of the body during invasion. Counter-clockwise torsion was also noted in free toxoplasmas. Host-cell responses to active invasion varied with experimental conditions and with the type of host cell. Under adverse culture conditions for phagocytosis, normal macrophages formed rudimentary filopodia or lamellipodia around the tips of in vading toxoplasmas; macrophages subjected to hyperthermia before similar incubation with toxoplasmas showed little or no response to invasion. Normal and heat-treated lymphocytes showed little surface reaction to invasion, but occa ionally a flocculent collar was seen around the tip of an invading toxoplasma. Scanning electron microscopy provides clues to possil'e mechanisms of toxoplasma locomotion and host-cell invasion.     

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.     

Cytoskeleton of Toxoplasma gondii1

The cytoskeleton of Toxoplasma gondii was studied by electron microscopy using 1) whole mounts of detergent-extracted parasites and 2) 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.     

Subcellular localization of cadmium in the root cells of<Emphasis Type="Italic">Allium cepa</Emphasis> by electron energy loss spectroscopy and cytochemistry

The ultrastructural investigation of the root cells ofAllium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver grains were abundantly localized in the vesicles, which were distributed in the cytoplasm along the cell wall.     

Itraconazole affects Toxoplasma gondii endodyogeny

The antifungal agent itraconazole is an effective drug against systemic mycoses inhibiting cytochrome P-450-mediated ergosterol synthesis, essential for fungal survival. In this work, we show the activity of this azole as a potential agent against Toxoplasma gondii, the causative agent of toxoplasmosis. Monolayers of LLC-MK2 epithelial cells infected with tachyzoites of RH strain were incubated with different concentrations of itraconazole for 24 and 48 h. The IC(50) values obtained were 114.0 and 53.6 nM for 24 and 48 h, respectively. Transmission electron microscopy (TEM) analysis of itraconazole-treated intracellular tachyzoites showed endoplasmic reticulum and nuclear envelope swelling. The drug also caused rupture of the parasite's surface membrane and affected the parasite's division by endodyogeny. This observation was confirmed both by fluorescence microscopy of cells labeled with diamidino-2-phenylindole and by three-dimensional reconstruction of serial thin sections analyzed by TEM. The treatment with itraconazole led to the formation of a mass of daughter cells, suggesting the interruption of the scission process during the parasite's cell division.     

Capsulation of in vitro and in vivo grown Bacteroides species

By centrifugation on a four step Percoll density gradient cells of Bacteroides species could be separated according to the size of extracellular structure. The difference in size was visible by both light and electron microscopy. Two structures were observed on Bacteroides fragilis by electron microscopy, namely a fibrous network and an electron dense layer. An electron dense layer was visible on Bacteroides ovatus only when stained with ruthenium red. B. fragilis cells grown in the mouse peritoneal cavity did not produce a large fibrous network. An electron dense layer was observed on some cells in the presence of ruthenium red stain and cells possessing this layer were phagocytosed in vivo.     

Mode of Action of Ponazuril Against Toxoplasma gondii Tachyzoites in Cell Culture

ABSTRACT. Toxoplasma gondii is an important apicomplexan parasite of humans and other warm-blooded animals. Ponazuril is a triazine anticoccidial recently approved for use in horses in the United States. We investigated the inode of action of ponazuril against developing RH strain T. gondii tachyzoites in African green monkey kidney cells. Host cells were infected with  2.0 × 10⁵  tachyzoites and treated with 5 μg/ml ponazuril. Cultures were fixed and examined by transmission electron microscopy 3 days after treatment. Ponazuril interfered with normal parasite division. This led to the presence of multinucleate schizonts stages. Up to six tachyzoites were observed partially budded from the surface of these schizonts. Large vacuoles developed in these schizonts and they eventually degenerated.     

Significance of electron dense microbodies in trap cells of the nematophagous fungus Arthrobotrys oligospora

We have studied the fate of electron dense microbodies in nematode-trapping organs (traps) of the fungus A. oligospora during the initial hours following nematode capture. The interaction studies were performed with isolated traps which had captured a nematode under conditions where the fungal cells had no access to external energy sources. Video enhanced contrast microscopy showed that under these conditions the number of dense bodies present in the trap cell that formed the penetration tube, rapidly decreased. During subsequent penetration and development of the infection bulb this decrease continued while at this time common cell organelles such as mitochondria and vacuoles were formed. This was confirmed by electron microscopy which also revealed that the dense bodies were degraded by means of an autophagic process. The organelles were degraded individually and finally turned into compartments which, based on ultrastructural criteria, were considered vacuoles. Fusion of such vacuoles into larger organelles frequently occurred. The degradation process was initiated early in the interaction since initial stages were already evident within 15 min after capture. Generally it took 1–2 h before the infection bulb had fully developed and trophic hyphae formation started. During this time the original trap cell, characterized by numerous dense bodies, was transformed into an active vegetative hyphal cell containing typical cell organelles such as nuclei, mitochondria, a strongly proliferated endoplasmic reticulum, vacuoles and normal microbodies but lacked dense bodies. This disappearance of dense bodies was confined to the cell that penetrated the nematode and—less frequently—its two neighbouring cells in the hyphal loop. In the other cells, constituting the trap, the dense bodies remained unaffected. As will be discussed, the present results support our current view that traps of A. oligospora contribute to the survival of the organism in its natural environment.     

Dynamin inhibitor impairs Toxoplasma gondii invasion

The protozoan parasite Toxoplasma gondii infects its host cells through an active mechanism. In this work, we obtained evidence that host cells also play a fundamental role during the infection process. We found that previous incubation of the host cells, but not the parasites, with Dynasore, a small molecule that inhibits dynamin GTPase activity, markedly reduced the penetration of T. gondii tachyzoites into LLC-MK2 cells. In contrast, parasite adhesion to the host cell surface increased, as observed both by light and electron microscopy. Intriguingly, the few parasites internalized by Dynasore-treated cells remained in vacuoles located at the periphery of the cell, in contrast to the perinuclear localization seen in the control.     

Effects of miltefosine treatment in fibroblast cell cultures and in mice experimentally infected with Neospora caninum tachyzoites

Miltefosine was investigated for its activity against Neospora caninum tachyzoites in vitro, and was shown to inhibit the proliferation of N. caninum tachyzoites cultured in human foreskin fibroblasts (HFF) with an IC50 of 5·2 μM. Treatment of infected cells with 25 μM miltefosine for a period of 10 h had only a parasitostatic effect, while after 20 h of treatment parasiticidal effects were observed. This was confirmed by transmission electron microscopy of N. caninum-infected and miltefosine-treated HFF. Administration of miltefosine to N. caninum-infected Balb/c female mice at 40 mg/kg/day for 14 days resulted in 6 out of 10 mice exhibiting weight loss, ruffled coat and apathy between days 7 and 13 post-infection. In the group that received placebo, only 2 out of 8 mice succumbed to infection, but the cerebral burden was significantly higher compared to the miltefosine treatment group. In a second experiment, the time-span of treatment was reduced to 5 days, and mice were maintained without further treatment for 4 weeks. Only 2 out of 9 mice in the miltefosine treatment group exhibited signs of disease, while 8 out of 10 mice succumbed to infection in the placebo group. These results showed that miltefosine hampered the dissemination of parasites into the CNS during experimental N. caninum infection in mice.     

“O_S-732人体成骨肉瘤细胞系”超微结构及细胞化学的研究

本文研究了 Os-732细胞株的超微结构及酶的特性。在 SEM 下细胞形态呈多样性,细胞表面形成树枝状伪足;TEM 下胞质内含丰富的扩张粗面内质网,发达的高尔基复合体及分泌泡;大量碱性磷酸酶作用的沉积物出现于胞膜表面。这些特征均显示它与成骨细胞的相似性。为证明此株细胞是威骨系统的肿瘤提供可靠的依据。结合细胞株的动物接种,染色体和免疫学特性等研究,证明此细胞株是一新的成骨肉瘤细胞株。     

Light- and electron-microscopic study of the endolymphatic sac of the tree frog,Hyla arborea japonica

Summary The endolymphatic sac of the tree frog and its crystals were observed by light- and electron microscopy. Scanning electron microscopy revealed that the crystals have a faceted body and two pointed ends. Light- and transmission electron microscopy revealed that the endolymphatic sac is composed of many small chambers. In their lumina, numerous ghosts of crystals that resulted from decalcification were observed. The ghosts were demarcated by a linear dense material or embedded in a flocculent substance. The epithelium of the endolymphatic sac is simple squamous or cuboidal and peculiar cytoplasmic granules are found in most cells. The granules are surrounded by a limiting membrane and have varying electron density. Some granules contain a core and/or tubular structures. Vacuoles containing large ghosts are also found in the epithelial cells. These ghosts were quite similar to those in the lumen and sometimes coexist with cell debris. The fine structure of the endolymphatic sac and its crystals is discussed.     

Fine Structure of Hepatozoon mocassini (Apicomplexa,Eucoccidiorida) Gamonts and Modifications of the Infected Erythrocyte Plasmalemma1

Transmission electron microscopy and scanning electron microscopy were used to investigate the fine structure of Hepatozoon mocassini gamonts and modifications of the infected erythrocyte plasmalemma. Intraerythrocytic gamonts were contained within a parasitophorous vacuole. An electron-lucid space observed between the gamont pellicle and the membrane of the vacuole corresponded to the unstained space described in light microscopy studies. Gamonts possessed a conoid, polar ring, subpellicular microtubules, four pairs of rhoptries, micronemes, ovoid granules, mitochondria with tubular cristae, and a pellicle composed of three individual unit membranes. The conoid had an anterior diameter of 320 nm, a posterior diameter of 360 nm, and a length of 150 nm. In contrast to a report on Hepatozoon aegypti, no micropore or “canopy-like structure” was observed. The plasmalemma of infected erythrocytes exhibited two types of modifications: gross membrane deformations and knobs with an electron-dense central mass. These knobs are structurally distinct from previously described membrane excrescences.     

Surface coat transformation and capsule formation by Leuconostoc mesenteroides NCDO 523 in the presence of sucrose

When Leuconostoc mesenteroides NCDO 523 was grown in MRS browth, electron microscopy of cells fixed in the presence of ruthenium red showed that the cell wall was covered with a thin layer of filamentous material. When MRS-grown cells were resuspended in the same medium supplemented with 3.6% sucrose, this surface coat doubled in thickness and a number of radial thickenings appeared within it. After 3 h the filamentous component of the surface coat had disappeared leaving only the radial projections. The progressive accumulation of polymer to produce a capsule visible by light microscopy was observed in only about 20% of the population. In this minority of cells, a dense globular dextran composed of fibrillar and particulate elements was always produced in the initial stages of synthesis. After 18 h, the dextran capsule was generally composed of an inner globular and outer fibrillar layer. It appeared that the outer layer was derived from the globular dextran of the capsule by a process of dispersion.     

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.