Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro.

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a 'cage' around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo.

Toxoplasma gondii is a protozoan that infects 30% of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.

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 4days 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.

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays.

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

Bacteroides fragilis adherence to Caco-2 cells.

The ability of ten Bacteroides fragilis strains isolated from intestinal and non-intestinal infections, normal flora and environment to adhere to human colon carcinoma cells, Caco-2, was examined. The adherence capacity varied among the strains tested from strongly adherent (76-100%) to non- or weakly adherent (0-25%). Negative staining with Indian ink showed that all the strains were capsulated, although strain 1032 (strongly adherent and originated from bacteremia) had the highest rate of capsulated cells in the culture. All strains studied presented an electron-dense layer and no fimbrial structures in their surface after PTA negative staining. The analysis of the strains with ruthenium red showed the presence of an acidic polysaccharide and also surface vesicles in all of them. The strain 1032 presented an aggregative adherence pattern toward Caco-2 cells monolayers. It could be seen trapped by elongated microvilli and surrounded by extracellular material in the scanning electron microscope. Treatment with sodium periodate (100 mM/1 h) reduced significantly its adherence capacity and also the expression of an electron-dense layer and of the capsule, detected with PTA and Indian ink staining, respectively. We suggest that the capsular polysaccharide might mediate the adherence of the B. fragilis to Caco-2 cells.

Surface charge and surface carbohydrates of Cyptobia salmositica virulent and avirulent forms and of C. bullocki (Kinetoplastida: Cryptobiidae).

The surface charge and surface carbohydrate residues of the virulent (freshly isolated from the fish blood) and avirulent forms (from culture) of Cryptobia salmositica and one strain of C. bullocki were studied. Measurements of the zeta potential of parasites showed that C. bullocki and the virulent form of C. salmositica had a net negative surface charge of about -15 mV, whereas the attenuated form of C. salmositica showed a surface charge of -7.9 mV. Enzymatic treatments of parasites with neuraminidase, trypsin, or phospholipase C indicated the presence of sialic acid residues, phosphate groups, and protein glycoconjugates as components of the Cryptobia surface that accounted for their surface charge. Residues of alpha-D-man, alpha- and beta-D-gal, alpha-D-galNAc, alpha-L-fuc, and D-glcNAc could be detected on the surface of all parasites by specific fluorescein isothiocyanate (FITC)- and colloidal gold-labeled lectins. The cell surface of the avirulent form of C. salmositica showed the strongest reactivity to almost all lectins tested. A remarkable binding pattern of lectins in the anterior region of parasites was observed.

Computer aided three-dimensional reconstruction of the free-living protozoan Bodo sp. (Kinetoplastida:Bodonidae).

Computer aided three-dimensional (3-D) reconstructions of Bodo sp., a free-living kinetoplastid, were made from ultrathin (100 nm) and semithin (200 nm) serial sections, which were observed by transmission electron microscopy. Organelles and membrane systems were digitized into a computer and three-dimensional images were generated using the SYNU software package. It was observed that the internal disposition of structures like the contractile vacuole, the Golgi complex, the mitochondrion, and the nucleus maintain a constant relationship relative to each other and to the cytostome and flagellar pocket. The Golgi complex and the contractile vacuole elements are not apparently connected. The contractile vacuole fills a significant volume in the cell. Volume alterations occurring during systole/diastole cycles of the contractile vacuole are compensated by cytoskeletal adaptations. There is a subpellicular microtubule-free area adjacent to the contractile vacuole. Morphological evidence indicates that the flagellar pocket may not be the only site of vacuolar content elimination. A better view of the cytoskeleton was obtained in detergent-extracted cells, where a set of curved microtubules was observed separating the flagellar pocket from the cytostome and surrounding the cytopharynx.

Freeze-fracture study of Bodo sp. (Kinetoplastida: Bodonidae).

Freeze-fracture technique was used to analyse the structure of conventionally fixed and quickly frozen Bodo sp., a free-living kinetoplastid. In the former method, chemically fixed and cryopreserved cells presented a corrugated membrane pattern in the flagella and cell body surfaces. In the latter, however, replicas from quickly frozen unfixed flagellates showed membranes with a smoother aspect, allowing the observation of intramembranous particles (IMPs) on the fracture faces, hardly detectable in previously fixed samples. The IMPs were randomly distributed throughout the cell surface, except in the sparsely seen short IMP rows.

The cell surface charge of Bodo sp. (Kinetoplastida: Bodonidae).

Cytochemistry, using cationized ferritin particles at pH 7.2 and colloidal iron particles at pH 1.8, and cell electrophoresis were used to analyze the surface charge of the protozoan Bodo sp. Surface anionic sites are homogeneously distributed throughout the protozoan surface, including the flagella, the cytostome and the flagellar pocket. Based on treatment of the cells with neuraminidase, trypsin and phospholipase C it was shown that surface exposed sialic acid residues and phosphate groups significantly contribute to the negative surface charge of Bodo sp.

Comparative study of the surface charge of some isolates of trypanosomatids of the genus Phytomonas.

The surface charge of different isolates of Phytomonas from Euphorbia hyssopifolia, Euphorbia pinea, Euphorbia characias and Manihot esculenta was analysed by the binding of cationic particles (colloidal iron hydroxide at pH 1.8 and cationized ferritin at pH 7.2) to the protozoan surface and by determination of the cell electrophoretic mobility (EPM). All the isolates had a net negative surface charge, and the isolate from E. hyssopifolia manifested the greatest negative charge. A good relationship between the electrophoretic mobility data and the density of the cationic particles on the parasite's surface, as seen in ultrathin sections, was observed. Neuraminidase treatment did not significantly reduce the mean EPM of the parasites analysed.