Dynamics of the orphan myosin MyoF over Trypanosoma cruzi life cycle and along the endocytic pathway.

Trypanosoma cruzi proliferative forms perform endocytosis through a specialized structure named the cytostome-cytopharynx complex (SPC). The SPC is a specialized invagination of the cell membrane that extends through the cell body towards the posterior regions, with its aperture close to the flagellar pocket. Recently, diverse proteins were found along the cytopharynx, including two myosin motors. One of these is the orphan myosin MyoF, that was proved to be essential for endocytosis in epimastigotes. However, the dynamics of MyoF localization along the endocytic pathway and through the T. cruzi life cycle remain unclear. Using CRISPR-Cas9 genome editing, we generated epimastigotes expressing MyoF fused to mNeonGreen from its endogenous locus. Using these cells, we observed that during the epimastigote cell cycle MyoF signal disappeared during G2, reappearing at early cytokinesis. Additionally, we show that MyoF localization during metacyclogenesis is compatible with the progressive disappearance of the SPC, being absent in metacyclic trypomastigotes. Detergent fractionation showed that MyoF was predominantly present in the insoluble fraction and immunolocalized at the SPC microtubules in whole-mount cytoskeleton preparations. Moreover, during tracer uptake through the SPC, MyoF followed the tracer along the endocytic pathway and was found in posterior compartments after 30 min. Taken together, the data suggest that MyoF may play a role not only at the cargo entry site but also along the endocytic pathway.

On the structural organization of the bacillary band of Trichuris muris under cryopreparation protocols and three-dimensional electron microscopy.

Whipworms of the genus Trichuris are nematode parasites that infect mammals and can lead to various intestinal diseases of human and veterinary interest. The most intimate interaction between the parasite and the host intestine occurs through the anterior region of the nematode body, inserted into the intestinal mucosa during infection. One of the most prominent structures of the nematode surface found at the infection site is the bacillary band, a surface domain formed by a number of cells, mostly stichocytes and bacillary glands, whose structure and function are still under debate. Here, we used confocal microscopy, field emission scanning electron microscopy, helium ion microscopy, transmission electron microscopy and FIB-SEM tomography to unveil the functional role of the bacillary gland cell. We analyzed the surface organization as well as the intracellular milieu of the bacillary glands of Trichuris muris in high pressure frozen/freeze-substituted samples. Results showed that the secretory content is preserved in all gland openings, presenting a projected pattern. FIB-SEM analysis showed that the lamellar zone within the bacillary gland chamber is formed by a set of lacunar structures that may exhibit secretory or absorptive functions. In addition, incubation of parasites with the fluid phase endocytosis marker sulforhodamine B showed a time-dependent uptake by the parasite mouth, followed by perfusion through different tissues with ultimate secretion through the bacillary gland. Taken together, the results show that the bacillary gland possess structural characteristics of secretory and absorptive cells and unequivocally demonstrate that the bacillary gland cell functions as a secretory structure.

Schiefer counter: An alternative method for clonogenic assay evaluation.

INTRODUCTION: Clonogenic assay evaluates the potential of cells to undergo division or generate clones following treatment with a chemical or other agent, thereby allowing the evaluation of cytotoxic and/or antiproliferative effects. Clonogenic assay analysis using traditional methods tends to be time-consuming and yield inconsistent results, whereas results from analyses conducted using automated image processing methods may be misleading or subject to misinterpretation. Thus, the aim of this work was to validate and demonstrate the applicability of a recently developed software. METHODS: Repeatability of measurements was evaluated by comparing results from 10 replicate images from a single well. To evaluate the viability of the software, results were compared with those obtained from manual counting, crystal violet optical density, and up-to-date automated methods. A clonogenic index was experimentally developed using the individual area occupied by colonies, while clone stratification was used to differentiate between antiproliferative and cytotoxic effects. RESULTS: The developed software showed to be a reliable and consistent tool for clonogenic assay evaluation, presenting a repeatability mean error of 0.79% for the number of colonies and 0.89% for the total area of colonies, as well as exhibiting a significant correlation (p < 0.05) with results obtained from widely adopted gold standard methods. The software was also able to detect an appropriate dose-dependent effect as well as a predominant cytotoxic effect of vincristine on MCF-7 cells and calculate the clonogenic index. DISCUSSION: Therefore, this software is adequate for the analysis of clonogenic assay images, differentiating between cytotoxic and antiproliferative trends.

Development of an in vitro system to study the developmental stages of Toxoplasma gondii using a genetically modified strain expressing markers for tachyzoites and bradyzoites.

Toxoplasma gondii, the agent of toxoplasmosis, is an intracellular parasite that can infect a wide range of vertebrate hosts. Toxoplasmosis causes severe damage to immunocompromised hosts and its treatment is mainly based on the combination of pyrimethamine and sulfadiazine, which causes relevant side effects primarily observed in AIDS patients, including bone marrow suppression and hematological toxicity (pyrimethamine) and/or hypersensitivity and allergic skin reactions (sulfadiazine). Thus, it is important to investigate new compounds against T. gondii, particularly those that may act on bradyzoites, which are present in cysts during the chronic disease phase. We propose an in vitro model to simultaneously study new candidate compounds against the two main causative stages of Toxoplasma infection in humans, using the EGS-DC strain that was modified from a type I/III strain (EGS), isolated from a case of human congenital toxoplasmosis in Brazil and engineered to express markers for both stages of development. One feature of this strain is that it presents tachyzoite and bradyzoite in the same culture system and in the same host cell under normal culture conditions. Additionally, this strain presents stage-specific fluorescent protein expression, allowing for easy identification of both stages, thus making this strain useful in different studies. HFF cells were infected and after 4 and 7 days post infection the cells were treated with 10 µM of pyrimethamine or atovaquone, for 48 or 72 h. We used high-throughput screening to quantify the extent of parasite infection. Despite a reduction in tachyzoite infection caused by both treatments, the atovaquone treatment reduced the bradyzoite infection while the pyrimethamine one increased it. Ultrastructural analysis showed that after treatment with both drugs, parasites displayed altered mitochondria. Fluorescence microscopy of cells labeled with MitoTracker CMXRos showed that the cysts present inside the cells lost their mitochondrial membrane potential. Our results indicate that this experimental model is adequate to simultaneously analyze new active compounds against tachyzoite and bradyzoite forms.

Taxonomy of Physaloptera mirandai (Nematoda: Physalopteroidea) based in three-dimensional microscopy and phylogenetic positioning.

Gastrointestinal nematodes are important ecological assets for the maintenance of the biodiversity in the Atlantic Forest in Brazil. They parasitize a number of animals of the local fauna, in which some species can promote serious injuries in the stomach wall of their hosts, which may lead to death. Among these nematodes, parasites of the genus Physaloptera are known to parasitize mammals (particularly carnivores and small rodents), birds and reptiles, being important for the local biodiversity. In this work, three hundred and sixty-two nematodes were recovered from the stomach of twenty-one Metachirus nudicaudatus (Didelphimorphia: Didelphidae) collected in Duas Bocas Biological Reserve, State of Espírito Santo, one of the largest Atlantic Forest remnants and important wildlife refuge of the Atlantic Forest in Brazil. Analysis using fluorescence and scanning electron microscopy as well as phylogenetic assessment using the mitochondrial cytochrome c oxidase subunit I gene showed that the parasites belong to the Physaloptera. Our results show details of the nematode morphology including the cloacal papillae distribution, cuticular topography details, 2D and 3D measurements of the structures with taxonomic importance. Molecular data confirmed the validity of P. mirandai and the phylogeny supported the monophyly of the assemblage formed by Physaloptera and Turgida. The use of a combination of quantitative and multidimensional microscopy tools, such as 3D reconstruction and modeling, allied to phylogenetic analysis may provide grounds for a new approach on helminth taxonomy and structural characterization.

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.

Candida albicans biofilms: comparative analysis of room-temperature and cryofixation for scanning electron microscopy.

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde-based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room-temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room-temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze-substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.

In vitro treatment of Toxoplasma gondii with copper(II) complexes induces apoptosis-like and cellular division alterations.

Toxoplasma gondii is the causative agent of toxoplasmosis, which is one of the most common parasitic diseases in the world. This pathogen causes severe damage to immunocompromised hosts, and the most frequently used therapy is the combination of pyrimethamine and sulfadiazine, which has side effects. Thus, there is a need for new therapies that target T. gondii. Herein, we present the anti-Toxoplasma effect of two new copper(II) complexes: [(H2L1) Cu (µ-Cl)2 Cu(H2L1)] Cl2·5H2O (1) and [(H2L2) Cu (µ-Cl)2 Cu(H2L2)] Cl2·6H2O (2). Complexes (1) and (2) irreversibly controlled parasite growth in vitro, with IC50 values of 0.78µM and 3.57µM, respectively, after 48h. These complexes induced part of the tachyzoite population to convert to bradyzoites, which eventually die. The cell death mechanism was unknown, but signs of apoptosis, such as membrane blebs and nuclear fragmentation, and necrosis, such as plasma membrane disruption, intense cytoplasm vesiculation and the release of cellular contents, were seen. In addition, complex (2) interfered with the correct disposition of the inner membrane complex of the parasite, affecting cell division. These results indicate that these copper complexes have potential effects against T. gondii and may be used as drugs in the future or serve as prototypes for the development of new drugs to treat toxoplasmosis.

Aquatic Insects from the Caatinga: checklists and diversity assessments of Ubajara (Ceará State) and Sete Cidades (Piauí State) National Parks, Northeastern Brazil.

BACKGROUND: Diversity and distribution of Neotropical aquatic insects is still poorly known, with many species to be recorded and many others to be described, due to the small number of taxonomists and sparse faunistic studies. This knowledge is especially poor in the Caatinga Domain in Northeastern Brazil, even though, this region may have played an important historical role in the spatial evolution of faunas of forested areas in northern South America. NEW INFORMATION: Aquatic insect checklists of 96 species from Parque Nacional de Ubajara (Ceará State, Brazil) and 112 species from Parque Nacional de Sete Cidades (Piauí State, Brazil) are presented, representing the following taxa: Elmidae, Epimetopidae, Hydrophilidae, and Torridincolidae (Coleoptera), Hemerodromiinae (Diptera: Empididae), Ephemeroptera, Gerromorpha and Nepomorpha (Hemiptera), Odonata, Plecoptera, and Trichoptera. Because of the scarce number of biological inventories in Northeastern Brazil, several new distributional records (of species, genera, and families) for Brazil, Northeastern Brazil, and Ceará and Piauí states are provided. In addition, several undescribed species were detected, being 26 from Ubajara and 20 from Sete Cidades. Results represent a significant increase to the known fauna of these states, ranging from 13%-70% increase for Ceará and 41% to 91% increase for Piauí. Although both parks are relatively close to each other and within the Caatinga domain, their aquatic fauna display a very high complementarity (89% species), possibly due to structural differences of water bodies sampled in each park. Rarefaction curves based on quantitative light trap samples suggest a much higher expected species richness of aquatic insects at Sete Cidades than at Ubajara National Park. Discussion on biogeographical affinities of this sample of the Caatinga fauna is provided.

Lipids and leukocytes in newborn umbilical vein blood, birth weight and maternal body mass index.

Maternal obesity during pregnancy may influence fetal development and possibly predispose offspring to cardiovascular disease. The aim of the present study was to evaluate the relationship between maternal pre-pregnancy body mass index (BMI) and weight gain during pregnancy, and newborn birth weight, with lipid profile, high-sensitivity C-reactive protein (hs-CRP) and leukocyte in newborns. We performed a cross-sectional study of 245 mothers and their children. Blood was collected from the umbilical vein and assayed for lipid profile, hs-CRP and leukocyte count. Newborns average weight was 3241 g, total cholesterol 53.9 mg/dl, high-density lipoprotein cholesterol (HDL-c) 21.9 mg/dl, low-density lipoprotein cholesterol (LDL-c) 26.2 mg/dl, triglyceride 29.5 mg/dl and leukocytes 13,777/mm3. There was a direct correlation of pre-pregnancy BMI of overweight mothers with total cholesterol (r=0.220, P=0.037) and LDL-c (r=0.268, P=0.011) of newborns. Total cholesterol, LDL-c and HDL-c were higher in pre-term newborns (66.3±19.7, 35.9±14.6 and 25.2±7.7 mg/dl, respectively) that in full-term (52.4±13.1, 25.0±8.7 and 21.5±6.0 mg/dl), with P=0.001, 0.001 and 0.003, respectively. Leukocyte counts were higher in full-term newborns (14,268±3982/mm3) compared with pre-term (9792±2836/mm3, P<0.0001). There was a direct correlation between birth weight and leukocyte counts of newborns (r=0.282, P<0.0001). These results suggest the possible interaction of maternal weight and fetal growth with lipid metabolism and leukocyte count in the newborn, which may be linked to programming of the immune system.

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.

Reduction of Toxoplasma gondii Development Due to Inhibition of Parasite Antioxidant Enzymes by a Dinuclear Iron(III) Compound.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan that can infect a wide range of vertebrate cells. Here, we describe the cytotoxic effects of the dinuclear iron compound [Fe(HPCINOL)(SO4)]2-µ-oxo, in which HPCINOL is the ligand 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol, on T. gondii infecting LLC-MK2 host cells. This compound was not toxic to LLC-MK2 cells at concentrations of up to 200 µM but was very active against the parasite, with a 50% inhibitory concentration (IC50) of 3.6 µM after 48 h of treatment. Cyst formation was observed after treatment, as indicated by the appearance of a cyst wall, Dolichos biflorus lectin staining, and scanning and transmission electron microscopy characteristics. Ultrastructural changes were also seen in T. gondii, including membrane blebs and clefts in the cytoplasm, with inclusions similar to amylopectin granules, which are typically found in bradyzoites. An analysis of the cell death pathways in the parasite revealed that the compound caused a combination of apoptosis and autophagy. Fluorescence assays demonstrated that the redox environment in the LLC-MK2 cells becomes oxidant in the presence of the iron compound. Furthermore, a reduction in superoxide dismutase and catalase activities in the treated parasites and the presence of reactive oxygen species within the parasitophorous vacuoles were observed, indicating an impaired protozoan response against these radicals. These findings suggest that this compound disturbs the redox equilibrium of T. gondii, inducing cystogenesis and parasite death.

Potent In Vitro Antiproliferative Synergism of Combinations of Ergosterol Biosynthesis Inhibitors against Leishmania amazonensis.

Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the Leishmania genus. Treatments available have limited safety and efficacy, high costs, and difficult administration. Thus, there is an urgent need for safer and more-effective therapies. Most trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. In previous studies, we showed that Leishmania amazonensis is highly susceptible to aryl-quinuclidines, such as E5700, which inhibit squalene synthase, and to the azoles itraconazole (ITZ) and posaconazole (POSA), which inhibit C-14α-demethylase. Herein, we investigated the antiproliferative, ultrastructural, and biochemical effects of combinations of E5700 with ITZ and POSA against L. amazonensis. Potent synergistic antiproliferative effects were observed against promastigotes, with fractional inhibitory concentration (FIC) ratios of 0.0525 and 0.0162 for combinations of E5700 plus ITZ and of E5700 plus POSA, respectively. Against intracellular amastigotes, FIC values were 0.175 and 0.1125 for combinations of E5700 plus ITZ and E5700 plus POSA, respectively. Marked alterations of the ultrastructure of promastigotes treated with the combinations were observed, in particular mitochondrial swelling, which was consistent with a reduction of the mitochondrial transmembrane potential, and an increase in the production of reactive oxygen species. We also observed the presence of vacuoles similar to autophagosomes in close association with mitochondria and an increase in the number of lipid bodies. Both growth arrest and ultrastructural/biochemical alterations were strictly associated with the depletion of the 14-desmethyl endogenous sterol pool. These results suggest the possibility of a novel combination therapy for the treatment of leishmaniasis.

Redescription of Spirura guianensis (Nematoda: Spiruridae) from a rare South American Gracile Opossum.

Spirura genus Blanchard, 1849 comprise of nematode parasites that infect primate and marsupial species. Although several taxonomical studies have shown that the infection by this species occurs primarily in the esophagus of primates, evidence for the occurrence of these parasites in other hosts (marsupials, rodents and bats) has become the subject of investigation by several groups. In this work, we describe the presence of Spirura guianensis Ortlepp, 1924 in the marsupial Gracilinanus agilis (Marsupialia: Didelphidae) found in the Pantanal of Mato Grosso do Sul state of Brazil. Structural characteristics of this nematode were identified using light microscopy (bright field and fluorescence stereomicroscopy) and scanning electron microscopy (SEM) approaches. Details of the surface topography such as cephalic projections, ventral boss, details of the caudal papillae and cuticular ornamentations were shown, providing taxonomic characteristics that may help in the establishment of diagnostic protocols. In addition, the presence of this species in a new host and new geographical area of Brazil provide grounds for a revision on the distribution of S. guianensis in South America.

Mitochondrial respiration and genomic analysis provide insight into the influence of the symbiotic bacterium on host trypanosomatid oxygen consumption.

Certain trypanosomatids co-evolve with an endosymbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges. Symbionts were able to respire for up to 4 h after isolation from Angomonas deanei. FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) similarly increased respiration in wild-type and aposymbiotic protozoa, though a higher maximal O2 consumption capacity was observed in the symbiont-containing cells. Rotenone, a complex I inhibitor, did not affect A. deanei respiration, whereas TTFA (thenoyltrifluoroacetone), a complex II activity inhibitor, completely blocked respiration in both strains. Antimycin A and cyanide, inhibitors of complexes III and IV, respectively, abolished O2 consumption, but the aposymbiotic protozoa were more sensitive to both compounds. Oligomycin did not affect cell respiration, whereas carboxyatractyloside (CAT), an inhibitor of the ADP-ATP translocator, slightly reduced O2 consumption. In the A. deanei genome, sequences encoding most proteins of the respiratory chain are present. The symbiont genome lost part of the electron transport system (ETS), but complex I, a cytochrome d oxidase, and FoF1-ATP synthase remain. In conclusion, this work suggests that the symbiont influences the mitochondrial respiration of the host protozoan.

New Aspidoderidae species parasite of Didelphis aurita (Mammalia: Didelphidae): a light and scanning electron microscopy approach.

Nematodes of the family Aspidoderidae (Nematoda: Heterakoidea) Skrjabin and Schikobalova, 1947, are widely distributed in the Americas. The family Aspidoderidae includes the subfamilies Aspidoderinae Skrjabin and Schikobalova, 1947, and Lauroiinae Skrjabin and Schikobalova, 1951. These two subfamilies are delineated by the presence or absence of cephalic cordons at the anterior region. The nematodes in the subfamily Aspidoderinae, which includes the genus AspidoderaRailliet and Henry, 1912, are represented by nematodes with anterior cephalic cordons at the anterior end. The nematodes of the genus AspidoderaRailliet and Henry, 1912, are found in the cecum and large intestine of mammals of the orders Edentata, Marsupialia and Rodentia. Species within this genus have many morphological similarities. The use of scanning electron microscopy allows the specific characterization of the species within this genus. In the present work, we describe a new species of Aspidodera parasite of the large intestine of Didelphis aurita (Mammalia: Didelphidae) Wied-Neuwied, 1826, collected from Cachoeiras de Macacu, Rio de Janeiro. The combination of light and scanning electron microscopy allowed us a detailed analysis of this nematode.

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.

New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4',6-diamidino-2-phenylindole (DAPI)-staining.

Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4',6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multi-photon microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.

Trypanosoma cruzi uses macropinocytosis as an additional entry pathway into mammalian host cell.

Several intracellular pathogens are internalized by host cells via multiple endocytic pathways. It is no different with Trypanosoma cruzi. Evidences indicate that T. cruzi entry may occur by endocytosis/phagocytosis or by an active manner. Although macropinocytosis is largely considered an endocytic process where cells internalize only large amounts of solutes, several pathogens use this pathway to enter into host cells. To investigate whether T. cruzi entry into peritoneal macrophages and LLC-MK2 epithelial cells can be also mediated through a macropinocytosis-like process, we used several experimental strategies presently available to characterize macropinocytosis such as the use of different inhibitors. These macropinocytosis' inhibitors blocked internalization of T. cruzi by host cells. To further support this, immunofluorescence microscopy and scanning electron microscopy techniques were used. Field emission scanning electron microscopy revealed that after treatment, parasites remained attached to the external side of host cell plasma membrane. Proteins such as Rabankyrin 5, tyrosine kinases, Pak1 and actin microfilaments, which participate in macropinosome formation, were localized at T. cruzi entry sites. We also observed co-localization between the parasite and an endocytic fluid phase marker. All together, these results indicate that T. cruzi is able to use multiple mechanisms of penetration into host cell, including macropinocytosis.