Changes in the Proliferation of the Neural Progenitor Cells of Adult Mice Chronically Infected with Toxoplasma gondii.

During Toxoplasma gondii chronic infection, certain internal factors that trigger the proliferation of neural progenitor cells (NPCs), such as brain inflammation, cell death, and changes in cytokine levels, are observed. NPCs give rise to neuronal cell types in the adult brain of some mammals. NPCs are capable of dividing and differentiating into a restricted repertoire of neuronal and glial cell types. In this study, the proliferation of NPCs was evaluated in CD-1 adult male mice chronically infected with the T. gondii ME49 strain. Histological brain sections from the infected mice were evaluated in order to observe T. gondii tissue cysts. Sagittal and coronal sections from the subventricular zone of the lateral ventricles and from the subgranular zone of the hippocampal dentate gyrus, as well as sagittal sections from the rostral migratory stream, were obtained from infected and non-infected mice previously injected with bromodeoxyuridine (BrdU). A flotation immunofluorescence technique was used to identify BrdU+ NPC. The scanning of BrdU+ cells was conducted using a confocal microscope, and the counting was performed with ImageJ® software (version 1.48q). In all the evaluated zones from the infected mice, a significant proliferation of the NPCs was observed when compared with that of the control group. We concluded that chronic infection with T. gondii increased the proliferation of NPCs in the three evaluated zones. Regardless of the role these cells are playing, our results could be useful to better understand the pathogenesis of chronic toxoplasmosis.

Rescue of Mitochondrial Function in Hutchinson-Gilford Progeria Syndrome by the Pharmacological Modulation of Exportin CRM1.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder caused by the expression of progerin, a mutant variant of Lamin A. Recently, HGPS studies have gained relevance because unraveling its underlying mechanism would help to understand physiological aging. We previously reported that the CRM1-mediated nuclear protein export pathway is exacerbated in HGPS cells, provoking the mislocalization of numerous protein targets of CRM1. We showed that normalization of this mechanism by pharmacologically inhibiting CRM1 with LMB (specific CRM1 inhibitor), mitigates the senescent phenotype of HGPS cells. Since mitochondrial dysfunction is a hallmark of HGPS, in this study we analyze the effect of LMB on mitochondrial function. Remarkably, LMB treatment induced the recovery of mitochondrial function in HGPS cells, as shown by the improvement in mitochondrial morphology, mitochondrial membrane potential, and ATP levels, which consequently impeded the accumulation of ROS but not mitochondrial superoxide. We provide evidence that the beneficial effect of LMB is mechanistically based on a combinatory effect on mitochondrial biogenesis via upregulation of PGC-1α expression (master transcription cofactor of mitochondrial genes), and mitophagy through the recovery of lysosomal content. The use of exportin CRM1 inhibitors constitutes a promising strategy to treat HGPS and other diseases characterized by mitochondrial impairment.

A comprehensive ultrastructural analysis of the Toxoplasma gondii cytoskeleton.

The invasive nature of Toxoplasma gondii is closely related to the properties of its cytoskeleton, which is constituted by a group of diverse structural and dynamic components that play key roles during the infection. Even if there have been numerous reports about the composition and function of the Toxoplasma cytoskeleton, the ultrastructural organization of some of these components has not yet been fully characterized. This study used a detergent extraction process and several electron microscopy contrast methods that allowed the successful isolation of the cytoskeleton of Toxoplasma tachyzoites. This process allowed for the conservation of the structures known to date and several new structures that had not been characterized at the ultrastructural level. For the first time, characterization was achieved for a group of nanofibers that allow the association between the polar apical ring and the conoid as well as the ultrastructural characterization of the apical cap of the parasite. The ultrastructure and precise location of the peripheral rings were also found, and the annular components of the basal complex were characterized. Finally, through immunoelectron microscopy, the exact spatial location of the subpellicular network inside the internal membrane system that forms the pellicle was found. The findings regarding these new structures contribute to the knowledge concerning the biology of the Toxoplasma gondii cytoskeleton. They also provide new opportunities in the search for therapeutic strategies aimed at these components with the purpose of inhibiting invasion and thus parasitism.

Protein Serine/Threonine Phosphatase Type 2C of Leishmania mexicana.

Protein phosphorylation and dephosphorylation are increasingly recognized as important processes for regulating multiple physiological mechanisms. Phosphorylation is carried out by protein kinases and dephosphorylation by protein phosphatases. Phosphoprotein phosphatases (PPPs), one of three families of protein serine/threonine phosphatases, have great structural diversity and are involved in regulating many cell functions. PP2C, a type of PPP, is found in Leishmania, a dimorphic protozoan parasite and the causal agent of leishmaniasis. The aim of this study was to clone, purify, biochemically characterize and quantify the expression of PP2C in Leishmania mexicana (LmxPP2C). Recombinant LmxPP2C dephosphorylated a specific threonine (with optimal activity at pH 8) in the presence of the manganese divalent cation (Mn+2). LmxPP2C activity was inhibited by sanguinarine (a specific inhibitor) but was unaffected by protein tyrosine phosphatase inhibitors. Western blot analysis indicated that anti-LmxPP2C antibodies recognized a molecule of 45.2 kDa. Transmission electron microscopy with immunodetection localized LmxPP2C in the flagellar pocket and flagellum of promastigotes but showed poor staining in amastigotes. Interestingly, LmxPP2C belongs to the ortholog group OG6_142542, which contains only protozoa of the family Trypanosomatidae. This suggests a specific function of the enzyme in the flagellar pocket of these microorganisms.

The Arp2/3 Inhibitory Protein Arpin Is Required for Intestinal Epithelial Barrier Integrity.

The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.

Proteomic characterization of the pellicle of Toxoplasma gondii.

Toxoplasma gondii is one of the most successful intracellular parasites in the world. The dynamic, adhesion, invasion, and even replication capabilities of Toxoplasma are based on dynamic machinery located in the pellicle, a three membrane complex that surrounds the parasite. Among the proteins that carry out these processes are inner membrane complex (IMC) proteins, gliding-associated proteins (GAP), diverse myosins, actin, tubulin, and SRS proteins. Despite the importance of the pellicle, the knowledge of its composition is limited. Broad protein identification from an enriched pellicle fraction was obtained by independent digestion with trypsin and chymotrypsin and quantified by mass spectrometry. By trypsin digestion, 548 proteins were identified, while by chymotrypsin digestion, additional 22 proteins were identified. Besides, a group of "sequences related to SAG1" proteins (SRS) were detected together with unidentified new proteins. From identified SRS proteins, SRS51 was chosen for analysis and modeling as its similarities with crystallized adhesion proteins, exhibiting the presence of a spatial groove that is apparently involved in adhesion and cell invasion. As SRS proteins have been reported to be involved in the activation of the host's immune response, further studies could consider them as targets in the design of vaccines or of drugs against Toxoplasma. SIGNIFICANCE: To date, the proteomic composition of the pellicle of Toxoplasma is unknown. Most proteins reported in Toxoplasma pellicle have been poorly studied, and many others remain unidentified. Herein, a group of new SRS proteins is described. Some SRS proteins previously described from pellicle fraction have adhesion properties to the host cell membrane, so their study would provide data related to invasion mechanism and to open possibilities for considering them as targets in the design of immunoprotective strategies or the design of new pharmacological treatments.

Toxoplasma gondii excreted/secreted proteases disrupt intercellular junction proteins in epithelial cell monolayers to facilitate tachyzoites paracellular migration.

Toxoplasma gondii shows high dissemination and migration properties across biological barriers infecting immunologically privileged organs. Toxoplasma uses different routes for dissemination; however, the mechanisms are not fully understood. Herein, we studied the effects of proteases present in excretion/secretion products (ESPs) of Toxoplasma on MDCK cell monolayers. Ultrastructural analysis showed that ESPs of Toxoplasma disrupt the intercellular junctions (IJ) of adjacent cells. The tight junction (TJ) proteins ZO-1, occludin, and claudin-1 suffered a progressive decrease in protein levels upon ESPs treatment. In addition, ESPs induced mislocalization of such TJ proteins, along with the adherent junction protein E-cadherin, and this was prevented by pre-treating the ESPs with protease inhibitors. Reorganisation of cytoskeleton proteins was also observed. Endocytosis inhibitors, Dyngo®-4a and Dynasore, impeded the modifications, suggesting that TJ proteins internalisation is triggered by the ESPs proteases hence contributing to the loss of IJ. The observed disruption in TJ proteins went in line with a decrease in the transepithelial electrical resistance of the monolayers, which was significantly blocked by pre-treating ESPs with metalloprotease and serine protease inhibitors. Moreover, exposure of cell monolayers to ESPs facilitated paracellular migration of tachyzoites. Our results demonstrate that Toxoplasma ESPs contain proteases that can disrupt the IJ of epithelial monolayers and this could facilitate the paracellular route for Toxoplasma tissue dissemination and migration.

Anti-toxoplasma, antioxidant and cytotoxic activities of Pleopeltis crassinervata (Fée) T. Moore hexane fraction.

The apicomplexan parasite Toxoplasma gondii (T. gondii) causes toxoplasmosis in humans. Pyrimethamine and sulfadiazine that are the drugs of choice to treat the disease, produce severe side effects as well as failure treatments because of drug resistance; thus, novel anti-Toxoplasma compounds are needed and natural compounds can be a good source to obtain them, as medicinal plants have been used to control other apicomplexan parasites. Pleopeltis crassinervata (P. crassinervata) is a fern used in some rural areas of Mexico to treat among other malaises, mouth ulcers, gastrointestinal problems and parasites. Therefore, the efficacy of extracts and fractions obtained from P. crassinervata fronds was evaluated on the viability of T. gondii RH strain tachyzoites by the Stytox green method. RH is the prototypical type 1 Toxoplasma strain, isolated for the first time from the brain of a patient boy named R. H. Its phytochemical profile, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Hep-2 cytotoxicity and antioxidant activity by ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) methods, were also assessed. Hexane fraction exhibited the highest anti-Toxoplasma activity with an IC50 of 16.90 µg/mL. This fraction did not show antioxidant activity and contained at least 2 terpenoid type compounds with retention factor (Rf) of 0.75 and 0.86. The fraction was not toxic to the host cells in doses up to 50 µg/mL. P. crassinervata frond hexane fraction seems to be a good candidate to obtain possible anti-Toxoplasma compounds. This study is the first to report the biological, antioxidant and cytotoxic activity of P. crassinervata fern.

Proteomic and structural characterization of self-assembled vesicles from excretion/secretion products of Toxoplasma gondii.

After the cell invasion, the parasite Toxoplasma gondii locates within a parasitophorous vacuole to proliferate. It continuously modifies the composition of the parasitophorous vacuole by the secretion of GRA and ROP proteins, some of which become inserted into the vacuole membrane, remain as soluble proteins or involved in the intravacuolar network. In this report, we analyze the excretion/secretion products and the vesicles released by extracellular tachyzoites, this structures were morphologically analyzed by electron microscopy and characterized by mass spectrometry. The structural analysis showed parasites secreting in vitro individual vesicles with similarities to ectosomes and exosomes and which characterized to self-assembly in vitro forming vesicle-tubular structures morphologically similar to the intravacuolar network from infected cells. The vesicle-tubular structures were recognized with antibodies against ROP2 and GRA2. In addition, analysis by Western blot evidenced proteins from the secretory organelles. A detailed proteomic analysis of exosomes, ectosomes and soluble proteins released in vitro is here reported. Presence of GRA proteins in secretions from resting extracellular parasites indicates that these molecules are not exclusively secreted within the parasitophorous vacuole of the infected cell as reported but they are constitutively excreted/secreted even in an extracellular condition. Data are available via ProteomeXchange with identifier PXD013767. SIGNIFICANCE: Extracellular tachyzoites constitutively secrete components that previously were considered be secreted only within the parasitophorous vacuole, suggesting that in the infected host these molecules are in direct interaction with cells and molecules of the host cell including those of the immune response.

Characterization of metalloproteases and serine proteases of Toxoplasma gondii tachyzoites and their effect on epithelial cells.

Toxoplasma gondii can infect all nucleated cells from warm-blooded organisms. After infection, Toxoplasma spreads throughout the body and migrates across biological barriers, such as the intestinal and blood-brain barriers, as well as the placenta in pregnant women. The mechanisms for parasite dissemination are still unknown; however, proteases could play a role as a virulence factor. The aim of this study was to detect and to characterize proteases in whole-cell extracts and in excretion/secretion products from tachyzoites of the RH strain isolated from infected mice. Both fractions were analyzed by gelatin and casein zymography and by azocasein degradation. The biochemical characterization of proteases included standardization of optimal conditions for their activation, such as pH, the presence of cofactors, and a reducing agent. In both fractions, we detected at least nine gelatin-degrading metalloproteases in the range of 50 to 290 kDa. The proteases present in the excretion/secretion products were found as soluble proteins and not associated with exosome-like vesicles or other secretory vesicles. Moreover, by using casein zymography, it was possible to detect three serine proteases. Exposure of MDCK cells to excretion/secretion products modified the organization of the cell monolayer, and this effect was reverted after washing thoroughly with PBS and inhibition by metalloprotease and serine protease inhibitors. Proteomic analysis of excretion/secretion products identified 19 proteases. These findings suggest that tachyzoites of a highly virulent strain of Toxoplasma use a battery of proteases to modify the epithelium, probably as a strategy to facilitate their tissue dissemination.

Isomers of conjugated linoleic acid induce insulin resistance through a mechanism involving activation of protein kinase Cε in liver cells.

Conjugated linoleic acid (CLA) constitutes a group of isomers derived from linoleic acid. Diverse studies have suggested that these unsaturated fatty acids have beneficial effects on human health. However, it has also been reported that their consumption can generate alterations in hepatic tissue. Thus, in the present study, we evaluated the effect of two of the major isomers of CLA, cis-9, trans-11-CLA and trans-10, cis-12-CLA, in the regulation of insulin signaling in a hepatic cell model, clone 9 (C9). We found that the two isomers decrease insulin-stimulated phosphorylation of the main proteins involved in insulin signaling, such as Akt at Ser473 and Thr308, the insulin receptor at Tyr1158, IRS-1 at Tyr632, and GSK-3 at Ser9/21. Protein expression, however, was unaffected. Interestingly, both isomers of CLA promoted phosphorylation and activation of PKCε. Inhibition of PKCε activity by a dominant-negative form or knockdown of endogenous PKCε prevented the adverse effects of CLA isomers on insulin-induced Akt phosphorylation. Additionally, we also found that both isomers of CLA increase phosphorylation of IRS-1 at Ser612, a mechanism that probably underlies the inhibition of IRS-1 signaling by PKCε. Using confocal microscopy, we found that both isomers of CLA induced lipid accumulation in C9 cells with the presence of spherical cytosolic vesicles, suggesting their identity as neutral lipid droplets. These findings indicate that cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers could have a significant role in the development of insulin resistance in hepatic C9 cells through IRS-1 serine phosphorylation, PKCε activation, and hepatic lipid accumulation.

The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection.

Candida albicans is an opportunistic fungus that is part of the normal microflora commonly found in the human digestive tract and the normal mucosa or skin of healthy individuals. However, in immunocompromised individuals, it becomes a serious health concern and a threat to their lives and is ranked as the leading fungal infection in humans worldwide. As existing treatments for this infection are non-specific or under threat of developing resistance, there is a dire necessity to find new targets for designing specific drugs to defeat this fungus. Some authors reported the presence of the transglutaminase activity in Candida and Saccharomyces, but its identity remains unknown. We report here the phenotypic effects produced by the inhibition of transglutaminase enzymatic activity with cystamine, including growth inhibition of yeast cells, induction of autophagy in response to damage caused by cystamine, alteration of the normal yeast division pattern, changes in cell wall, and inhibition of the yeast-to-mycelium transition. The latter phenomenon was also observed in the C. albicans ATCC 26555 strain. Growth inhibition by cystamine was also determined in other Candida strains, demonstrating the importance of transglutaminase in these species. Finally, we identified enolase 1 as the cell wall protein responsible for TGase activity. After studying the inhibition of enzymatic activities with anti-CaEno1 antibodies and through bioinformatics studies, we suggest that the enolase and transglutaminase catalytic sites are localized in different domains of the protein. The aforementioned data indicate that TGase/Eno1 is a putative target for designing new drugs to control C. albicans infection.

Mycophenolic acid induces differentiation of Toxoplasma gondii RH strain tachyzoites into bradyzoites and formation of cyst-like structure in vitro.

The biochemical and structural changes that occur during the conversion of Toxoplasma gondii tachyzoites to bradyzoites and the formation of tissue cyst are not well understood. Maintaining cells infected with T. gondii type II and III strains under stress conditions induces the tachyzoite-bradyzoite in vitro differentiation, along with the formation of cyst-like structures. However, due to the long exposure to such conditions required to induce the differentiation, the severe damages in the host cell and the low encystation frequency, it has been difficult to dissect in more detail these processes. Here, we successfully induced the in vitro formation of Toxoplasma cysts-like structures from tachyzoites of the type I RH strain by treating with mycophenolic acid, an inhibitor of the inosine monophosphate dehydrogenase. Mycophenolic acid is a drug widely used for HXGPRT positive selection of Toxoplasma mutant strains along with xanthine incubation in the culture medium; under such conditions, formation of tissue cysts has not been reported. We show that the exposure of extracellular tachyzoites to mycophenolic acid in absence of xanthine, followed by host cell invasion, triggered their differentiation into cyst-like structures. The differential expression of CST1, BAG1, and SAG1 molecules, as well as the structural modifications of infected cells, was characterized during the formation of cyst-like structures in vitro. These findings will allow the characterization of signaling pathways involved in tachyzoite to bradyzoite conversion and formation of tissue cysts.

Isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives induce regulated necrosis-like cell death on Leishmania (Leishmania) mexicana.

Leishmaniasis is a neglected tropical disease caused by the parasite of the genus Leishmania. About 13 million people are infected worldwide, and it is estimated that 350 million are at risk of infection. Clinical manifestations depend on the parasite species and factors related to the host such as the immune system, nutrition, housing, and financial resources. Available treatments have severe side effects; therefore, research currently focuses on finding more active and less toxic compounds. Quinoxalines have been described as promising alternatives. In this context, 17 isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives were evaluated as potential leishmanicidal agents. Their effect on the cell metabolism of Leishmania mexicana promastigotes and their cytotoxic effects on the J774.A1 cell line and on erythrocytes were evaluated, and their selectivity index was calculated. Compounds T-069 (IC50 = 1.49 µg/mL), T-070 (IC50 = 1.71 µg/mL), T-072 (IC50 = 6.62 µg/mL), T-073 (IC50 = 1.25 µg/mL), T-085 (IC50 = 0.74 µg/mL), and T-116 (IC50 = 0.88 µg/mL) were the most active against L. mexicana promastigotes and their mechanism of action was characterized by flow cytometry and microscopy. Compound T-073, the most selective quinoxaline derivative, induced cell membrane damage, phosphatidylserine exposition, reactive oxygen species production, disruption of the mitochondrion membrane potential, and DNA fragmentation, all in a dose-dependent manner, indicating the induction of regulated necrosis. Light and transmission electron microscopy showed the drastic morphological changes induced and the mitochondrion as the most sensitive organelle in response to T-073. This study describes the mechanism by which active isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide quinoxalines affect the parasite.

Antibacterial and cell penetrating effects of LFcin17-30, LFampin265-284, and LF chimera on enteroaggregative Escherichia coli.

Lactoferrin (LF) is a protein with antimicrobial activity, which is conferred in part by 2 regions contained in its N-terminal lobe. These regions have been used to develop the following synthetic peptides: lactoferricin17-30, lactoferrampin265-284, and LF chimera (a fusion of lactoferricin17-30 and lactoferrampin265-284). We have reported that these LF peptides have antibacterial activity against several pathogenic bacteria; however, the exact mechanism of action has not been established. Here, we report the effects of LF peptides on the viability of enteroaggregative Escherichia coli (EAEC) and the ability of these peptides to penetrate into the bacteria cytoplasm. The viability of EAEC treated with LF peptides was determined via enumeration of colony-forming units, and the binding and internalization of the LF peptides was followed via immunogold labeling and electron microscopy. Treatment of EAEC with 20 and 40 µmol/L LF peptides reduced bacterial growth compared with untreated bacteria. Initially the peptides associated with the plasma membrane, but after 5 to 30 min of incubation, the peptides were found in the cytoplasm. Remarkably, bacteria treated with LF chimera developed cytosolic electron-dense structures that contained the antimicrobial peptide. Our results suggest that the antibacterial mechanism of LF peptides on EAEC involves their interaction with and penetration into the bacteria.

A new type of quinoxalinone derivatives affects viability, invasion, and intracellular growth of Toxoplasma gondii tachyzoites in vitro.

Quinoxalinone derivatives, identified as VAM2 compounds (7-nitroquinoxalin-2-ones), were evaluated against Toxoplasma gondii tachyzoites of the RH strain. The VAM2 compounds were previously synthesized based on the design obtained from an in silico prediction with the software TOMOCOMD-CARDD. From the ten VAM2 drugs tested, several showed a deleterious effect on tachyzoites. However, VAM2-2 showed the highest toxoplasmicidal activity generating a remarkable decrease in tachyzoite viability (in about 91 %) and a minimal alteration in the host cell. An evident inhibition of host cell invasion by tachyzoites previously treated with VAM2-2 was observed in a dose-dependent manner. In addition, remarkable alterations were observed in the pellicle parasite, such as swelling, roughness, and blebbing. Toxoplasma motility was inhibited, and subpellicular cytoskeleton integrity was altered, inducing a release of its components to the soluble fraction. VAM2-2 showed a clear and specific deleterious effect on tachyzoites viability, structural integrity, and invasive capabilities with limited effects in host cells morphology and viability. VAM2-2 minimum inhibitory concentration (MIC50) was determined as 3.3 µM ± 1.8. Effects of quinoxalinone derivatives on T. gondii provide the basis for a future therapeutical alternative in the treatment of toxoplasmosis.

Recombinant Dengue virus protein NS2B alters membrane permeability in different membrane models.

BACKGROUND: One of the main phenomena occurring in cellular membranes during virus infection is a change in membrane permeability. It has been observed that numerous viral proteins can oligomerize and form structures known as viroporins that alter the permeability of membranes. Previous findings have identified such proteins in cells infected with Japanese encephalitis virus (JEV), a member of the same family that Dengue virus (DENV) belongs to (Flaviviridae). In the present work, we investigated whether the small hydrophobic DENV protein NS2B serves a viroporin function. METHODS: We cloned the DENV NS2B sequence and expressed it in a bacterial expression system. Subsequently, we evaluated the effect of DENV NS2B on membranes when NS2B was overexpressed, measured bacterial growth restriction, and evaluated changes of permeability to hygromycin. The NS2B protein was purified by affinity chromatography, and crosslinking assays were performed to determine the presence of oligomers. Hemolysis assays and transmission electron microscopy were performed to identify structures involved in permeability changes. RESULTS: The DENV-2 NS2B protein showed similitude with the JEV viroporin. The DENV-2 NS2B protein possessed the ability to change the membrane permeability in bacteria, to restrict bacterial cell growth, and to enable membrane permeability to hygromycin B. The NS2B protein formed trimers that could participate in cell lysis and generate organized structures on eukaryotes membranes. CONCLUSIONS: Our data suggest that the DENV-2 NS2B viral protein is capable of oligomerizing and organizing to form pore-like structures in different lipid environments, thereby modifying the permeability of cell membranes.

Proteomic characterization of the subpellicular cytoskeleton of Toxoplasma gondii tachyzoites.

Toxoplasma, the causative agent of toxoplasmosis in animals and humans, has a subpellicular cytoskeleton that is involved in motility, cell shape and invasion. Knowledge of components of the cytoskeleton is necessary to understand the invasion mechanisms as well as for the identification of possible therapeutic targets. To date, most cytoskeletal components of Toxoplasma remain unidentified due mainly to the lack of reproducible methods for their isolation. Based on the successful isolation of the cytoskeleton, it was possible to report for the first time, the proteomic characterization of the subpellicular cytoskeleton of Toxoplasma formed by 95 cytoskeletal proteins through proteomic analysis by tandem mass spectrometry of one dimension SDS PAGE. By bioinformatic analysis of the data, proteins were classified as: 18 conventional cytoskeletal proteins; 10 inner membrane complex proteins, including 7 with alveolin repeats; 5 new proteins with alveolin like repeats; 37 proteins associated with other organelles and 25 novel proteins of unknown function. One of the alveolin like proteins not previously described in Toxoplasma named TgArticulin was partially characterized with a specific monoclonal antibody. Presence of TgArticulin was exclusively associated with the cytoskeleton fraction with a cortical distribution. Functions for the several molecules identified are proposed. BIOLOGICAL SIGNIFICANCE: This manuscript describes, for the first time, the proteome of the subpellicular cytoskeleton of Toxoplasma gondii. The importance of this study is related to the role of the cytoskeleton in the highly invasive capability of a parasite that causes abortion, blindness, and death by encephalitis in immunocompromised patients. Proteomic characterization of the cytoskeleton of T. gondii tachyzoites was possible by the development of a successful procedure for the isolation of the subpellicular cytoskeleton. Knowledge of the composition of the cytoskeleton of Toxoplasma is fundamental for the understanding of the motility and host cell invasion mechanisms, and for the future design and development of toxoplasmicidal drugs with effects against specific components of the cytoskeleton of this parasite that are absent in mammal host cells.

Osteopontin expression and localization of Ca++ deposits in early stages of osteoarthritis in a rat model.

Calcium deposits have been related to articular cartilage (AC) degeneration and have been observed in late stages of osteoarthritis (OA). However, the role of those deposits, whether they induce the OA pathogenesis or they appear as a consequence of such process, is still unknown. In this work, we present the kinetics of expression and tissue localisation of osteopontin (OPN), a mineralisation biomarker, and calcium deposits in samples from (normal, sham) and osteoarthritic cartilage (in a rat model). Immunohistochemical and Western blot assays for OPN, as well as Alizarin red staining for calcium deposits were performed; superficial, middle, and deep zones of AC were analysed. An increased expression of OPN and calcium deposits was found in the osteoarthritic cartilage compared with that of control groups, particularly in the superficial zone of AC in early stages of OA. In addition, the expression and localisation of OPN and calcium deposits during the OA pathogenesis suggest that the pathological AC mineralisation starts in the superficial zone during OA pathogenesis.

Mycobacterium tuberculosis H37Rv induces ectosome release in human polymorphonuclear neutrophils.

Ectocytosis, the cellular process by which ectosomes (Ects) are released, is an important phenomenon by which eukaryotic cells exchange molecular information. Ects released from N-formylmethionyl-leucyl-phenylalanine (fMLP)-activated human polymorphonuclear neutrophils (PMNs) have recently been characterized. Molecules such as CD35 and phosphatidylserine (PS), and enzymes such as myeloperoxidase and elastase were found in these vesicles, suggesting that Ects from PMNs could function as ecto-organelles with anti-microbial activity. Here we show for the first time that human PMNs release ectosomes in response to Mycobacterium tuberculosis H37Rv infection. We found that the release of ectosomes was not associated exclusively with mycobacterial infection since infection with other microorganisms (e.g., Leishmania mexicana, Staphylococcus aureus, and Escherichia coli or activation with phorbol myristate acetate (PMA)) also induced ectocytosis. Ects release started as early as 10min after infection or activation. Expression of CD35, PS, Rab5, Rab7 and gp91(Phox), a subunit of Cyt b555 was demonstrated on the Ects membrane. Based on our observations we conclude that Ects are released from human neutrophils in response to cell activation and that this process is not related to apoptosis.