Monitoring of dynamin during the Toxoplasma gondii cell cycle

The obligate intracellular protozoan parasite Toxoplasma gondii actively invades virtually all warm-blooded nucleated cells. This process results in a non-fusogenic vacuole, inside which the parasites replicate continuously until egress signaling is triggered. In this work, we investigated the role of the large GTPase dynamin in the interaction of T. gondii with the host cell by using laser and electron microscopy during three key stages: invasion, development and egress. The detection of dynamin during invasion indicates the occurrence of endocytosis, while T. gondii egress appeared to be independent of dynamin participation. However, the presence of dynamin during T. gondii development suggests that this molecule plays undescribed roles in the tachyzoite's cell cycle

Phosphatidylserine Exposure by Toxoplasma gondii Is Fundamental to Balance the Immune Response Granting Survival of the Parasite and of the Host

Phosphatidylserine (PS) exposure on the cell surface indicates apoptosis, but has also been related to evasion mechanisms of parasites, a concept known as apoptotic mimicry. Toxoplasma gondii mimics apoptotic cells by exposing PS, inducing secretion of TGF-beta1 by infected activated macrophages leading to degradation of inducible nitric oxide (NO) synthase, NO production inhibition and consequently persisting in these cells. Here PS+ and PS− subpopulation of tachyzoites were separated and the entrance mechanism, growth and NO inhibition in murine macrophages, and mice survival and pathology were analyzed. Infection index in resident macrophages was similar for both PS subpopulations but lower when compared to the total T. gondii population. Growth in resident macrophages was higher for the total T. gondii population, intermediate for the PS+ and lower for the PS− subpopulation. Production of NO by activated macrophages was inhibited after infection with the PS+ subpopulation and the total populations of tachyzoites. However, the PS− subpopulation was not able to inhibit NO production. PS+ subpopulation invaded macrophages by active penetration as indicated by tight-fitting vacuoles, but the PS− subpopulation entered macrophages by phagocytosis as suggested by loose-fitting vacuoles containing these tachyzoites. The entrance mechanism of both subpopulations was confirmed in a non-professional phagocytic cell line where only the PS+ tachyzoites were found inside these cells in tight-fitting vacuoles. Both subpopulations of T. gondii killed mice faster than the total population. Clear signs of inflammation and no tachyzoites were seen in the peritoneal cavity of mice infected with the PS− subpopulation. Moreover, mice infected with the PS+ subpopulation had no sign of inflammation and the parasite burden was intense. These results show that PS+ and PS− subpopulations of T. gondii are necessary for a successful toxoplasma infection indicating that both subpopulations are required to maintain the balance between inflammation and parasite growth

A Window to Toxoplasma gondii Egress

The Toxoplasma gondii cellular cycle has been widely studied in many lifecycle stages; however, the egress event still is poorly understood even though different types of molecules were shown to be involved. Assuming that there is no purpose or intentionality in biological phenomena, there is no such question as “Why does the parasite leaves the host cell”, but “Under what conditions and how?”. In this review we aimed to summarize current knowledge concerning T. gondii egress physiology (signalling pathways), structures, and route

Calcium ionophore-induced egress of toxoplasma gondii shortly after host cell invasion

11 p. : il.Calcium plays crucial roles in important events of Toxoplasma gondii life cycle, including motility, invasion and egress from the host cell. Calcium ionophore has been used to artificially trigger release of the parasites from infected cells. In this report we describe that calcium ionophore A21387 induced T. gondii egress from LLC-MK2 cells at times as early as 2 h after entry. Addition of kinase inhibitors as staurosporine, wortmanine and genistein to the incubation medium significantly reduced ionophore-induced egress. The same occurred when the actin inhibitor cytochalasin D was used. Parasites egressed 2 h post-infection from ionophoretreated cultures were unable of establishing infection in a new cell. S-VHS recording of egressing parasites showed that they assume an hourglass shape as they cross the plasma membrane, similar to the moving junction constriction observed during active invasion, and extrudes the conoid, similarly to what is also observed during invasion. Transmission and high resolution scanning electron microscopy revealed that the egressing tachyzoites are free from host cell derived membranes. These include plasma membrane and parasitophorous vacuole membranes as well as associated endoplasmic reticulum membranes. Taken together, these results indicate that although invasion and egress may share similar signaling pathways, as indicated by the effect of kinase and actin inhibitors, the tachyzoites move freely in the cytosol, a phenomenon very distinctive from invasion and that deserves attention

Dynamin inhibitor impairs Toxoplasma gondii invasion

6 p. : il.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

Prostaglandin A1 Inhibits Replication of Classical Swine Fever Virus

Prostaglandins (Pgs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report the effect of prostaglandin (PgA1) on the multiplication of a positive strand RNA virus, Classical Swine Fever Virus (CSFV) in PK15 cells. PgA1 was found to inhibit the multiplication of CSFV. At a concentration of 5 g/ml, which was nontoxic to the cells, PgA1 inhibitis virus production in 99%. In PgA1 treated cells the size and number of characteristic Classical Swine Fever focus decreased in amount

Analysis of <i>Toxoplasma gondii</i> infection <i>in vivo</i>.

<p>C57/BL6 mice were infected with PS⁺, PS⁻ subpopulations or the total population of <i>T. gondii</i>. (A) Survival curve of mice after the infection with <i>T. gondii</i>. Kaplan Meier analysis <i>p</i> = 0.0273. (B) Light microscopy of spleen and liver tissue of C57/BL6 mice after interaction with PS⁺ or PS⁻ subpopulation of <i>T. gondii</i>. Bars = 100 µm. (B1–3) Spleen images after interaction with the PS⁻ (B1), PS⁺ (B2) or the total population of <i>T. gondii</i> (B3). Note the presence of inflammatory cells in B1 (arrows) and the presence of parasites in B2 and B3 (arrows). (B4–6) Liver images after interaction with PS⁻ (B4), PS⁺ (B5) or the total population of <i>T. gondii</i> (B6). Note similar results obtained for the spleen tissue. Inset – Transmission electron microscopy: B2, Bar = 2 µm; B4, Bar = 16 µm. Results are from two independent experiments with 6 animals per group.</p

Phosphatidylserine (PS)⁺ and PS⁻ subpopulations of <i>Toxoplasma gondii</i> were magnetically separated and analyzed by flow cytometry after annexin-V staining.

<p>(A) PS⁺ subpopulation of <i>T. gondii</i>. (B) PS⁻ subpopulation of <i>T. gondii</i>. The black lines represent control parasites without Annexin – V and the gray lines refer to the isolated subpopulations. Results from one representative experiment out of five.</p

Thieno[2,3-b]pyridine derivatives: a new class of antiviral drugs against Mayaro virus

Mayaro virus (MAYV) is an arthropod-borne virus and a member of the family Togaviridae, genus Alphavirus. Its infection leads to an acute illness accompanied by long-lasting arthralgia. To date, there are no antiviral drugs or vaccines against infection with MAYV and resources for the prevention or treatment of other alphaviruses are very limited. MAYV has served as a model to study the antiviral potential of several substances on alphavirus replication. In this work we evaluated the antiviral effect of seven new derivatives of thieno[2,3-b]pyridine against MAYV replication in a mammalian cell line. All derivatives were able to reduce viral production effectively at concentrations that were non-toxic for Vero cells. Molecular modeling assays predicted low toxicity risk and good oral bioavailability of the substances in humans. One of the molecules, selected for further study, demonstrated a strong anti-MAYV effect at early stages of replication, as it protected pre-treated cells and also during the late stages, affecting virus morphogenesis. This study is the first to demonstrate the antiviral effect of thienopyridine derivatives on MAYV replication in vitro, suggesting the potential application of these substances as antiviral molecules against alphaviruses. Additional in vivo research will be needed to expand the putative therapeutic applications.Fil: Amorim, Raquel. Universidade Federal do Rio de Janeiro; BrasilFil: Meneses, Marcelo Damião Ferreira de. Universidade Federal do Rio de Janeiro; BrasilFil: Borges, Julio Cesar. Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro; BrasilFil: Pinheiro, Luiz Carlos da Silva. Universidade Federal do Rio de Janeiro; BrasilFil: Caldas, Lucio Ayres. Universidade Federal do Rio de Janeiro; BrasilFil: Cirne Santos, Claudio Cesar. Universidade Federal do Rio de Janeiro; BrasilFil: Mello, Marcos Vinícius Palmeira de. Universidade Federal Fluminense; BrasilFil: Souza, Alessandra Mendonça Teles de. Universidade Federal do Rio de Janeiro; BrasilFil: Castro, Helena Carla. Universidade Federal Fluminense; BrasilFil: Paixão, Izabel Christina Nunes de Palmer. Universidade Federal Fluminense; BrasilFil: Campos, Renata de Mendonça. Universidade Federal do Rio de Janeiro; BrasilFil: Bergmann, Ingrid Evelyn. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología ; ArgentinaFil: Malirat, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología ; ArgentinaFil: Bernardino, Alice Maria Rolim. Universidade Federal Fluminense; BrasilFil: Rebello, Moacyr Alcoforado. Universidade Federal do Rio de Janeiro; BrasilFil: Fernandes Ferreira, Davis. Universidade Federal do Rio de Janeiro; Brasi