A glycosylphosphatidylinositol-anchored carbonic anhydrase-related protein of Toxoplasma gondii is important for rhoptry biogenesis and virulence

Carbonic anhydrase-related proteins (CARPs) have previously been described as catalytically inactive proteins closely related to α-carbonic anhydrases (α-CAs). These CARPs are found in animals (both vertebrates and invertebrates) and viruses as either independent proteins or domains of other proteins. We report here the identification of a new CARP (TgCA_RP) in the unicellular organism Toxoplasma gondii that is related to the recently described η-class CA found in Plasmodium falciparum. TgCA_RP is posttranslationally modified at its C terminus with a glycosylphosphatidylinositol anchor that is important for its localization in intracellular tachyzoites. The protein localizes throughout the rhoptry bulbs of mature tachyzoites and to the outer membrane of nascent rhoptries in dividing tachyzoites, as demonstrated by immunofluorescence and immunoelectron microscopy using specific antibodies. T. gondii mutant tachyzoites lacking TgCA_RP display a growth and invasion phenotype in vitro and have atypical rhoptry morphology. The mutants also exhibit reduced virulence in a mouse model. Our results show that TgCA_RP plays an important role in the biogenesis of rhoptries

New details on the fine structure of the rhoptry of Toxoplasma gondii

Rhoptries are organelles that have important, complex roles in Apicomplexa biology. During Toxoplasma gondii infection, these organelles take part in several essential and complex processes that include host cell entry and parasite development. Using different electron microscopy techniques, we characterized the fine morphology of the rhoptries of two of the most important life stages of T. gondii : the tachyzoite and the bradyzoite forms. The observed tachyzoite and bradyzoite rhoptries had delimited regions characterized by a dark and electron‐dense neck, an amorphous and less electron‐dense bulb, and a region of intermediate electron density, which connects the bulb to the neck. Metal replicas of frozen‐fractured tachyzoites showed intramembranous particles of different densities and sizes on the fractured faces of rhoptry membranes. Both in tachyzoites and bradyzoites, the intramembranous particles were arranged in distinctive parallel arrays that decorated most part of these organelles. Tubulo‐vesicular subcompartments and free particles within the rhoptry lumen were observed on freeze‐fractured replicas. Cryo‐fixed, deep‐etched samples showed several pore‐like structures localized in the bulb portion. No obvious evidence was found of a possible connection between rhoptries and micronemes

Toxoplasma gondii: Fluconazole and itraconazole activity against toxoplasmosis in a murine model

Toxoplasma gondii is an important opportunistic pathogen affecting immunocompromised patients with AIDS. Toxoplasmic encephalitis is responsible for high morbidity and mortality. In this study, we investigated the activity of the antifungals fluconazole (FLZ) and itraconazole (ITZ) against T. gondii in mice infected with the Me49 strain. As previously reported for ITZ, FLZ also demonstrated a selective effect against T. gondii in vitro; the IC50 values obtained for FLZ were 8.9 μM and 3.1 μM after 24 h and 48 h of treatment, respectively. A 10-day treatment of mice with orally or intraperitoneally administered 20 mg/kg/day FLZ showed a significant survival difference compared to untreated mice. The administration of 20 mg/kg/day ITZ significantly reduced the brain cyst burden compared to untreated mice but did not exert significant protection against death. The results obtained in this work are rather promising as ITZ and FLZ are safe and low-cost drugs available on the market

Intravacuolar network may act as a mechanical support for Toxoplasma gondii inside the parasitophorous vacuole

The intravacuolar network inside the parasitophorous vacuole of Toxoplasma gondii consists of an intricate system of membrane‐limited tubules of uncertain role in parasite development. We propose that it is an important structural support to the maintenance of the parasites in the characteristic rosette arrangement of parasites inside the vacuole, rather than being associated with the nutrient acquisition from the host cell, as previously suggested. We based our assumptions on observations made by field emission scanning electron microscopy of an epithelial cell line (LLCMK2) infected at various time intervals. Scraping the surface of infected monolayers with Scotch tape exposed the inner organization of the parasitophorous vacuole. Ultrathin sections and freeze‐fracture replicas of analogous samples were correlated with field emission observations and added new data on tubular membranes and general organization of the parasitophorous vacuole

The organization of the wall filaments and characterization of the matrix structures of Toxoplasma gondii cyst form

The encystation process is a key step in Toxoplasma gondii life cycle, allowing the parasite to escape from the host immune system and the transmission among the hosts. A detailed characterization of the formation and structure of the cyst stage is essential for a better knowledge of toxoplasmosis. Here we isolated cysts from mice brains and analysed the cyst wall structure and cyst matrix organization using different electron microscopy techniques. Images obtained showed that the cyst wall presented a filamentous aspect, with circular openings on its surface. The filaments were organized in two layers: a compact one, facing the exterior of the whole cyst and a more loosen one, facing the matrix. Within the cyst wall, we observed tubules and a large number of vesicles. The cyst matrix presented vesicles of different sizes and tubules, which were organized in a network connecting the bradyzoites to each other and to the cyst wall. Large vesicles, with a granular material in their lumen of glycidic nature were observed. Similar vesicles were also found associated with the posterior pole of the bradyzoites and in proximity to the cyst wall

Cipro derivatives and Cipro affect MORN1 localization during parasite division.

<p>In parasites presenting normal division process MORN1 localizes at the basal complex of the mother cell (arrow) and also caps the final end of the IMC in budding daughter cells (arrowheads) (A), a single strong point in the middle of the cell was also observed in normal non-dividing parasites (B rosette 2 curved arrow). Tethered daughter cells resulting from the treatment with 5 μM Et-Cipro (B-C) and Adam-Cipro (D-E) for 24h, and 20 μM Cipro for 72h (F) showed wide MORN1 basal caps (B and D large arrows) and lack or a weak deposition of MORN1, at the basal end of mother (arrow) and budding daughter cells (arrowheads) (B-F)</p

Effect of 7-day treatment with different doses of Cipro derivatives on the survival mice.

<p>Swiss Webster mice were infected i.p. with 5x10³ tachyzoites of <i>T</i>. <i>gondii</i> (RH strain) one day prior to the start of treatment. Results were evaluated by the Kaplan-Meier product limit method, and compared using the log-rank (Mantel-Cox) test. * <i>P</i><0.05 vs. untreated controls. The numbers of treated mice in each group were: untreated, 10 (A), 14 (B and D) and 15 (C); 50 and 100 mg Cipro, 11 (three groups each); 150 mg Cipro, 8 (two groups); 50 and 100 mg Et-Cipro, 11 (three groups); 50 and 100 mg Ph-Cipro, 8 (two groups); 150 mg Ph-Cipro, 11 (three groups); 50 mg Adam-Cipro, 3 (one group); 100 mg Adam-Cipro, 12 (three groups).</p

Ciprofloxacin Derivatives Affect Parasite Cell Division and Increase the Survival of Mice Infected with <i>Toxoplasma gondii</i>

<div><p>Toxoplasmosis, caused by the protozoan <i>Toxoplasma gondii</i>, is a worldwide disease whose clinical manifestations include encephalitis and congenital malformations in newborns. Previously, we described the synthesis of new ethyl-ester derivatives of the antibiotic ciprofloxacin with ~40-fold increased activity against <i>T</i>. <i>gondii in vitro</i>, compared with the original compound. Cipro derivatives are expected to target the parasite’s DNA gyrase complex in the apicoplast. The activity of these compounds <i>in vivo</i>, as well as their mode of action, remained thus far uncharacterized. Here, we examined the activity of the Cipro derivatives <i>in vivo</i>, in a model of acute murine toxoplasmosis. In addition, we investigated the cellular effects <i>T</i>. <i>gondii</i> tachyzoites <i>in vitro</i>, by immunofluorescence and transmission electron microscopy (TEM). When compared with Cipro treatment, 7-day treatments with Cipro derivatives increased mouse survival significantly, with 13–25% of mice surviving for up to 60 days post-infection (vs. complete lethality 10 days post-infection, with Cipro treatment). Light microscopy examination early (6 and 24h) post-infection revealed that 6-h treatments with Cipro derivatives inhibited the initial event of parasite cell division inside host cells, in an irreversible manner. By TEM and immunofluorescence, the main cellular effects observed after treatment with Cipro derivatives and Cipro were cell scission inhibition - with the appearance of ‘tethered’ parasites – malformation of the inner membrane complex, and apicoplast enlargement and missegregation. Interestingly, tethered daughter cells resulting from Cipro derivatives, and also Cipro, treatment did not show MORN1 cap or centrocone localization. The biological activity of Cipro derivatives against <i>C</i>. <i>parvum</i>, an apicomplexan species that lacks the apicoplast, is, approximately, 50 fold lower than that in <i>T</i>. <i>gondii</i> tachyzoites, supporting that these compounds targets the apicoplast. Our results show that Cipro derivatives improved the survival of mice acutely infected with <i>T</i>. <i>gondii</i> and inhibited parasite replication early in the first cycle of infection <i>in vitro</i>, highlighting their therapeutic potential for the treatment of toxoplasmosis.</p></div