The apicomplexan protozoan Toxoplasma gondii, the causative agent of toxoplasmosis, harbors an apicoplast, a plastid-like organelle with essential metabolic functions. Although the FASII fatty acid biosynthesis pathway located in the apicoplast is essential for parasite survival, the cellular effects of FASII disruption in T. gondii had not been examined in detail. Here, we combined light and electron microscopy techniques – including focused ion beam scanning electron microscopy (FIB-SEM) – to characterize the effect of FASII disruption in T. gondii, by treatment with the FASII inhibitor triclosan or by inducible knockdown of the FASII component acyl carrier protein. Morphological analyses showed that FASII disruption prevented cytokinesis completion in T. gondii tachyzoites, leading to the formation of large masses of ‘tethered’ daughter cells. FIB-SEM showed that tethered daughters had a mature basal complex, but a defect in new membrane addition between daughters resulted in incomplete pellicle formation. Addition of exogenous fatty acids to medium suppressed the formation of tethered daughter cells and supports the notion that FASII is essential to generate lipid substrates required for the final step of parasite division.

The redirectioning of drugs in the pharmaceutical market is a well-known practice to identify new therapies for parasitic diseases. The histone deacetylase inhibitors Tubastatin A (TST) and Suberoylanilide Hydroxamic Acid (SAHA), firstly developed for cancer treatment, are effective against protozoa parasites. In this work, we aimed to demonstrate the activity of these drugs as potential agents against Toxoplasma gondii, the causative agent of toxoplasmosis. TST and SAHA were active against different genotypes of Toxoplasma gondii, such as, RH (type I), EGS (I/III) and ME49 (type II) strains. The IC₅₀ values for the RH strain were 19 ± 1 nM and 520 ± 386 nM for TST and 41 ± 3 nM and 67 ± 36 nM for SAHA, for 24 and 48 h, respectively. Both compounds were highly selective for T. gondii and their anti-proliferative effect was irreversible for 8 days. The calculated selectivity indexes (39 for TST and 30 for SAHA) make them lead compounds for the future development of anti-Toxoplasma molecules. Western blotting showed TST led to a significant increase of the nuclear histone H4 and a decrease of H3 acetylation levels. Treatment with 1 μM TST and 0.1 μM SAHA for 48 h decreased the amount of global α-tubulin. Fluorescence and electron microscopy showed that both drugs affected the endodyogeny process impairing the budding of daughter cells. The drugs led to the formation of large, rounded masses of damaged parasites with several centrosomes randomly dispersed and incorrect apicoplast division and positioning. TST-treated parasites showed a rupture of the mitochondrial membrane potential and led to a failure of the IMC assembling of new daughter cells. SAHA and TST possibly inhibit HDAC3 and other cytoplasmic or organelle targeted HDACs involved in the modification of proteins other than histones.

The new complexes Zn(ITZ) ₂ Cl ₂ (1) and Zn(ITZ) ₂ (OH) ₂ (2) were synthetized by a reaction of itraconazole with their respective zinc salts under reflux. These Zn-ITZ complexes were characterized by elemental analyses, molar conductivity, mass spectrometry, ¹ H and ¹³ C{ ¹ H} nuclear magnetic resonance, and UV-vis and infrared spectroscopies. The antiparasitic and antifungal activity of Zn-ITZ complexes was evaluated against three protozoans of medical importance, namely, Leishmania amazonensis , Trypanosoma cruzi , and Toxoplasma gondii , and two fungi, namely, Sporothrix brasiliensis and Sporothrix schenckii .

Coordination between nucleus and mitochondria is essential for cell survival, and thus numerous communication routes have been established between these two organelles over eukaryotic cell evolution. One route for organelle communication is via membrane contact sites, functional appositions formed by molecular tethers. We describe a novel nuclear-mitochondrial membrane contact site in the protozoan Toxoplasma gondii. We have identified specific contacts occurring at the nuclear pore and demonstrated an interaction between components of the nuclear pore and the mitochondrial protein translocon, highlighting them as molecular tethers. Genetic disruption of the nuclear pore or the TOM translocon components, TgNup503 or TgTom40, respectively, result in contact site reduction, supporting their potential involvement in this tether. TgNup503 depletion further leads to specific mitochondrial morphology and functional defects, supporting a role for nuclear-mitochondrial contacts in mediating their communication. The discovery of a contact formed through interaction between two ancient mitochondrial and nuclear complexes sets the ground for better understanding of mitochondrial-nuclear crosstalk in eukaryotes.

Apicomplexans are the causative agents of numerous important infectious diseases including malaria and toxoplasmosis. Most of them harbour a chloroplast-like organelle called the apicoplast that is essential for the parasites' metabolism and survival. While most apicoplast proteins are nuclear encoded, the organelle also maintains its own genome, a 35 kb circle. In this study we used Toxoplasma gondii to identify and characterise essential proteins involved in apicoplast genome replication and to understand how apicoplast genome segregation unfolds over time. We demonstrated that the DNA replication enzymes Prex, DNA gyrase and DNA single stranded binding protein localise to the apicoplast. We show in knockdown experiments that apicoplast DNA Gyrase A and B, and Prex are required for apicoplast genome replication and growth of the parasite. Analysis of apicoplast genome replication by structured illumination microscopy in T. gondii tachyzoites showed that apicoplast nucleoid division and segregation initiate at the beginning of S phase and conclude during mitosis. Thus, the replication and division of the apicoplast nucleoid is highly coordinated with nuclear genome replication and mitosis. Our observations highlight essential components of apicoplast genome maintenance and shed light on the timing of this process in the context of the overall parasite cell cycle.