Synthesis and characterization of latex-protein complexes from different antigens of Toxoplasma gondii for immunoagglutination assays

The acute phase recombinant protein of Toxoplasma gondii P22Ag was expressed and purified and the homogenate of the parasite was obtained from an infected mouse. These antigens were used to produce latex-protein complexes (LPC) through physical adsorption and chemical coupling onto different latexes, with the aim of producing immunoagglutination (IA) reagents able to detect recently acquired toxoplasmosis. Polystyrene and “core-shell” latexes where employed, exhibiting varied particle size, functionality (carboxyl or epoxy), and charge density. In sensitization experiments for producing LPC, the recombinant protein showed better coupling efficiency onto the particles surface than the homogenate and this could be explained by the complex mixture of the homogenate, which includes a large number of proteins of different molecular mass, isoelectric points, and hydrophobicity. The synthesized LPC were employed in IA assays. To this effect, the agglutination reaction was followed by measuring the changes in the optical absorbance by turbidimetry. Experiments against control sera were performed to evaluate the performance of various LPC and it was observed that the IA test based on P22Ag and the carboxylated latex of 350 nm of particle diameter allowed a good discrimination between acute sera and chronic/negative ones. The proposed test is cheap, rapid, and easy to implement.     

Biochemical Studies of Mitochondrial Malate: Quinone Oxidoreductase from Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC₅₀ of 0.822 μM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites.     

Genetic Disruption of Toxoplasma gondii peroxiredoxin (TgPrx) 1 and 3 Reveals the Essential Role of TgPrx3 in Protecting Mice from Fatal Consequences of Toxoplasmosis

Toxoplasma gondii is a worldwide protozoan parasite that endangers human health and causes enormous economic losses to the animal production sector. A safe and effective vaccine or treatment is needed to reduce these hazards. In this study, we revealed the cyto-nuclear and mitochondrial localization of TgPrx1 and TgPrx3 proteins, respectively. We knocked out the T. gondii peroxiredoxin (TgPrxKO) 1 and 3 genes using a parental type II Prugniaud strain lacking KU80 and HXGPRT genes (PruΔku80Δhxgprt) via CRISPR-Cas9 technology. The successful KO was confirmed using PCR, IFAT, and Western blotting in two clones of both target genes, named TgPrx1KO and TgPrx3KO. Regarding in vitro assays, no significant variations between any of the knocked-out clones in TgPrx1KO or TgPrx3KO parasite strains, or even PruΔku80Δhxgprt, were obtained in rates of infection, proliferation, or egress. Nevertheless, mice that were infected with tachyzoites of the TgPrx3KO strain showed a marked decrease in survival rate compared with TgPrx1KO- and PruΔku80Δhxgprt-infected mice. This effect was confirmed using different mouse strains (ICR and C57BL/6J mice), sexes (male and female), and immunological backgrounds (ICR and SCID mice). In addition, TgPrx1KO and TgPrx3KO induced high levels of interferon gamma (IFN-γ) in infected mice at 8 days post infection, and increased IL-6 and IL-12p40 production from murine macrophages cultivated in vitro. The results of the present study suggested that TgPrx3 can induce anti-T. gondii immune responses that protect the mice from fatal consequences of toxoplasmosis. The results of our current and previous studies represent TgPrx3 as an excellent candidate for sub-unit vaccines, suggesting it may contribute to the control of toxoplasmosis for susceptible humans and animals.     

Potent Fluoro‐oligosaccharide Probes of Adhesion in Toxoplasmosis

Unnatural, NMR‐ and MRI‐active fluorinated sugar probes, designed and synthesised to bind to the pathogenic protein TgMIC1 from Toxoplasma gondii, were found to display binding potency equal to and above that of the natural ligand. Dissection of the binding mechanism and modes, including the first X‐ray crystal structures of a fluoro‐oligosaccharide bound to a lectin, demonstrate that it is possible to create effective fluorinated probe ligands for the study of, and perhaps intervention in, sugar–protein binding events.