Abstract
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.
Highlights
Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, an obligate intracellular apicomplexan protozoan parasite that can infect almost all nucleated cells [1,2,3,4]
Compared to the HEPES buffer, we found that the specific activity of TgMQO prepared with MOPS buffer was lower in the membrane fraction but higher after purification and stable for several months (Table S1)
Same experiment performed by varying malate concentrations yielded similar results as purification of PfMQO
Summary
Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, an obligate intracellular apicomplexan protozoan parasite that can infect almost all nucleated cells [1,2,3,4]. Recent estimations are that 30–50% of the global population is seropositive for T. gondii [5]. It has long been considered a mild pathogen compared to other deadly apicomplexan parasites such as Plasmodium falciparum [6], the pathogen of malaria, though in many aspects T. gondii exhibits metabolic traits similar to those of Plasmodium species, with regard to the hepatic stage [7]. Toxoplasmosis chemotherapy options are limited, and all drugs currently used have severe side effects, are solely active against tachyzoites, and not able to clear cystic chronic infection [19,20]. Metabolic pathways or molecular targets from the parasites that are absent or different from the corresponding host pathways are attractive drug targets [19,23]
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