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Inhibition of the Toxoplasma gondii replication by inhibition of the mitochondrial respiratory chain

Toxoplasma gondii is an obligate intracellular parasite which belongs to the apicomplexan pathogens including Plasmodium falciparum, the caustic agent of Malaria. There is a need for research on new drug targets of these parasites because of the resistance to current therapeutics that has been reported recently. The mitochondrial respiratory chain of Toxoplasma and Plasmodium contains a component which is not present in mammalian cells and therefore it is considered to be a promising drug target for anti-parasitic agents: T. gondii and P. falciparum possess an alternative NADH dehydrogenase instead of a conventional complex I. Recently, the compound 1-hydroxy-2-dodecyl-4(1)-quinolone (HDQ) has been identified as a high affinity inhibitor of the alternative NADH dehydrogenase of the yeast Yarrowia lipolytica in enzymatic assays. Moreover, HDQ inhibited the growth of P. falciparum and genotype I T. gondii parasites in tissue culture. The aim of this dissertation was to further characterise the mode of action of HDQ in different T. gondii strains and mutants. Two independent types of growth assays based on vacuole size as well as on s-galactosidase activity showed that HDQ effectively inhibits the growth of T. gondii in the nanomolar range in both, genotype I and II parasites. The inhibitor was parasitostatic in short-term experiments up to 24 hours. Unexpectedly, the parasites recovered from inhibition in long-term experiments up to 96 hours, although they were continuously treated with HDQ. A critical feature of a 1-hydroxy-2-alkyl-4(1)-quinolone derivate is the length of the alkyl side chain at position two. HDQ, as a derivate with a long alkyl side chain (C12) showed excellent inhibition of T. gondii replication whereas a derivate with a shorter alkyl side chain (C6) failed to inhibit parasite growth. Most likely, a defined minimal length of the alkyl side chain is necessary to diffuse into the inner mitochondrial membrane or to act as a competitor with ubiquinones. The combined treatment of T. gondii infected cells with HDQ and the anti-malarial drug atovaquone, a complex III inhibitor of the mitochondrial respiratory chain, resulted in a synergism which was present independently of the parasite s genotype. This synergism could be explained by the simultaneous inhibition of the ubiquinone/ubiquinol cycle, by HDQ at the reduction side and by atovaquone at the oxidation side. In contrast, the combined treatment of T. gondii infected cells with HDQ and the complex III inhibitor antimycin A showed rather an additive effect. Another objective of this thesis was the comparison between wild type parasites and UPRT deficient mutants. Wild type parasites are able to generate pyrimidines by the de novo biosynthesis and in addition by the pyrimidine salvage pathway, while UPRT deficient mutants lack the salvage pathway. UPRT deficient mutants displayed a significant higher sensitivity towards HDQ as wild type parasites. This hypersensitivity to HDQ might be explained by a direct or indirect inhibition of de novo pyrimidine biosynthesis, which is linked with the mitochondrial respiratory chain via the enzyme dihydroorotate dehydrogenase that uses ubiquinone as an electron acceptor. This dissertation further strengthens the concept that the T. gondii respiratory chain is an excellent target for the development of specific anti-parasitic drugs and thus is an interesting object for further research.