| dc.description.abstracteng | The single subunit mitochondrial type II NADH dehydrogenases (NDH2s) in apicomplexan parasites have been considered as potential drug targets due to the absence in mammalian cells. Toxoplasma gondii encodes two isoforms of NDH2s, TgNDH2-I and TgNDH2-II, whose functions are not known. Therefore, this study is explicitly focusing on drug target validation of these enzymes as well as revealing their specific roles in both mitochondrial functions and energy metabolism in T. gondii.The validity of TgNDH2s as putative targets was firstly addressed in this study. Previously, the quinolone-like compound 1-hydroxy-2-alkyl-4(1)quinolone (HDQ), known as a high affinity inhibitor for Yarrowia lipolytica NDH2, has been shown to effectively inhibit the replication of T. gondii. To obtain biochemical evidence that TgNDH2s are the targets for HDQ, both TgNDH2 isoforms were heterologously expressed in a Yarrowia NDH2 deletion strain. TgNDH2-I was able to display oxidoreductase activities by using NADH and n-decylubiquinone as substrates. Additionally, TgNDH2-I could rescue the loss of complex I activity in Yarrowia, indicating that TgNDH2-I is expressed as an active enzyme. Furthermore, TgNDH2-I activity was effectively inhibited by HDQ with an IC50 at nanomolar concentration. Steady-state kinetics analyses for TgNDH2-I are in accordance with a ping-pong mechanism. Moreover, the mode of inhibition of HDQ on TgNDH2-I revealed that HDQ is a non-competitive inhibitor for NADH. Collectively, these biochemical data provide direct evidence that TgNDH2-I is a target enzyme of HDQ in T. gondii.Having validated TgNDH2 activity as a target of HDQ, the physiological impact of HDQ activity on mitochondrial functions was examined next. Intracellular parasites exposed to HDQ displayed a significant collapse of the mitochondrial membrane potential (∆Ψm). Strikingly, ∆Ψm in living parasites was depolarized within minutes, as recorded by time-lapse microscopy. The effect was diminished by adding substrates for mitochondrial dehydrogenases located downstream in the respiratory chain, which is in agreement with a specific inhibition of TgNDH2 activity by HDQ. Further experiments demonstrated that ∆Ψm in the presence of HDQ was stabilized with the F0-ATPase inhibitor oligomycin, indicating that T. gondii is likely to possess a conventional-like mitochondrial F0F1-ATPase. Additional subcellular localization of a membrane-associated F1-ATPase β subunit strongly supports this location, which is different to the location previously suggested to Plasmodium as a soluble matrix protein. Moreover, the collapse of ∆Ψm mediated by HDQ is followed by a significant depletion of the intracellular parasitic ATP level. Taken together, these findings indicate that HDQ is an effective inhibitor of oxidative phosphorylation in tachyzoites. Interestingly, the percentage of ∆Ψm-positive bradyzoites is significantly lower as compared to tachyzoites, implying that the importance of oxidative phosphorylation varies in these two stages.Moreover, the integration of TgNDH2s in the parasitic energy metabolism was explored. A split GFP complementation approach was used in order to determine whether the active sites of the TgNDH2 isoforms are facing to the mitochondrial matrix or to the intermembrane space. A parasite line expressing a matrix-localized succinyl-CoA synthetase beta subunit (TgSCSβ)-GFP1-10 fusion protein was generated and transfected with TgNDH2-I-GFP11 or TgNDH2-II-GFP11 fusion constructs. Full-length constructs of TgNDH2-II and truncated versions of TgNDH2-I were able to reconstitute GFP fluorescence, suggesting an internal, matrix-oriented localization of both enzymes. This indicates that both enzymes are specifically using NADH contributed from the matrix but not from the cytosol. Further investigations on the specific functional roles of TgNDH2s were focused on phenotypic analyses of conditional TgNDH2-I and TgNDH2-II depletion mutants. Unexpectedly, the phenotypic studies on these mutants showed that either TgNDH2-I or TgNDH2II is non-essential for replication, suggesting that TgNDH2s may be highly functional redundant and in turn complement each other.The mode of action of HDQ together with the biochemical and molecular findings on TgNDH2s presented here may contribute to the ongoing strategic drug development to combat the emerging drug resistance of apicomplexan parasites. | de |