Characterization of alternative NADH dehydrogenases in the respiratory chain of Toxoplasma gondii as a novel drug targets

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علوم بحتة وطبيعية

الملخص

The single mitochondrion of Toxoplasma gondii generates a membrane potential in theproliferative tachyzoite stage. However, its contribution to energy metabolism indormant bradyzoites is less clear.The steady state mRNA level of 11 nuclear genes coding for key subunits of the electrontransport chain was quantified by quantitative real-time RT-PCR and compared intachyzoite versus bradyzoites. No significant difference on the transcript levels betweenboth stages was found when normalized to the expression of β-tubulin.To facilitate expression studies of energy-associated components, the open reading frameof the following genes was determined and verified by RT-PCR: (i) alternative NADHdehydrogenase I and II; (ii) flavoprotein subunit of succinate dehydrogenase; (iii)cytochrome c1; and (iv) β-subunit of ATP synthase. These genes display highestsimilarities to orthologs in Plasmodium. They are single copy, nuclear encoded geneswith their coding sequence interrupted by multiple introns. The N-terminus of theprecursor proteins has characteristics of a mitochondrial targeting pre-sequence. Theopen reading frame of the five genes was fused with a c-myc tag and brought undercontrol of a tetracycline regulatable promoter. These plasmids were stably transfectedinto the T. gondii TATi-1 line. Co-localization studies showed that the encoded proteinsare expressed and transported correctly into the mitochondrion. Expression andlocalization of TgNDH2-I and TgATP-β were confirmed by mouse antiserum raisedagainst their recombinant proteins. The over-expression of these proteins has noinfluence on the growth rate of the parasites.The T. gondii genome predicts the presence of typical components of the respiratorychain, except complex I (NADH:Q reductase). Instead, two isoforms of a single subunit,non-proton pumping alternative NADH dehydrogenases, TgNDH2-I and TgNDH2-II,have been identified. The total DNA sequences encompass 2793 and 7686 base pairs andcontain open reading frames of 1875 and 1974 base pairs, respectively. The respectivegenes code for a precursor polypeptides of 618 and 657 amino acid residues with acalculated MW of 67 and 72 KDa, respectively. The open reading frames encoding theisoforms show 58% nucleotide sequence identity, and their gene products share 43%amino acid sequence identity.VIIIThe fact that alternative (type II) NADH dehydrogenases of T. gondii and P. falciparumare absent in mammalian cells makes them promising antimicrobial drug targets. Thequinolone-like compound 1-hydroxy-2-dodecyl-4(1)quinolone (HDQ) was recentlydescribed as a high affinity inhibitor of fungal alternative NADH-dehydrogenases inenzymatic assays, probably by interfering with the ubiquinol binding site of the enzyme.In this study, HDQ effectively inhibited the replication rate of T. gondii in tissue culture.The IC50 of HDQ was determined for T. gondii with two independeant types of growthassays at ~2-8 nM. An important feature of the HDQ structure is the length of the alkylsite chain at position 2. Derivatives with alkyl site chains of C6, C8, C12 (HDQ) and C14all displayed excellent anti-T. gondii activity, while a C5 derivative completely failed toinhibit parasite replication. Due to the structural similarities, it is most likely that 1-hydroxy-2-alkyl-4(1)quinolones compete with ubiquinones for the same binding site inalternative NADH dehydrogenases. HDQ treatment of T. gondii-infected culturesresulted in an induction of bradyzoite differentiation. This effect is well known fromother compounds which also inhibit parasite replication and simultaneously induce theexpression of bradyzoite specific genes in T. gondii. A combined treatment of T. gondiiinfected cells with HDQ and the antimalarial agent atovaquone, which blocks theubiquinol oxidation site of cytochrom b in complex III, resulted in a strong synergism.An interference of the mitochondrial ubiquinone/ubiquinol cycle at two differentlocations appears thus to be a highly effective strategy to inhibit parasite replication.HDQ and derivatives represent particularly in combination with atovaquone promisingcompounds with high potential for anti-malarial and anti-toxoplasmal therapy.In the background of the regulatable additional copies, the endogenous genes weretargeted for disruption by homologous recombination to generate inducible knock-outmutants. Depletion of TgNDH2-I in a conditional knock-out mutant has neither influenceon the replication rate nor on the in vitro stage conversion, suggesting that TgNDH2-IIactivity is sufficient for intracellular development. However, slight upregulation ofTgNDH2-II has been shown on the steady state mRNA in the knock-out mutantscompared to the wild type parasites. Moreover, TgNDH2-I depleted parasites display astrongly increased susceptibility for HDQ treatment, where 1 nM HDQ was sufficient tocompletely inhibit parasite replication. Taken together, we tempt to speculate that theover-expression of the second isoform i.e. TgNDH2-II compensates the loss ofTgNDH2-I activity.

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0b3e3d52-33d1-4f07-8edd-9b38d4fa0be7

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0

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Crawler

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الرسائل العلمية

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رسائل دكتوراة

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pdf text

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1697153_1.pdf

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[{"name_ar":"أحمد محمود حسن صالح","title_ar":"اعداد","title_en":"Preparation"}]

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English

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اسم المصدر

Characterization of alternative NADH dehydrogenases in the respiratory chain of Toxoplasma gondii as a novel drug targets

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0

كلمات الباحثين

Electron Transport Chain, HDQ, RT-PCR, Toxoplasma gondii

إشراف وإعداد

الإعداد

أحمد محمود حسن صالح

الاقتباسات الببليوغرافية