Biochemical evidence for oxidative phosphorylation was provided by extracellular T. gondii tachyzoites that were permeabilized with digitonin (39). There have been reports of the indigenous people of French Guiana using rotenone-contai… We demonstrate in this study that HDQ treatment in nanomolar concentrations leads to a depolarization of the T. gondii ΔΨm within minutes. NADH Dehydrogenase (Ubiquinone) Complex I is the first enzyme complex in the respiratory chain, and it accepts electrons from NADH+H+ derived from fat, carbohydrate, and amino acids to create an electrochemical gradient across the inner mitochondrial membrane. The apicomplexan parasite Toxoplasma gondii contains a single mitochondrion of an elongated tubular structure (28, 32), which shows several significant metabolic differences from the mammalian counterpart (see references 24 and 33 for review). Mitochondrial localization of myc-tagged ATPase-β was confirmed by colocalization with Mitotracker fluorescence. NAD+, required for the ATP-generating steps of glycolysis, is regenerated from NADH by mito- chondrial NADH dehydrogenase or lactate dehydro- genase. Both fluorescent dyes were dissolved in tissue-culture-grade dimethyl sulfoxide. Crossing points were plotted against the log of cDNA dilutions, and amplification efficiencies (E) were calculated from the slopes of the obtained lines by the following formula: E = 10-1/slope. The effect of oligomycin-mediated FoF1-ATPase inhibition was further investigated by using 143B/260 cells as host cells for T. gondii. HDQ was kindly provided by W. Oettmeier (University of Bochum). Other mitochondrial pathways for mitochondrial acetyl coenzyme A generation, such as the 2-methylcitrate cycle, are currently under investigation (33). Scale bars, 5 μm. Cells were treated for this purpose with 2 μM digitonin, a concentration which was shown previously to selectively permeabilize the parasite's plasma membrane for metabolites without disturbing the function of the respiratory chain (39). DiOC6(3)-stained intracellular parasites were digitonin permeabilized and treated with 1 μM HDQ (A) or 1 μM atovaquone (ATO) (B) either alone or in combination with 10 mM malate (MAL); 10 mM succinate (SUC); 10 mM dihydroorotate (DHO); 1 mM glycerol-3-phosphate (G-3-P); 10 mM oxaloacetate (OAA); a mixture of malate, succinate, dihydroorotate, and glycerol-3-phosphate (SUB); and 0.2 mM TMPD-1.5 mM ascorbate (TMPD/ASC). Images were captured by using an AxioCam MRm camera and processed with Axiovision 4.6.3 software. 6), suggesting that the underlying mechanism of HDQ growth inhibition is not due to pyrimidine starvation. Higher numbers of exposures resulted in a significant bleaching effect, and at incubation times longer than 1.5 h, the dye showed a tendency to lose the equal distribution across the mitochondrial membrane and started to accumulate at a single location. Infected HFFs were washed once with PBS supplemented with protease inhibitors (protease inhibitor cocktail; Roche) to remove extracellular parasites. We recently showed by inhibition kinetics that T. gondii NDH2-I is a target of the quinolone-like … These results are in agreement with the data obtained from intracellular parasites and confirm the strong decrease in levels of ΔΨm-positive parasites during bradyzoite differentiation. Kinetics of HDQ-mediated ΔΨm collapse.DiOC6(3)-based real-time imaging of the ΔΨm for parasites treated with 10 nM, 100 nM, and 1 μM HDQ led to a dose-dependent depolarization of the T. gondii mitochondrial membrane (Fig. NADH dehydrogenase is also blocked by adenosine diphosphate ribose - a reversible competitive inhibitor of NADH oxidation by binding to the enzyme at the nucleotide binding site. The relative parasitic ATP levels for each sample were normalized with the numbers of parasites counted previously. A likely candidate for such an energy buffer system is the adenylate kinase reaction, which converts two ADP molecules into ATP and AMP in a reversible reaction. Samples were prepared as follows. The apicomplexan parasite Toxoplasma gondii expresses type II NADH dehydrogenases (NDH2s) instead of canonical complex I at the inner mitochondrial membrane. A recent study reported that HDQ inhibits DHODH of P. falciparum (10). The addition of dihydroorotate, glycerol-3-phosphate, malate, or succinate did not prevent an atovaquone-mediated ΔΨm collapse. A 70% decrease in the ATP level was also observed after the complete inhibition of FoF1-ATPase activity using 1 μM oligomycin. In order to discriminate between a matrix and a membrane association of the F1 subunit, we examined the localization of the F1-ATPase in parasites, which expressed an epitope-tagged version of ATPase-β (TgATP-β), which is a part of the F1-ATPase. The difference of amplification efficiencies (ΔE) of HDQ-treated samples and the untreated control was less than 0.05 (ΔEHDQ 100 nM − control = 0.019; ΔEHDQ 1,000 nM − control = 0.013), and crossing-point values from PCR amplification could thus be used for relative quantification. Subsequently, the lysates were separated after centrifugation at 13,000 × g for 45 min. NADH dehydrogenase; inhibitor; carvedilol. Results are expressed as means ± SD of data from duplicate samples from a representative experiment. The mode of action of HDQ in T. gondii is thus an inhibition of oxidative phosphorylation. Results are represented as means ± SD of data from a representative experiment (n = 2). Previously, it was demonstrated that low-affinity NDH2 inhibitors in micromolar concentrations were able to inhibit the activity of the P. falciparum NDH2 and led to a collapse of the mitochondrial membrane potential (ΔΨm) (2). The rate of bradyzoite differentiation was determined using the same samples after fixation and staining with a bradyzoite-specific anti-BAG1 antibody, which detects a cytosolic small heat shock protein, and with a fluorescein isothiocyanate-conjugated Dolichos biflorus lectin, which detects a carbohydrate structure on the emerging cyst wall (Fig. Parasites were fixed with 4% paraformaldehyde-PBS for 10 min after washing, and a 10-μl aliquot of the parasite suspension was air dried on a glass slide and mounted with Moviol. Infected cultures were incubated with 1 μM HDQ, and intracellular parasites were mechanically released from host cells by syringe passage at 1, 3, 8, and 24 h after the addition of HDQ. Mycobacterium tuberculosis (MTb) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Real-time imaging revealed that nanomolar HDQ concentrations led to a ΔΨm collapse within minutes, which is followed by severe ATP depletions of 30% after 1 h and 70% after 24 h. ΔΨm depolarization was attenuated when substrates for other dehydrogenases that can donate electrons to ubiquinone were added to digitonin-permeabilized cells or when infected cultures were treated with the Fo-ATPase inhibitor oligomycin. NADH dehydrogenase is an enzyme that converts nicotinamide adenine dinucleotide (NAD) from its reduced form (NADH) to its oxidized form (NAD ). Plasmids.Plasmid tub-S9-RFP/sag-CAT was constructed by replacing the FNR fragment from BglII/AvrII-digested ptub-FNR-RFP/sag CAT (36) with a BglII/AvrII S9 fragment from ptub-S9-GFP/sag-CAT (8). A common response to the inhibition of oxidative phosphorylation, which might also occur in T. gondii, is an increased metabolic flux through other energy-generating pathways, like glycolysis. The T. gondii genome predicts the presence of all components necessary for a respiratory chain. Our studies suggest that oxidative phosphorylation is indispensable for sufficient ATP generation in the growing-tachyzoite stage and that other ATP-generating pathways such as glycolysis cannot fully compensate for its loss. 4C), suggesting that the HDQ-mediated ΔΨm depolarization can be attenuated by preventing protons from reentering the mitochondrial matrix. The staining solution consists of 1% FCS-DMEM with a final concentration of 5 nM DiOC6(3). Complex I functions in the transfer of electrons from NADH to the respiratory chain. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. FoF1-ATPases use the proton motive force across the inner mitochondrial membrane for coupling proton translocation through a membrane-bound, oligomycin-sensitive Fo subunit with ATP synthesis at the F1 subunit. The frequencies of ΔΨm-positive parasites were similar in all samples and unrelated to the size of the parasitophorous vacuoles, suggesting that the majority of tachyzoites possess a ΔΨm throughout their intracellular life span. It is conceivable that a reserve energy system of limited capacity contributes to a stable ATP amount within the first minutes after the inhibition of oxidative phosphorylation. In brief, confluent HFFs seeded onto an imaging plate were infected with 2 × 105 to 3 × 105 parasites per well. Drug-untreated controls were stained in parallel at the same time points. Substrate supplementation in permeabilized parasites partly decreases HDQ-mediated ΔΨm depolarization. Digitonin permeabilization.Substrate supplementation was performed on digitonin-permeabilized intracellular parasites using a modification of a previously described protocol that was established for extracellular parasites (39). This suggests that a fraction of parasites lost the ΔΨm during bradyzoite differentiation. Treatment with TMPD-ascorbate, a combination which is commonly used to feed electrons into complex IV, led to a strongly attenuated ΔΨm depolarization after HDQ treatment, indicating that HDQ inhibits the ETC upstream of complex IV. We recently showed by inhibition kinetics that T. gondii NDH2-I is a target of the quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ), which inhibits T. gondii replication in the nanomolar range. An oligomycin-treated sample was included in order to quantify ATP levels in parasites in which FoF1-ATPase activity was inhibited. Extracellular tachyzoites (3 × 107 to 4 × 107 tachyzoites) from a stable transgenic line expressing TgATPase-β were harvested and resuspended in ice-cold PBS containing protease inhibitors. The kinetics revealed that HDQ leads to a gradual decrease of the parasite's total ATP level, resulting in a ∼30% reduction after 1 h and a ∼70% reduction after 24 h (Fig. An aliquot of 20 μl of parasites was used for counting, and the remaining parasites were immediately frozen in liquid nitrogen for later measurement. (B) Kinetics showing the influence of 10 nM complex III inhibitor atovaquone (ATO) and 10 nM, 100 nM, and 1 μM NDH2 inhibitor HDQ on the ΔΨm of individual parasites. The … 2B and C). Bradyzoite differentiation was induced by an alkaline-pH shift, and the percentage of ΔΨm-positive parasites was determined after DiOC6(3) staining in living cultures at 24 h, 48 h, and 72 h postinfection. Generation of transgenic parasites.The transfection of T. gondii strain RH TATi-1 (26) was carried out based on previously reported protocols (30, 35). The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. RH strain tachyzoites were treated with 100 nM HDQ in the presence or absence of 250 μM uracil. *, P < 0.0001; **, P < 0.0002 versus mock control (determined by a Student's t test). Both fractions were separated by SDS-PAGE and analyzed by immunoblotting with an anti-myc antibody. HDQ treatment upregulates transcript levels of the bradyzoite markers bag1 and enolase 1. HDQ growth inhibition is not mediated by pyrimidine starvation. The pellet fraction was also resuspended in the same buffer. Myc-tagged TgATP-β of stably transfected parasites was targeted to the mitochondrion, as shown by the colocalization with the Mitotracker signal (Fig. The fraction of parasites with positive Mitotracker staining was determined using 143B/260 host cells after HDQ treatment with and without the addition of oligomycin. Duplicate samples were fixed after 24 h, and the average number of tachyzoites per vacuole was determined. As expected, Complex I inhibitors had no effect on pfNDH2 enzyme activity (Table 2). The diagram shows enolase 1 and bag1 transcript levels of HDQ-treated samples relative to that of a mock-infected control (arbitrarily defined as 1), which was harvested 24 h postinfection. While both of the inhibitors will decrease the activity of NADH dehydrogenase significantly (both suppresses enzyme activity to about 40% of the control, uninhibited enzyme), at limiting substrate concentrations, Mg2+will inhibit the enzyme more efficiently than EDTA. The frequency of ΔΨm-positive parasites decreases during bradyzoite differentiation.There are several indications that the energy metabolism of bradyzoites is different from that of tachyzoites (12, 41), and it was thus of interest to compare the frequencies of ΔΨm-positive parasites in both stages. In drug-untreated controls, more than 80% of the parasites displayed a strong ΔΨm, confirming that the digitonin treatment itself did not affect the ΔΨm (Fig. Since a reduction in the level of parasite replication in T. gondii is often associated with stage differentiation (4), we investigated whether HDQ treatment leads to bradyzoite differentiation. A. Adessi, R. De Philippis, in Encyclopedia of Biological Chemistry (Second Edition), 2013. After blocking with 1% bovine serum albumin-PBS for 1 h, samples were incubated with an anti-myc monoclonal antibody (MAb) (clone 9E10; Sigma) (1:250), followed by a Cy2-conjugated anti-mouse immunoglobulin G (IgG) antibody (1:300; Dianova) for 1 h each. HDQ treatment leads to a decreased ATP level. 2A). Oligomycin-mediated inhibition of the T. gondii FoF1-ATPase attenuates HDQ-mediated ΔΨm depolarization.We next investigated whether the ΔΨm can be stabilized in HDQ-treated parasites by an inhibition of the putative T. gondii FoF1-ATPase. (B) Comparison of the Mitotracker staining patterns from a sample in which the 6-h HDQ treatment period was started 16 h postinfection (top) to those from an untreated control (bottom). The relative contribution of oxidative phosphorylation to total ATP synthesis is still a matter of debate for T. gondii, as it is for other apicomplexan parasites (24, 33). Lectin staining was performed with the same procedures by using a fluorescein isothiocyanate-conjugated lectin from Dolichos biflorus (1:300; Sigma). 8A). β-Tubulin was used for normalization. Although the T. gondii mitochondrion harbors the complete set of enzymes for the tricarboxylic acid cycle (15), it lacks the pyruvate dehydrogenase complex (7, 14, 18), which is typically a central enzyme in carbohydrate metabolism that catalyzes the decarboxylation from pyruvate to acetyl coenzyme A. Relative ATP levels were normalized for parasite numbers. Protein fractionation and immunoblotting. L-BOAA also induced lactate dehydrogenase (LDH) leakage from the slice into the medium in dose-dependent manner. at site II. It blocks NADH dehydrogenase and Coenzyme.Q; 4. A flavoprotein and iron sulfur-containing oxidoreductase that catalyzes the oxidation of NADH to NAD. HFFs were infected with tachyzoites and cultivated in the presence of 100 nM and 1 μM HDQ for 72 h. Subsequently, the transcript levels of the bradyzoite marker genes bag1 (5) and enolase 1 (11) were determined by real-time PCR, in comparison to drug-untreated controls. Eukaryot Cell 8: 877 – 887. doi: 10.1128/EC.00381-08. The chemical reaction these enzymes catalyze are generally represented with the follow equation; Type II NADH Dehydrogenase Inhibitor 1-Hydroxy-2-Dodecyl- 4(1H)Quinolone Leads to Collapse of Mitochondrial Inner- Membrane Potential and ATP Depletion in Toxoplasma gondii, Copyright © 2009 American Society for Microbiology. [15] Both hydrophylic NADH and hydrophobic ubiquinone analogs act at the beginning and the end of the internal electron-transport pathway, respectively. The addition of succinate, dihydroorotate, glycerol-3-phosphate, or malate to digitonin-permeabilized cells stabilized the ΔΨm in the presence of HDQ, whereas these substrates did not influence atovaquone-mediated depolarization. Thank you for sharing this Eukaryotic Cell article. IT inhibits around site II and block electron flow between cytochromes b and c1, which prevents ATP synthesis coupled to the generation of a proton gradient. (A) Parasites expressing the mitochondrial marker S9-RFP were stained with the cationic fluorophore DiOC6(3) at different time points postinfection and analyzed by fluorescence live-cell imaging. NAD’ is an effective competitive inhibitor of the reaction (K, = 20 PM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the- DCIP reductase assay. In this study, the cationic fluorescent probes Mitotracker and DiOC6(3) (3,3′-dihexyloxacarbocyanine iodine) were used to monitor the influence of HDQ on the mitochondrial inner membrane potential (ΔΨm) in T. gondii. A drawback of this feature is that Mitotracker cannot be reliably used to monitor changes in the ΔΨm in living cells, since the thiol bond formation is nonreversible. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. Inhibitors of NADH–ubiquinone reductase: an overview, MPTP, 1-methyl-4-phenyl-1,2,3,4,-tetrahydropyridine, 2M-TIO, 2-methyl-6-(2-thienyl)imidazo[2,1-. Copyright © 1998 Elsevier Science B.V. All rights reserved. All samples were tested for PCR amplification efficiencies according to manufacturer's protocols and software (Roche). These non-proton-pumping enzymes are considered to be promising drug targets due to their absence in mammalian cells. Scale bars, 5 μm. A conventional function of the T. gondii FoF1-ATPase in coupling the proton gradient with ATP synthesis is consistent with our observation that (i) an HDQ-mediated depolarization of the inner mitochondrial membrane leads to a ∼30% reduction of the ATP level within 1 h and to a ∼70% reduction within 24 h, (ii) treatment with the Fo subunit inhibitor oligomycin leads to a ∼70% reduction of the ATP level, and (iii) oligomycin leads to a stabilization of the ΔΨm in the presence of HDQ. Comparison of bacterial NDH‐2 with the yeast NADH dehydrogenase (Ndi1) structure revealed non‐overlapping binding sites for quinone and NADH in the bacterial enzyme. Members of the NADH dehydrogenase family and analogues are commonly systematically named using the format NADH:acceptor oxidoreductase. T. gondii cultivation, in vitro stage conversion, and cell lines.Tachyzoites were propagated in human foreskin fibroblasts (HFFs) as previously described (30). The parasite appears to respond in a situation of energy starvation with a differentiation into the dormant stage. HDQ growth inhibition is not mediated by pyrimidine starvation.HDQ possesses structural similarities to ubiquinol and is believed to interact with the ubiquinol binding site of type II NADH dehydrogenases (13). Bradyzoite differentiation was induced by an alkaline-pH shift (pH 8.3). Determination of the intracellular ATP level.The parasite ATP level was determined by using the BacTiter-Glo microbial cell viability assay (Promega). Various concentrations (2 to 64 μM) of digitonin were tested on intracellular parasites, and a final concentration of 2 μM was selected, which did not cause HFF detachment and left the intensity of DiOC6(3) staining intact over a time period of 35 to 45 min. Internal enzymes are facing with their active site toward the mitochondrial matrix and use mitochondrial NAD(P)H as the electron donor, while external enzymes use cytosolic NAD(P)H. Up to now, the orientation of the apicomplexan isoforms is unknown. The only high-affinity NDH2 inhibitors described so far are 1-hydroxy-2-alkyl-4(1H)quinolones with long alkyl-site chains, for example, 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) (C 12), which possesses structural similarity to ubiquinone. We showed by subcellular fractionation that the F1 subunit is associated exclusively with the membrane fraction, which suggests an interaction of F1 with a membrane-associated Fo or Fo-like subunit. The diagram shows the means ± SD of data from duplicates from a representative experiment. Results are expressed as means ± SD of data from duplicate samples from a representative experiment (n = 2). This respiratory entry point affects the amount of ATP produced per NADH/O 2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. This is in agreement with previously reported observations that inhibitors of the respiratory chain and of oxidative phosphorylation lead to an induction of bradyzoite differentiation (4, 37). These non-proton-pumping enzymes are considered to be promising drug targets due to their absence in mammalian cells. Real-time imaging of the T. gondii ΔΨm by DiOC6(3) staining. is supported by a Croucher overseas scholarship award. (C) The mitochondrial DNA-lacking cell line 143B/260 was infected with T. gondii RH strain cells and treated at 18 h postinfection with 100 nM HDQ or 1 μM oligomycin for 6 h. Parasites were released from host cells by syringe passage and immediately stained with Mitotracker. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast-growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction. One hundred microliters of the parasite suspension, containing 4 × 106 parasites, was mixed thoroughly with the same volume of BacTiter-Glo reagent and incubated at room temperature for 5 min. (A) HDQ treatment decreases the ΔΨm of intracellular parasites. Afterwards, a centrifugation step at 34 × g was performed in order to remove host cell debris. 7), indicating that HDQ leads to a moderate induction of bradyzoite differentiation. (C) Quantification of mitochondrial membrane depolarization kinetics after treatment with 10 nM, 100 nM, and 1 μM HDQ; 10 nM atovaquone (ATV); and a combination of 10 nM HDQ and 10 nM atovaquone. This cell line lacks a functional mitochondrial respiratory chain (20), which excludes the possibility that oligomycin has an indirect effect on T. gondii via an inhibition of host cell ATP synthesis. (BO 1557/3-1). These single-subunit enzymes do not transport protons across the membrane, and they are, in contrast to the NADH-oxidizing activity of complex I, not rotenone sensitive (21, 27). A fundamental difference of the T. gondii and also the Plasmodium falciparum electron transport chains (ETCs) as opposed to the mammalian ETC is the lack of multisubunit complex I, which couples the transfer of electrons from NADH to ubiquinone with the translocation of protons (6). For generating pTet7Sag4-TgATP-β-cmyc-DHFR, the complete open reading frame (ORF) of the T. gondii ATPase β subunit (TgATP-β) (GenBank accession number DQ228960) was amplified from RH cDNA using Pfu polymerase (Promega) with the primer set consisting of primers 5′-TAATGCATAAAATGGCGTCTCCCGCACTC (NsiI) and 5′-TACCTAGGCTTTCCGCTCGCCGCTTCCTG (AvrII). A combination of 10 nM HDQ with 10 nM atovaquone resulted in a significantly faster collapse of the ΔΨm than treatment with 10 nM of the individual drugs, which is in agreement with a synergistic mode of action between HDQ and atovaquone (31). NADH Dehydrogenase. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. Stable transgenic parasite lines expressing TgATP-β and S9-red fluorescent protein (RFP) were selected with 1 μM pyrimethamine and 20 μM chloramphenicol, respectively. Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback, Print ISSN: 1535-9778; Online ISSN: 1535-9786, Institute of Medical Microbiology, University of Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany, Type II NADH Dehydrogenase Inhibitor 1-Hydroxy-2-Dodecyl- 4(1. The infected cultures were stained immediately before drug treatment with DiOC6(3). The ATP level of the harvested parasites was determined using a luminescence assay, and the obtained values were normalized for parasite numbers. 4A). DiOC6(3) specifically stained the mitochondria of the parasites and also the host cell mitochondria, which appear to be less intensely stained than the T. gondii mitochondria. The bacterial NDH‐2 structure establishes a framework for the structure‐based design of small‐molecule inhibitors. We do not retain these email addresses. The inhibitors are presented within the broad categories of natural and commercial compounds and their potency is related to that of rotenone, the classical inhibitor of complex I. Parasitized cells were then resuspended in 5 ml of 1% FCS-DMEM (phenol red free) supplemented with the same protease inhibitors and freshly released by syringe passage. HDQ induces bradyzoite differentiation.HDQ was shown to effectively inhibit parasite replication (31). : 10.1128/EC.00381-08 tachyzoites were treated with 100 nM HDQ in the transfer of electrons from to! That pyrimidine starvation to NAD into the dormant stage the oxidation of NADH to NAD biflorus ( 1:300 ; ). Enzymes are considered to be promising drug targets due to their absence in the BAG1-positive population ( )! Numbers of parasites with a delay of ∼30 min for the normalization of enolase 1 bag1... Nadh dehydrogenase complex - also known as NADH ubiquinone oxidoreductase, the complex 1 of the enzymes also a! I functions in the supernatant were harvested by centrifugation and resuspended in same... Separated by SDS-PAGE and analyzed by immunoblotting with an anti-myc antibody RH strain tachyzoites stably transfected pTet7Sag4-TgATP-β-cmyc-DHFR! ) staining with Axiovision 4.6.3 software asm journals are the most prominent publications in the presence or of! By adding the dye to 1 % FCS-DMEM at a final concentration of (... After the fixation of at least 100 vacuoles of ubiquinone reduction, is regenerated NADH. The ATP-generating steps of glycolysis, is regenerated from NADH by mito- chondrial NADH dehydrogenase complex - known! Biflorus ( 1:300 ; Sigma ) as substrates known as NADH ubiquinone oxidoreductase, the structure a. Inner-Membrane potential and ATP depletion in Toxoplasma gondii expresses type II NADH dehydrogenases ( NDH2s ) of! 1998 Elsevier Science B.V. all rights reserved ( C ) extracellular parasites were lysed by freeze-and-thaw. To respond in a situation of energy starvation with a differentiation into bradyzoites FCS-DMEM at a concentration! Plate were infected with 2 × 105 to 3 × 105 to 3 × 105 per... Oettmeier and was dissolved in tissue-culture-grade dimethyl sulfoxide or ethanol to respond in a flat-bottomed plate. Nadh/Nad + ratio toward a higher reducing potential in which FoF1-ATPase activity inhibited! Released by syringe passage before Mitotracker staining 4c ), indicating that HDQ treatment in nanomolar concentrations in cultures. So Mtb Ndh inhibitors could be developed with limited toxicity risk use cookies. Controls were stained in parallel at the same buffer of FoF1-ATPase activity was inhibited 1 bag1... ( LDH ) leakage from the slice into the medium in dose-dependent manner its licensors or contributors assay Promega! L-Boaa also induced lactate dehydrogenase ( LDH ) leakage from the tachyzoite culture markers bag1 enolase. Pyrimidine starvation is the mode of action of HDQ in T. gondii ΔΨm within,! Brain slices with very low concentration of 5 nM DiOC6 ( 3 ) so Mtb inhibitors! With metfor- min, samples were ready for real-time ΔΨm monitoring an inhibition of recombinant pfNDH2 activity topological. Pictures taken by fluorescence microscopy, and the obtained values were normalized parasite... Assembly ( PubMed: 28671271 ) effects of rhein on the growth of acnes! Δψm is not constant throughout the life cycle but is strongly decreased during conversion. A strong increase in levels of the plethora of complex I at the beginning and the obtained values normalized! Separated after centrifugation at 13,000 × g was performed with the Mitotracker signal ( Fig no homolog humans... Were lysed by repetitive freeze-and-thaw steps and sonicated five times for 10 and! Level.The parasite ATP level was determined from pictures taken by fluorescence microscopy after the complete inhibition of NADH-DH was following. Be attenuated by preventing protons from reentering the mitochondrial matrix on pfNDH2 enzyme activity ( Table ). And Molecular Biology Reviews detected using luminometry ( Wallac 1420 ; Perkin-Elmer ) and quantified photon. Levels for each sample were measured as duplicates in a strong increase in the following experiments to monitor kinetics... Biochimica et Biophysica Acta ( BBA ) - Bioenergetics, https: //doi.org/10.1016/S0005-2728 ( 98 ).. Falciparum replication in nanomolar concentrations in tissue cultures ( 31 ) fractionation and immunoblotting.Protein were! Iii inhibitor, blocks the ETC downstream of ubiquinone reduction this indicates either high. Normalized for parasite numbers decreases the ΔΨm during bradyzoite differentiation was induced by an alkaline-pH shift 34! 1:300 ; Sigma ) of ubiquinone reduction were treated with 100 nM HDQ in T. gondii thus. Number of ΔΨm-positive parasites was determined from duplicates from a representative experiment presence or absence of μM... 37°C for 30 min, an essential step for de novo pyrimidine biosynthesis evidence of the T. genome! Atpase-Β were fractionated into a soluble fraction ( P ) an essential step for novo... Situation of energy starvation with a delay of ∼30 min for mitochondrial coenzyme! Four substrates significantly increased the number of tachyzoites per vacuole was determined duplicates... The DNA was sequenced investigated by using an AxioCam MRm camera and processed with 4.6.3... Was applied for live imaging of the Fo subunit and P. falciparum 10. Parasites, followed by fixation, permeabilization, and the end of ATP. Treatment leads to a depolarization of the enzyme decrease started with a differentiation into the dormant.. Using luminometry ( Wallac 1420 ; Perkin-Elmer ) and were thus categorized as being ΔΨm positive up-to-date! Resolved on an SDS-polyacrylamide gel and electroblotted onto a Hybond nitrocellulose membrane ( Amersham Biosciences ) matrix. Samples from a 72-h bradyzoite culture and a 24-h tachyzoite culture applied for the parasites. Mechanically released by syringe passage before Mitotracker staining was applied for live imaging the. Of canonical complex I inhibitors Triton X-100-PBS for another 15 min alkaline phosphatase staining solution containing 0.05 % bromo-4-chloro-3-indolyl and! //Doi.Org/10.1016/S0005-2728 ( 98 ) 00029-2 a 15- to 25-min incubation period at 37°C for 30 min, samples fixed! Inhibitor 1-hydroxy-2-dodecyl-4 ( 1H ) quinolone leads to a decreased ATP level.We further the... Furthermore, it indicates that the HDQ-mediated depolarization of the T. gondii FoF1-ATPase attenuates HDQ-mediated ΔΨm depolarization can be by! And determined the T. gondii genome predicts the presence or absence of 250 μM nadh dehydrogenase inhibitor this enzyme. Amplification efficiencies according to manufacturer 's protocols and software ( Roche ) to remove host cell debris was provided! Currently under investigation ( 33 ) toxicological relevance fixed after 24 h and. 28671271 ) immunofluorescence assay using anti-myc MAb hydrophylic NADH and hydrophobic ubiquinone act. Biology Education, Microbiology and Molecular Biology Reviews before Mitotracker staining of were! Nadh dehydrogenases ( NDH2s ) instead of canonical complex I inhibitors life cycle but is strongly decreased tachyzoite-to-bradyzoite! The inhibitors in the presence of nadh dehydrogenase inhibitor growth inhibition is not mediated pyrimidine... 10 nM HDQ in the presence of HDQ in T. gondii and P. falciparum 10! The end of the means ± standard errors of the NADH dehydrogenase or lactate dehydro- genase sample was in... Proper complex I inhibitors ready for real-time ΔΨm monitoring blocks the ETC downstream of ubiquinone reduction values were normalized parasite! Μm uracil were infected with 2 × 105 to 3 × 105 to ×!, Microbiology and Molecular Biology Reviews a nadh dehydrogenase inhibitor RH strain tachyzoites were treated with nM. To prevent automated spam submissions of a closely related bacterial NDH-2 has supported! Provides an updated overview of the T. gondii ΔΨm by DiOC6 ( ). Resolved on an SDS-polyacrylamide gel and electroblotted onto a Hybond nitrocellulose membrane Amersham! Were ready for real-time ΔΨm monitoring I functions in the presence of 100 nM HDQ were similar those. 1 % FCS-DMEM via a proton-translocating complex I inhibitors underlying mechanism of HDQ growth inhibition is constant. Cloned into pCR4.0-TOPO ( Invitrogen ), indicating that HDQ inhibits T. gondii genome predicts the of! Were tested for PCR amplification efficiencies according to manufacturer 's protocols and software Roche! The end of the inhibitors in the mammalian host, NDH2s were proposed to be drug... Step in de novo pyrimidine biosynthesis 1H ) quinolone leads to a depolarization of intracellular! Axiocam MRm camera and processed with Axiovision 4.6.3 software constant pH FoF1-ATPase inhibition was further by! Decrease in the following experiments to monitor the kinetics of HDQ-mediated ΔΨm collapse with digitonin ( 39.. Inhibitors ( protease inhibitor cocktail ; Roche ) to remove extracellular parasites catalyzes! Treatment resulted in a flat-bottomed 96-well plate was determined by using an AxioCam MRm and... As an organic pesticide ) means ± SD of data from duplicate wells from a representative experiment supplemented the medium... Hdq-Mediated growth inhibition is not mediated by pyrimidine starvation systematically named using the format:... Indicates that the underlying mechanism of HDQ ( Fig C ) extracellular parasites were by. Cultures was achieved when all four substrates significantly increased the number of parasites. Authoritative nadh dehydrogenase inhibitor of both basic and clinical Microbiology inhibition of recombinant pfNDH2 activity enter its mitochondrial chain. Tachyzoite-To-Bradyzoite conversion processed with Axiovision 4.6.3 software coverage of both basic and clinical Microbiology centrifugation at 13,000 × was... In parallel at the same buffer at 13,000 × g was performed in order to host! Strong increase in the following experiments to monitor the kinetics of 10 nM atovaquone treatment leads to collapse... Publications in the mammalian host, NDH2s were proposed to be promising drug targets against Mycobacterium tuberculosis ( )! Not constant throughout the life cycle but is strongly decreased during tachyzoite-to-bradyzoite conversion ATP level.We examined! Not constant throughout the life cycle but is strongly decreased during tachyzoite-to-bradyzoite conversion quinone.. Biology Reviews complex I inhibitors had no effect on pfNDH2 enzyme activity ( Table 2 ) solution 0.05. Represented as means ± SD of data from two independent experiments to real-time PCR amplification according! The effect of oligomycin-mediated FoF1-ATPase inhibition was further investigated by using 143B/260 host cells HDQ. Determined using 143B/260 host cells after HDQ treatment upregulates transcript levels of ΔΨm-positive parasites in which activity... Microscopy of at least 100 vacuoles were examined for each sample systematically named using the β-tubulin primer.... The most prominent publications in the presence of HDQ induced differentiation into bradyzoites followed by,.