Complex effects of macrolide venturicidins on bacterial F-ATPases likely contribute to their action as antibiotic adjuvants.

Bacterial energy metabolism is now recognized as a critical factor for the efficacy of antibiotics. The F-type ATPase/ATP synthase (FOF1) is a central player in cellular bioenergetics of bacteria and eukaryotes, and its potential as a selective antibiotic target has been confirmed by the success of bedaquiline in combatting multidrug-resistant tuberculosis. Venturicidin macrolides were initially identified for their antifungal properties and were found to specifically inhibit FOF1 of eukaryotes and bacteria. Venturicidins alone are not effective antibacterials but recently were found to have adjuvant activity, potentiating the efficacy of aminoglycoside antibiotics against several species of resistant bacteria. Here we discovered more complex effects of venturicidins on the ATPase activity of FOF1 in bacterial membranes from Escherichia coli and Pseudomonas aeruginosa. Our major finding is that higher concentrations of venturicidin induce time- and ATP-dependent decoupling of F1-ATPase activity from the venturicidin-inhibited, proton-transporting FO complex. This dysregulated ATPase activity is likely to be a key factor in the depletion of cellular ATP induced by venturicidins in prior studies with P. aeruginosa and Staphylococcus aureus. Further studies of how this functional decoupling occurs could guide development of new antibiotics and/or adjuvants that target the F-type ATPase/ATP synthase.

Discovery of Venturicidin Congeners and Identification of the Biosynthetic Gene Cluster from Streptomyces sp. NRRL S-4.

Chemical screening of Streptomyces sp. NRRL S-4 with liquid chromatography-mass spectrometry (LC-MS) and the following chromatographic isolation led to the discovery of four 20-membered macrolides, venturicidin A (4) and three new congeners venturicidins D-F (1-3). Genome sequencing of strain S-4 revealed the presence of a biosynthetic gene cluster (BGC) encoding glycosylated type I polyketides (PKS). The BGC designated to venturicidin biosynthesis (ven) was supported by the proposed biosynthetic pathway and confirmed by inactivation of the core PKS gene of venK. Bioinformatic analyses on the conserved motifs and known stereospecificities in PKS modules are consistent with the structure and absolute configuration. This is the first report of venturicidin BGC since the discovery of the macrolide in 1961. In the biological assays, venturicidin A (4) and E (2) displayed a high selective cytotoxicity against acute monocytic leukemia MV-4-11 cells with IC50 values of 0.09 and 0.94 µM, respectively. Venturicidin A (4) also showed a weak inhibitory activity on FMS-like-tyrosine kinase.

Venturicidin A, A Membrane-active Natural Product Inhibitor of ATP synthase Potentiates Aminoglycoside Antibiotics.

Despite the remarkable advances due to the discovery and development of antimicrobials agents, infectious diseases remain the second leading cause of death worldwide. This fact underlines the importance of developing new therapeutic strategies to address the widespread antibiotic resistance, which is the major contributing factor for clinical failures of the current therapeutics. In a screen for antibiotic adjuvants, we identified a natural product from actinomycetes, venturicidin A (VentA), that potentiates the aminoglycoside antibiotic gentamicin against multidrug-resistant clinical isolates of Staphylococcus, Enterococcus, and Pseudomonas aeruginosa. Furthermore, the combination of gentamicin and VentA was bactericidal and rapidly eradicated methicillin-resistant S. aureus (MRSA). The molecular mechanism of gentamicin potentiation activity is attributed to uncoupling of ATP synthesis by VentA from electron transport presumably by blocking the proton flow through ATP synthase, which results in an elevated concentration of extracellular protons and subsequent anticipated raise in gentamicin uptake. The disruption of the proton flux was characterized by perturbed membrane potential in MRSA. These results demonstrate that inhibition of ATP synthase along with the subsequent membrane dysregulation, as shown here with VentA, complements aminoglycoside antibiotics against MDR bacteria, and that this approach may be employed to combat bacterial resistance.

Time-resolved ATP measurements during vesicle respiration.

In vitro synthesis of ATP catalyzed by the ATP-synthase requires membrane vesicles, in which the ATP-synthase is present within the bilayer membrane. Inverted vesicle prepared from Gram negative cells (e.g., Escherichia coli or Pseudomonas putida) can be readily obtained and used for in vitro ATP-synthesis. Up to now, quantification of ATP synthesized by membrane vesicles has been mostly analyzed via bioluminescence-based assays. Alternatively, vesicle respiration and the associated ATP level can be determined using biosensors, which not only provide high selectivity, but allow ATP measurements without the sample being illuminated. Here, we present a microbiosensor for ATP in combination with scanning electrochemical microscopy (SECM) using an innovative two-compartment electrochemical cell for the determination of ATP levels at E.coli or P. putida inverted vesicles. For a protein concentration of 22 mg/ml, a total amount of 0.29 ±â€¯0.03 µM/µl ATP per vesicle was determined in case of E.coli; in turn, P. putida derived vesicles yielded 0.48 ±â€¯0.02 µM/µl ATP per vesicle at a total protein concentration of 25.2 mg/ml. Inhibition experiments with Venturicidin A clearly revealed that the respiratory chain enzyme complex responsible for ATP generation is effectively involved.

Selective and potent in vitro antitrypanosomal activities of ten microbial metabolites.

More than 400 compounds isolated from soil microorganisms, and catalogued in the antibiotic library of the Kitasato Institute for Life Sciences, were screened against African trypanosomes. Ten compounds were found to have selective and potent antitrypanosomal activity in vitro: aureothin, cellocidin, destomycin A, echinomycin, hedamycin, irumamycin, LL-Z 1272beta, O-methylnanaomycin A, venturicidin A and virustomycin A. Results of the in vitro assays using the GUTat 3.1 strain of Trypanosomal brucei brucei and the STIB900 strain of T. b. rhodesiense are presented. Cytotoxicity was determined using a human MRC-5 cell line. This is the first report of antitrypanosomal activities of the 10 microbial metabolites listed above.

Rotation of a complex of the gamma subunit and c ring of Escherichia coli ATP synthase. The rotor and stator are interchangeable.

ATP synthase (F0F1) transforms an electrochemical proton gradient into chemical energy (ATP) through the rotation of a subunit assembly. It has been suggested that a complex of the gamma subunit and c ring (c(10-14)) of F0F1 could rotate together during ATP hydrolysis and synthesis (Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T., Wada, Y., and Futai, M. (1999) Science 286, 1722-1724). We observed that the rotation of the c ring with the cI28T mutation (c subunit cIle-28 replaced by Thr) was less sensitive to venturicidin than that of the wild type, consistent with the antibiotic effect on the cI28T mutant and wild-type ATPase activities (Fillingame, R. H., Oldenburg, M., and Fraga, D. (1991) J. Biol. Chem. 266, 20934-20939). Furthermore, we engineered F0F1 to see the alpha(3)beta(3) hexamer rotation; a biotin tag was introduced into the alpha or beta subunit, and a His tag was introduced into the c subunit. The engineered enzymes could be purified by metal affinity chromatography and density gradient centrifugation. They were immobilized on a glass surface through the c subunit, and an actin filament was connected to the alpha or beta subunit. The filament rotated upon the addition of ATP and generated essentially the same frictional torque as one connected to the c ring. These results indicate that the gammaepsilonc(10-14) complex is a mechanical unit of the enzyme and that it can be used as a rotor or a stator experimentally, depending on the subunit immobilized.

Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact.

F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.

Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation.

F0F1, found in mitochondria or bacterial membranes, synthesizes adenosine 5'-triphosphate (ATP) coupling with an electrochemical proton gradient and also reversibly hydrolyzes ATP to form the gradient. An actin filament connected to a c subunit oligomer of F0 was able to rotate by using the energy of ATP hydrolysis. The rotary torque produced by the c subunit oligomer reached about 40 piconewton-nanometers, which is similar to that generated by the gamma subunit in the F1 motor. These results suggest that the gamma and c subunits rotate together during ATP hydrolysis and synthesis. Thus, coupled rotation may be essential for energy coupling between proton transport through F0 and ATP hydrolysis or synthesis in F1.

Presence of two enzymes, different from the F1F0-ATPase, hydrolyzing nucleotides in human term placental mitochondria.

The hydrolysis of ATP, ADP or GTP was characterized in mitochondria and submitochondrial particles since a tightly-bound ATPase associated with the inner mitochondrial membrane from the human placenta has been described. Submitochondrial particles, which are basically inner membranes, were used to define the location of this enzyme. Mitochondria treated with trypsin and specific inhibitors were also used. The oxygen consumption stimulated by ATP or ADP was 100% inhibited in intact mitochondria by low concentrations of oligomycin (0.5 microgram/mg) or venturicidine (0.1 microgram/mg), while the hydrolysis of ATP or ADP was insensitive to higher concentrations of these inhibitors but it was inhibited by vanadate. Oligomycin or venturicidine showed a different inhibition pattern in intact mitochondria in relation to the hydrolysis of ATP, ADP or GTP. When submitochondrial particles were isolated from mitochondria incubated with oligomycin or venturicidine, no further inhibition of the nucleotide hydrolysis was observed, contrasting with the partial inhibition observed in the control. By incubating the placental mitochondria with trypsin, a large fraction of the hydrolysis of nucleotides was eliminated. In submitochondrial particles obtained from mitochondria treated with trypsin or trypsin plus oligomycin, the hydrolysis of ATP was 100% sensitive to oligomycin at low concentrations, resembling the oxygen consumption; however, this preparation still showed some ADP hydrolysis. Native gel electrophoresis showed two bands hydrolyzing ADP, suggesting at least two enzymes involved in the hydrolysis of nucleotides, besides the F1F0-ATPase. It is concluded that human placental mitochondria possesses ADPase and ATP-diphosphohydrolase activities (247).

Proton coupling is preserved in membrane-bound chloroplast ATPase activated by high concentrations of tentoxin.

The effect of tentoxin at high concentrations was investigated in thylakoids and proteoliposomes containing bacteriorhodopsin and CF0CF1. Venturicidin-sensitive ATP hydrolysis, ATP-generated delta pH and ATP synthesis were practically 100% inhibited at 2 microM tentoxin, and restored to various extents beyond 50 microM. With respect to the native enzyme, tentoxin-reactivated ATPase had the following properties: (i) a higher delta pH requirement to synthetise ATP; (ii) a decreased futile proton flow through CF0CF1 (without ADP), which remains 100% blocked by ADP. It is concluded that despite its altered kinetic performances, tentoxin-modified CF0CF1 preserves its mechanism and remains a tightly coupled proton pump.

ATP synthase: activating versus catalytic proton transfer.

ATP synthase (F-ATPase) of chloroplasts, CF0CF1, is both activated and driven by transmembrane protonmotive force. We dichotomized between activating and driving proton transfer by specific inhibitors, tentoxin and venturicidin. Thylakoids membranes were submitted to voltage steps (by flashing light) superimposed to a steady pH-difference. Transient proton intake, transfer and release by CF0CF1 was monitored by spectroscopic probes. Both activities, activation and catalysis, required all three partial reactions of the proton, however, activating proton transfer rose first (monophasically, tau 1/2 approximately 15 ms) followed by another phase of equal magnitude with a time lag of about 15 ms. Both types of consecutive proton transfer reactions contribute free energy for ATP synthesis.

Potassium selective and venturicidin sensitive conductances of Fo purified from bovine heart mitochondria, reconstituted in planar lipid bilayers.

Purified F(o), isolated from bovine heart mitochondria was reconstituted into planar lipid bilayers. Two cation selective conductances could be identified. Most frequently, incorporation of F(o) resulted in a voltage sensitive K+ channel of 18 pS. Venturicidin drastically decreased the open probability of this channel. The second conductance of 47 pS had lost its voltage sensitivity but had retained the sensitivity to venturicidin. The F(o) preparation is very pure and will be used in crystallography studies. However, despite the high degree of biochemical purity, the preparation gives rise to several distinct conductances. Therefore, we conclude that for structure/function analysis of the F(o) part a biophysical characterization is required to assess the homogeneity of the preparation.

Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from pathway specificity.

Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex (OE33) are two of several thylakoid lumen-located proteins that are made in the cytosol, imported into chloroplasts, and subsequently transported into thylakoids. Recently, competition studies showed that there are two pathways for protein transport into the thylakoid lumen and that plastocyanin and OE33 are on the same pathway (Cline, K., Henry, R., Li, C., and Yuan, J. (1993) EMBO J. 12, 4105-4114). Our expectation is that transport requirements reflect the steps of the process and that proteins on the same pathway share similar requirements. Unfortunately, the transport requirements for plastocyanin and OE33 are not well established. Here, we investigated transport in a reconstituted system with isolated thylakoids. Efficient transport of OE33 and plastocyanin was only obtained when stromal extract was included in the assay. Heat or protease treatment of stromal extract eliminated its ability to stimulate transport. Transport was abolished by treatments designed to deplete ATP or to prevent its formation and was greatly reduced in the presence of ionophores that dissipate the trans-thylakoidal proton gradient. These results show that transport of OE33 and plastocyanin requires ATP and is stimulated by stromal protein(s) and the trans-thylakoidal proton gradient. Taken together, these and previous results suggest that there are two mechanistically distinct pathways for protein transport into the thylakoid lumen.

Deactivation of F0F1 ATPase in intact plant mitochondria. Effect of pH and inhibitors.

By using a method especially adapted to intact (pea leaf) mitochondria, we studied the regulation of the F0F1 ATPase by the electrochemical proton gradient (delta mu H+) and by the matricial pH. The kinetics of decay of the ATP hydrolase activity was studied immediately after the collapse of the electrochemical proton gradient by an uncoupler. At pH 7.5, three inhibitors of the ATPase (venturicidin, tri-n-butyl tin and aurovertin), used at non-saturating concentrations, inhibited ATP hydrolysis to the same extent throughout the decay. This showed that the activity was totally controlled by the ATPase during all the decay and rules out any involvement of the phosphate or nucleotide carriers. This interpretation was confirmed by the fact that carboxyatractyloside, an inhibitor of the ATP/ADP antiporter, had a strong effect only on the initial rate of ATP hydrolysis, but not on the rate measured after some tens of seconds of decay. Oligomycin, at variance with the other ATPase inhibitors, interfered with the deactivation process, suggesting that its effect depends on the conformational state of the enzyme. Between pH 6.5 and 7.5, the hydrolase activity rose continuously and was still kinetically controlled by the ATPase. At higher pH value, the activity slightly decreased and appeared limited by at least one of the carriers. The activity of the ATPase itself, free of any transport process, seemed to increase monotonously with pH from 6.5 to 8. The electrochemical proton gradient is required to maintain the ATPase active, whereas no effect can be observed on transport processes. Matricial pH, while modulating the apparent catalytic turnover, has no marked effect on the rate of deactivation. These results, obtained with intact mitochondria, extend previous observations on the isolated enzyme and question the binding of IF1 as a rate-limiting step for ATPase deactivation.

The prokaryotic thermophilic TF1-ATPase is functionally compatible with the eukaryotic CFo-part of the chloroplast ATP-synthase.

The ATP synthase from chloroplasts, CFo.F1, was reconstituted into liposomes, from which most of CF1 was removed by a short treatment with guanidinium chloride. ATP-dependent proton uptake was restored with these CFo-liposomes even better by the addition of the bacterial TF1-than of the related CF1-part. This proton uptake was prevented by tentoxin, a specific inhibitor of the CF1-ATPase, in these CFo.F1-liposomes, but not in the hybrid CFo.TF1-liposomes. Venturicidin, a specific inhibitor of proton flow through CFo, was able to block it in both the hybrid CFo.TF1-liposomes and reconstituted CFo.F1-liposomes. These results indicate that the bacterial TF1-part binds to the eukaryotic CFo-part of four subunits forming a functional CFo.TF1-ATPase.

Catalytic and activating protons follow different pathways in the H(+)-ATPase of potato tuber mitochondria.

The effect of some F0F1 inhibitors on the activation of the H(+)-ATPase by the electrochemical proton gradient was investigated in mitochondria extracted from potato tubers. Transient activated state of the ATPase was revealed by addition of ATP and of the detergent lauryldimethylamine oxide (LDAO) to energized mitochondria. Venturicidin, tri-n-butyltin and aurovertin at high concentrations did not affect the process of delta mu H(+)-activation, whereas oligomycin fully blocked it. The results support the idea of separate pathways or binding sites for catalytic and activating protons.

ATP synthesis energized by delta pNa and delta psi in proteoliposomes containing the F0F1-ATPase from Propionigenium modestum.

After incorporation of the purified Na(+)-translocating F0F1-ATPase from Propionigenium modestum into preformed phospholipid vesicles the synthesis of ATP from ADP and inorganic phosphate could be observed under conditions where a valinomycin-mediated K+ diffusion potential (delta psi) and/or a Na+ concentration gradient (delta pNa) were imposed. This reaction was not inhibited by the protonophore carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP). Furthermore, the delta pNa-driven ATP synthesis was stimulated by FCCP. In contrast, the addition of the Na+/H+ antiporter monensin or of the F0F1 inhibitors N,N'-dicyclohexylcarbodiimide and venturicidin abolished the synthesis of ATP completely. Finally, delta pNa alone was able to elicit ATP synthesis, when a Na+ concentration gradient of sufficient magnitude was applied. In this case ATP synthesis occurred above a threshold level of approximately 120 mV and, furthermore, delta psi and delta pNa appear to be equivalent as driving forces for this process. Therefore, the data provide firm evidence for the concept that delta"mu Na+ is the primary driving force for the synthesis of ATP in P. modestum.

Studies on the mechanism of oxidative phosphorylation. ATP synthesis by submitochondrial particles inhibited at F0 by venturicidin and organotin compounds.

Oligomycin,N,N'-dicyclohexylcarbodiimide (DCCD), venturicidin, and tetracoordinate organotin compounds (R3SnX) are potent inhibitors of the mitochondrial ATP synthase complex, all acting on the membrane sector, F0. Oligomycin and DCCD inhibit proton translocation through F0 and energy transfer between F0 and the catalytic sector, F1, of the ATP synthase complex. Our results have shown that venturicidin and organotin compounds (tributyltin and triphenyltin chloride were used) greatly attenuate these processes, but do not cause complete inhibition. As a result, bovine submitochondrial particles (SMP) treated with venturicidin or tributyltin chloride were shown to be capable of ATP hydrolysis and synthesis, albeit at very slow rates. We had shown previously that in ATP synthesis Vmax and apparent Km for ADP and Pi increase or decrease, respectively, as the steady-state membrane potential is elevated or lowered (Matsuno-Yagi, A., and Hatefi, Y. (1986) J. Biol. Chem. 261, 14031-14038). These changes occurred at constant Vmax/Km, suggesting that the apparent Km changes were due mainly to kcat changes. Results presented here show that, in respiring SMP treated with venturicidin or organotin compounds, the membrane potential is near the static-head level, but the slow rate of ATP synthesis takes place with a low KmADP value of 2-3 microM. In agreement with our previous conclusions, these results indicate that it is not the membrane potential per se that affects KmADP during ATP synthesis, but rather it is the rate of energy transfer from F0 to F1 that influences both Vmax and KmADP. Further conclusions from the above studies have been discussed in relation to the possible mechanism of energy transfer between F0 and F1 and the manner in which venturicidin and organotin compounds might attenuate this process.

Studies on the mechanism of oxidative phosphorylation. Different effects of F0 inhibitors on unisite and multisite ATP hydrolysis by bovine submitochondrial particles.

Bovine submitochondrial particles prepared in the presence of GTP (G-SMP), as well as G-SMP washed in 150 mM KCl, catalyzed unisite ATP hydrolysis with a first order rate constant of 0.12 s-1. This rate constant remained unchanged at ATP concentrations < 0.06 microM but increased sharply at higher ATP concentrations, presumably because of ATP binding to other catalytic or regulatory sites. Pretreatment of the particles with oligomycin greatly inhibited unisite ATP binding, in agreement with previous findings. Pretreatment of the particles with N,N'-dicyclohexylcarbodiimide had a slight effect on unisite ATP binding, whereas pretreatment with the inhibitors venturicidin and tributyl(or triphenyl)tin chloride had no effect. Titration of unisite ATPase activity with increasing concentrations of oligomycin or efrapeptin resulted in sigmoidal inhibition curves, as though more than a single inhibition site was being titrated by each inhibitor. Venturicidin and organotin compounds had little effect on the ATPase activity of SMP at [ATP] < or = [F1] and did not cause 100% inhibition at [ATP] >> [F1]. By analogy to our previous studies on the inhibition of the ubiquinol-cytochrome c reductase complex by antimycin (Hatefi, Y., and Yagi, T. (1982) Biochemistry 24, 6614-6618), it is proposed that venturicidin and organotin compounds freeze the structure of the F0 sector of the ATP synthase complex in such a manner that prevents the subunit molecular motions required for rapid proton flux but allows a slow proton flux generated by ATPase activity at low ATP concentrations.