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.

Functional heterogeneity of Fo·F1H+-ATPase/synthase in coupled Paracoccus denitrificans plasma membranes.

Fo·F1H+-ATPase/synthase in coupled plasma membrane vesicles of Paracoccus denitrificans catalyzes ATP hydrolysis and/or ATP synthesis with comparable enzyme turnover. Significant difference in pH-profile of these alternative activities is seen: decreasing pH from 8.0 to 7.0 results in reversible inhibition of hydrolytic activity, whereas ATP synthesis activity is not changed. The inhibition of ATPase activity upon acidification results from neither change in ADP(Mg2+)-induced deactivation nor the energy-dependent enzyme activation. Vmax, not apparent KmATP is affected by lowering the pH. Venturicidin noncompetitively inhibits ATP synthesis and coupled ATP hydrolysis, showing significant difference in the affinity to its inhibitory site depending on the direction of the catalysis. This difference cannot be attributed to variations of the substrate-enzyme intermediates for steady-state forward and back reactions or to possible equilibrium between ATP hydrolase and ATP synthase Fo·F1 modes of the opposite directions of catalysis. The data are interpreted as to suggest that distinct non-equilibrated molecular isoforms of Fo·F1 ATP synthase and ATP hydrolase exist in coupled energy-transducing membranes.

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.

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.