Purinergic ligands induce extracellular acidification and increased ATP turnover in HepG2 cells.

Nucleosides and nucleotides at µM concentrations stimulated a 300% increase in acid secretion in HepG2 cells, which was quantitatively accounted for as increased export of lactate generated by glycogenolysis. Agonist selectivity encompassed nucleosides and nucleotides for all 5 natural nucleobases and, along with antagonist profiles, was inconsistent with a role for purinergic receptors in mediating this activity. Agonist catabolism did not contribute significantly to either low selectivity or lactate production. Lactate production was driven by an increase in ATP turnover of as much as 56%. For some agonists, especially adenosine, ATP turnover decreased precipitously at mM concentrations, correlating with known adenosine-stimulated apoptosis. We propose that nucleoside/nucleotide agonists induce a futile energy cycle via a novel mechanism, which results in increased ATP turnover and initiates a continuum of events that for some agonists culminates in apoptosis.

Mathermycin, an anti-cancer molecule that targets cell surface phospholipids.

Mathermycin, a lantipeptide isolated from marine actinomycete Marinactinospora thermotolerans, is an antibiotic that has been shown to disrupt bacterial plasma membrane. We now provide evidences that mathermycin can also disrupt cancer, but not normal, cell plasma membranes through targeting phosphatidylethanolamine (PE), which is located only in the inner leaflet of the plasma membrane in normal cells but in both the inner and outer leaflets of the membrane in tumor cells. Our data shows that mathermycin inhibits the metabolic activity and induces mainly necrotic death of all cancer cell lines with EC50 between 4.2 and 16.9 µM, while normal cell lines have EC50 between 113 and 129 µM. The cytotoxicity of mathermycin could be inhibited by exogenous PE, but not phosphoserine and phosphocholine. The formation of mathermycin-PE complexes was confirmed by in silico analysis, HPLC and MS spectrometer. Furthermore, mathermycin exhibited similar cytotoxicity toward cancer and multidrug resistant cancer cells, which could be due to its ability to inhibit mitochondrial function, as shown by our data from the Seahorse™ metabolic analyzer. This study demonstrates that mathermycin is a potentially effective class of anti-tumor chemotherapeutics that do not easily develop resistance due to a mechanism of action targeting PE.

Oligomycin-induced proton uncoupling.

Oligomycin is a classical mitochondrial reagent that binds to the proton channel on the Fo component of ATP synthase. As a result, oligomycin blocks mitochondrial ATP synthesis, proton translocation, and O2 uptake. Here we show that oligomycin induces proton uncoupling subsequent to inhibition of ATP synthesis, as evidenced by recovery of O2 uptake to near baseline levels. Uncoupling is uniquely rapid and readily observed in HepG2 cells but is also observed at longer times in the unrelated H1299 cell line. Proton fluxes plateau at oligomycin concentrations in the region 0.25-5 µM. At the plateau, fluxes are lower than expected for the classical mitochondrial permeability transition pore, although in H1229 cells, fluxes increase to levels consistent with pore opening at higher oligomycin concentrations. Uncoupling is observed in cells metabolizing either pyruvate or lactate and reversed by addition of glucose to restore ATP synthesis. Uncoupling is not sensitive to cyclosporin A and is not reversed by the ANT inhibitor bongkrekic acid. However, bongkrekic acid inhibits uncoupling if added before oligomycin, which we interpret in terms of maintenance of mitochondrial ATP levels.

Shellmycin A-D, Novel Bioactive Tetrahydroanthra-γ-Pyrone Antibiotics from Marine Streptomyces sp. Shell-016.

Four novel bioactive tetrahydroanthra-γ-pyrone compounds, shellmycin A-D (1-4), were isolated from the marine Streptomyces sp. shell-016 derived from a shell sediment sample collected from Binzhou Shell Dike Island and Wetland National Nature Reserve, China. The structures of these four compounds were established by interpretation of 1D and 2D NMR and HR-MS data, in which the absolute configuration of 1 was confirmed by single crystal X-ray diffraction, and compound 3 and 4 are a pair of stereoisomers. Compound 1-4 exhibited cytotoxic activity against five cancer cell lines with the IC50 value from 0.69 µM to 26.3 µM. Based on their structure-activity relationship, the putative biosynthetic pathways of these four compounds were also discussed.

Singing Proficiency of Members of a Choir Formed by Prelingually Deafened Children With Cochlear Implants.

Purpose A group of 10 prelingually deafened children with cochlear implants (CIs) formed a choir and received 21 months of formal music training. The purpose of this study was to evaluate the singing proficiency of these children. Method The participants included all choir members (7 girls and 3 boys, mean age of 9.5 years old) who were unilateral CI users. Meanwhile, 8 age-matched children with normal hearing were recruited as controls and were trained on 1 song for 2 weeks. Individual singing samples without instrument accompaniment were recorded from all participants. The singing samples were subject to acoustic analysis in which the fundamental frequency (F0) of each note was extracted and the duration was measured. Five metrics were developed and computed to quantify the accuracy of their pitch and rhythm performance. The 5 metrics included (a) percent correct of F0 contour direction of adjacent notes, (b) mean deviation of the normalized F0 across the notes, (c) mean deviation of the pitch intervals, (d) mean deviation of adjacent note duration ratio, and (e) mean absolute deviation of note duration. Results The choir members with CIs demonstrated high accuracy in both pitch and tempo measures and performed on par with the children with normal hearing. Early start of music training after implantation and use of bimodal hearing contributed to the development of better music ability in these children with CIs. Conclusion These findings indicated that rigorous music training could facilitate high singing proficiency in prelingually deafened children with CIs.

Indole-Induced Reversion of Intrinsic Multiantibiotic Resistance in Lysobacter enzymogenes.

Lysobacter species are a group of environmental bacteria that are emerging as a new source of antibiotics. One characteristic of Lysobacter is intrinsic resistance to multiple antibiotics, which had not been studied. To understand the resistance mechanism, we tested the effect of blocking two-component regulatory systems (TCSs) on the antibiotic resistance of Lysobacter enzymogenes, a prolific producer of antibiotics. Upon treatment with LED209, an inhibitor of the widespread TCS QseC/QseB, L. enzymogenes produced a large amount of an unknown metabolite that was barely detectable in the untreated culture. Subsequent structural elucidation by nuclear magnetic resonance (NMR) unexpectedly revealed that the metabolite was indole. Indole production was also markedly induced by adrenaline, a known modulator of QseC/QseB. Next, we identified two TCS genes, L. enzymogenesqseC (Le-qseC) and Le-qseB, in L. enzymogenes and found that mutations of Le-qseC and Le-qseB also led to a dramatic increase in indole production. We then chemically synthesized a fluorescent indole probe that could label the cells. While the Le-qseB (cytoplasmic response regulator) mutant was clearly labeled by the probe, the Le-qseC (membrane sensor) mutant was not labeled. It was reported previously that indole can enhance antibiotic resistance in bacteria. Therefore, we tested if the dramatic increase in the level of indole production in L. enzymogenes upon blocking of Le-qseC and Le-qseB would lead to enhanced antibiotic resistance. Surprisingly, we found that indole caused the intrinsically multiantibiotic-resistant bacterium L. enzymogenes to become susceptible. Point mutations at conserved amino acids in Le-QseC also led to antibiotic susceptibility. Because indole is known as an interspecies signal, these findings may have implications.IMPORTANCE The environmental bacterium Lysobacter is a new source of antibiotic compounds and exhibits intrinsic antibiotic resistance. Here, we found that the inactivation of a two-component regulatory system (TCS) by an inhibitor or by gene deletion led to a remarkable increase in the level of production of a metabolite in L. enzymogenes, and this metabolite was identified to be indole. We chemically synthesized a fluorescent indole probe and found that it could label the wild type and a mutant of the TCS cytoplasmic response regulator but not a mutant of the TCS membrane sensor. Indole treatment caused the intrinsically multidrug-resistant bacterium L. enzymogenes to be susceptible to antibiotics. Mutations of the TCS sensor also led to antibiotic susceptibility. Because indole is known as an interspecies signal between gut microbiota and mammalian hosts, the observation that indole could render intrinsically resistant L. enzymogenes susceptible to common antibiotics may have implications.

Heterocyclic Aromatic N-Oxidation in the Biosynthesis of Phenazine Antibiotics from Lysobacter antibioticus.

Heterocyclic aromatic N-oxides often have potent biological activities, but the mechanism for aromatic N-oxidation is unclear. Six phenazine antibiotics were isolated from Lysobacter antibioticus OH13. A 10 gene cluster was identified for phenazine biosynthesis. Mutation of LaPhzNO1 abolished all N-oxides, while non-oxides markedly increased. LaPhzNO1 is homologous to Baeyer-Villiger flavoproteins but was shown to catazlye phenazine N-oxidation. LaPhzNO1 and LaPhzS together converted phenazine 1,6-dicarboxylic acid to 1,6-dihydroxyphenazine N5,N10-dioxide. LaPhzNO1 also catalyzed N-oxidation of 8-hydroxyquinoline.

Iterative polyketide biosynthesis by modular polyketide synthases in bacteria.

Modular polyketide synthases (type I PKSs) in bacteria are responsible for synthesizing a significant percentage of bioactive natural products. This group of synthases has a characteristic modular organization, and each module within a PKS carries out one cycle of polyketide chain elongation; thus each module is non-iterative in function. It was possible to predict the basic structure of a polyketide product from the module organization of the PKSs, since there generally existed a co-linearity between the number of modules and the number of chain elongations. However, more and more bacterial modular PKSs fail to conform to the canonical rules, and a particularly noteworthy group of non-canonical PKSs is the bacterial iterative type I PKSs. This review covers recent examples of iteratively used modular PKSs in bacteria. These non-canonical PKSs give rise to a large array of natural products with impressive structural diversity. The molecular mechanism behind the iterations is often unclear, presenting a new challenge to the rational engineering of these PKSs with the goal of generating new natural products. Structural elucidation of these synthase complexes and better understanding of potential PKS-PKS interactions as well as PKS-substrate recognition may provide new prospects and inspirations for the discovery and engineering of new bioactive polyketides.

Synthesis of a 2,4,6,8,10-dodecapentanoic acid thioester as a substrate for biosynthesis of Heat Stable Antifungal Factor (HSAF).

The N-acetylcystamine (SNAC) thioester of dodecapentaenoic acid, an analog of a putative intermediate in the biosynthesis of Heat Stable Antifungal Factor (HSAF), is synthesized. Key steps include sequential Horner-Emmons homologations with the Weinreb amide of diethylphosponoacetic acid, and thioesterification of an aldol-derived 3-hydroxyalkanoate, which serves as a stable precursor of the sensitive polyenoate. The thioester was investigated as a biosynthetic substrate using a purified nonribosomal peptide synthetase and was not incorporated in the observed products.

Fatty Acyl Incorporation in the Biosynthesis of WAP-8294A, a Group of Potent Anti-MRSA Cyclic Lipodepsipeptides.

WAP-8294A is a family of at least 20 cyclic lipodepsipeptides exhibiting potent anti-MRSA activity. These compounds differ mainly in the hydroxylated fatty acyl chain; WAP-8294A2, the most potent member of the family that reached clinical trials, is based on (R)-3-hydroxy-7-methyloctanoic acid. It is unclear how the acyl group is incorporated because no acyl-CoA ligase (ACL) gene is present in the WAP-8294A gene cluster in Lysobacter enzymogenes OH11. Here, we identified seven putative ACL genes in the OH11 genome and showed that the yield of WAP-8294A2 was impacted by multiple ACL genes with the ACL6 gene having the most significant effect. We then investigated several (R)-3-hydroxy fatty acids and their acyl SNAC (N-acetylcysteamine) thioesters as substrates for the ACLs. Feeding (R)-3-hydroxy-7-methyloctanoate-SNAC to the ACL6 gene deletion mutant restored the production of WAP-8294A2. Finally, we heterologously expressed the seven ACL genes in E. coli and purified six of the proteins. While these enzymes exhibit a varied level of activity in vitro, ACL6 showed the highest catalytic efficiency in converting (R)-3-hydroxy-7-methyloctanoic acid to its CoA thioester when incubated with coenzyme A and ATP. These results provided both in vivo and in vitro evidence to support the fact that ACL6 is the main player for fatty acyl activation and incorporation in WAP-8294A2 biosynthesis. The results also suggest that the molecular basis for the acyl chain diversity in the WAP-8294A family is the presence of functionally overlapping ACLs.

Iterative assembly of two separate polyketide chains by the same single-module bacterial polyketide synthase in the biosynthesis of HSAF.

Antifungal HSAF (heat-stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two "clean" strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl-coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene-PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS-NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide-peptide natural products.

Unusual activities of the thioesterase domain for the biosynthesis of the polycyclic tetramate macrolactam HSAF in Lysobacter enzymogenes C3.

HSAF is an antifungal natural product with a new mode of action. A rare bacterial iterative PKS-NRPS assembles the HSAF skeleton. The biochemical characterization of the NRPS revealed that the thioesterase (TE) domain possesses the activities of both a protease and a peptide ligase. Active site mutagenesis, circular dichroism spectra, and homology modeling of the TE structure suggested that the TE may possess uncommon features that may lead to the unusual activities. The iterative PKS-NRPS is found in all polycyclic tetramate macrolactam gene clusters, and the unusual activities of the TE may be common to this type of hybrid PKS-NRPS.

Identification and characterization of the anti-methicillin-resistant Staphylococcus aureus WAP-8294A2 biosynthetic gene cluster from Lysobacter enzymogenes OH11.

Lysobactor enzymogenes strain OH11 is an emerging biological control agent of fungal and bacterial diseases. We recently completed its genome sequence and found it contains a large number of gene clusters putatively responsible for the biosynthesis of nonribosomal peptides and polyketides, including the previously identified antifungal dihydromaltophilin (HSAF). One of the gene clusters contains two huge open reading frames, together encoding 12 modules of nonribosomal peptide synthetases (NRPS). Gene disruption of one of the NRPS led to the disappearance of a metabolite produced in the wild type and the elimination of its antibacterial activity. The metabolite and antibacterial activity were also affected by the disruption of some of the flanking genes. We subsequently isolated this metabolite and subjected it to spectroscopic analysis. The mass spectrometry and nuclear magnetic resonance data showed that its chemical structure is identical to WAP-8294A2, a cyclic lipodepsipeptide with potent anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and currently in phase I/II clinical trials. The WAP-8294A2 biosynthetic genes had not been described previously. So far, the Gram-positive Streptomyces have been the primary source of anti-infectives. Lysobacter are Gram-negative soil/water bacteria that are genetically amendable and have not been well exploited. The WAP-8294A2 synthetase represents one of the largest NRPS complexes, consisting of 45 functional domains. The identification of these genes sets the foundation for the study of the WAP-8294A2 biosynthetic mechanism and opens the door for producing new anti-MRSA antibiotics through biosynthetic engineering in this new source of Lysobacter.

Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes.

HSAF was isolated from Lysobacter enzymogenes , a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here we describe the HSAF biosynthetic gene cluster, which contains only a single-module polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/NRPS are six genes that encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activates L-ornithine and that the four-domain NRPS is able to catalyze the formation of a tetramic acid-containing product from acyl-S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms.