Biosynthesis and Heterologous Production of Argyrins.

Argyrins represent a family of cyclic octapeptides exhibiting promising antimicrobial, antitumorigenic and immunosuppressant activities. They derive from a nonribosomal peptide synthetase pathway, which was identified and characterized in this study from the myxobacterial producer strain Cystobacter sp. SBCb004. Using the native biosynthetic gene cluster (BGC) sequence as template synthetic BGC versions were designed and assembled from gene synthesis fragments. A heterologous expression system was established after chromosomal deletion of a well-expressed lipopeptide pathway from the host strain Myxococcus xanthus DK1622. Different approaches were applied to engineer and improve heterologous argyrin production, which was finally increased to 160 mg/L, around 20-fold higher yields compared to the native producer. Heterologous production platform also led to identification of several novel argyrin derivatives (A2, F3, G3, I, J, K, and L). The optimized production system provides a versatile platform for future supply of argyrins and novel derivatives thereof.

Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.

Correlating chemical diversity with taxonomic distance for discovery of natural products in myxobacteria.

Some bacterial clades are important sources of novel bioactive natural products. Estimating the magnitude of chemical diversity available from such a resource is complicated by issues including cultivability, isolation bias and limited analytical data sets. Here we perform a systematic metabolite survey of ~2300 bacterial strains of the order Myxococcales, a well-established source of natural products, using mass spectrometry. Our analysis encompasses both known and previously unidentified metabolites detected under laboratory cultivation conditions, thereby enabling large-scale comparison of production profiles in relation to myxobacterial taxonomy. We find a correlation between taxonomic distance and the production of distinct secondary metabolite families, further supporting the idea that the chances of discovering novel metabolites are greater by examining strains from new genera rather than additional representatives within the same genus. In addition, we report the discovery and structure elucidation of rowithocin, a myxobacterial secondary metabolite featuring an uncommon phosphorylated polyketide scaffold.

Soil myxobacteria as a potential source of polyketide-peptide substances.

Myxobacteria, a group of antimicrobial producing bacteria, have been successfully cultured and characterized from ten soil samples collected from different parts of Slovakia. A total of 79 myxobacteria belonging to four genera (Myxococcus, Corallococcus, Sorangium, and Polyangium) were isolated based on aspects of their life cycle. Twenty-five of them were purified, fermented, and screened for antimicrobial activities against 11 test microorganisms. Results indicated that crude extracts showed more significant activities against Gram-positive than against Gram-negative bacteria or fungi. Based on a higher degree and broader range of antimicrobial production, the two most potential extracts (K9-5, V3-1) were selected for HPLC fractionation against Micrococcus luteus and Staphylococcus aureus and LC/MS analysis of potential antibiotic metabolites. The analysis resulted in the identification of polyketide-peptide antibiotics, namely corallopyronin A and B (K9-5) and myxalamid B and C (V3-1), which were responsible for important Gram-positive activity in the observed strains. A sequence similarity search through BLAST revealed that these strains showed the highest sequence similarity to Corallococcus coralloides (K9-5, NCBI accession number KX256198) and Myxococcus xanthus (V3-1, NCBI accession number KX256197). Although screening of myxobacteria is laborious, due to difficulties in isolating cultures, this research represented the first report covering the isolation and cultivation of this challenging bacterial group from Slovakian soils as well as the screening of their antimicrobial activity, cultural identification, and secondary metabolite identification.

Isolation, Structure Elucidation, and (Bio)Synthesis of Haprolid, a Cell-Type-Specific Myxobacterial Cytotoxin.

Myxobacteria are well-established sources for novel natural products exhibiting intriguing bioactivities. We here report on haprolid (1) isolated from Byssovorax cruenta Har1. The compound exhibits an unprecedented macrolactone comprising four modified amino acids and a polyketide fragment. As configurational assignment proved difficult, a bioinformatic analysis of the biosynthetic gene cluster was chosen to predict the configuration of each stereocenter. In-depth analysis of the corresponding biosynthetic proteins established a hybrid polyketide synthase/nonribosomal peptide synthetase origin of haprolid and allowed for stereochemical assignments. A subsequent total synthesis yielded haprolid and corroborated all predictions made. Intriguingly, haprolid showed cytotoxicity against several cell lines in the nanomolar range whereas other cells were almost unaffected by treatment with the compound.

Isolation, Structure Elucidation, Biosynthesis, and Synthesis of Antalid, a Secondary Metabolite from Polyangium species.

The isolation, structure elucidation, and synthesis of antalid (1), a novel secondary metabolite from Polyangium sp., is described herein. The structure elucidation of 1 was performed with the aid of mass spectrometry, high field NMR experiments, and crystal structure analysis. The absolute configuration of antalid was confirmed through the Mosher ester method and ultimately by total synthesis. In addition, the biosynthetic origin of this hybrid PKS-NRPS natural product was unraveled by the in silico analysis of its biosynthetic gene cluster.

The Mechanism of Action of Lysobactin.

Lysobactin, also known as katanosin B, is a potent antibiotic with in vivo efficacy against Staphylococcus aureus and Streptococcus pneumoniae. It was previously shown to inhibit peptidoglycan (PG) biosynthesis, but its molecular mechanism of action has not been established. Using enzyme inhibition assays, we show that lysobactin forms 1:1 complexes with Lipid I, Lipid II, and Lipid II(A)(WTA), substrates in the PG and wall teichoic acid (WTA) biosynthetic pathways. Therefore, lysobactin, like ramoplanin and teixobactin, recognizes the reducing end of lipid-linked cell wall precursors. We show that despite its ability to bind precursors from different pathways, lysobactin's cellular mechanism of killing is due exclusively to Lipid II binding, which causes septal defects and catastrophic cell envelope damage.

Characterization of Antimycins - Producing Streptomycete Strain VY46 Isolated from Slovak Soil

ABSTRACT The strain no. VY46 was isolated from agricultural soil of Slovak republic and tested for potential antimicrobial activity against various human pathogens. On the basis of results, strain VY46 significantly inhibited growth of yeast Candida albicans and therefore was used for further characterization. In order to explore the potential bioactivities, extract of the fermented broth culture was prepared with organic solvent extraction method. The ethylacetate extract was subjected to HPLC fractionation against Candida albicans and followed by LC/MS analysis for potential production of anticandidal substances. The analysis resulted in the identification of two antimycins antibiotics, which may be responsible for important anticandidal activity of the strain. On the basis of liquid chromatography and mass spectrometry the antibiotics were identified as Urauchimycin A and Kitamycin A. According tothe results from cultural, morphological, physiological, biochemical and 16S rRNA gene sequence methods, the strain was identified as Streptomyces albidoflavus. In addition, neighbor-joining phylogenetic tree confirmed the relationships of this strain to other members of Streptomyces genera.

Cystobactamids: myxobacterial topoisomerase inhibitors exhibiting potent antibacterial activity.

The development of new antibiotics faces a severe crisis inter alia owing to a lack of innovative chemical scaffolds with activities against Gram-negative and multiresistant pathogens. Herein, we report highly potent novel antibacterial compounds, the myxobacteria-derived cystobactamids 1-3, which were isolated from Cystobacter sp. and show minimum inhibitory concentrations in the low µg mL(-1) range. We describe the isolation and structure elucidation of three congeners as well as the identification and annotation of their biosynthetic gene cluster. By studying the self-resistance mechanism in the natural producer organism, the molecular targets were identified as bacterial type IIa topoisomerases. As quinolones are largely exhausted as a template for new type II topoisomerase inhibitors, the cystobactamids offer exciting alternatives to generate novel antibiotics using medicinal chemistry and biosynthetic engineering.

Revealing the macromolecular targets of complex natural products.

Natural products have long been a source of useful biological activity for the development of new drugs. Their macromolecular targets are, however, largely unknown, which hampers rational drug design and optimization. Here we present the development and experimental validation of a computational method for the discovery of such targets. The technique does not require three-dimensional target models and may be applied to structurally complex natural products. The algorithm dissects the natural products into fragments and infers potential pharmacological targets by comparing the fragments to synthetic reference drugs with known targets. We demonstrate that this approach results in confident predictions. In a prospective validation, we show that fragments of the potent antitumour agent archazolid A, a macrolide from the myxobacterium Archangium gephyra, contain relevant information regarding its polypharmacology. Biochemical and biophysical evaluation confirmed the predictions. The results obtained corroborate the practical applicability of the computational approach to natural product 'de-orphaning'.

Targeting V-ATPase in primary human monocytes by archazolid potently represses the classical secretion of cytokines due to accumulation at the endoplasmic reticulum.

The macrolide archazolid inhibits vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, and potently suppresses cancer cell growth at low nanomolar concentrations. In view of the growing link between inflammation and cancer, we investigated whether inhibition of V-ATPase by archazolid may affect primary human monocytes that can promote cancer by sustaining inflammation through the release of tumor-promoting cytokines. Human primary monocytes express V-ATPase, and archazolid (10-100nM) increases the vesicular pH in these cells. Archazolid (10nM) markedly reduced the release of pro-inflammatory (TNF-α, interleukin-6 and -8) but also of anti-inflammatory (interleukin-10) cytokines in monocytes stimulated with LPS, without affecting cell viability up to 1000nM. Of interest, secretion of interleukin-1ß was increased by archazolid. Comparable effects were obtained by the V-ATPase inhibitors bafilomycin and apicularen. The phosphorylation of p38 MAPK and ERK-1/2, Akt, SAPK/JNK or of the inhibitor of NFκB (IκBα) as well as mRNA expression of IL-8 were not altered by archazolid in LPS-stimulated monocytes. Instead, archazolid caused endoplasmic reticulum (ER) stress response visualized by increased BiP expression and accumulation of IL-8 (and TNF-α) at the ER, indicating a perturbation of protein secretion. In conclusion, by interference with V-ATPase, archazolid significantly affects the secretion of cytokines due to accumulation at the ER which might be of relevance when using these agents for cancer therapy.

Resistance mechanisms of cancer cells to the novel vacuolar H(+)-ATPase inhibitor archazolid B.

Resistance of cancer cells towards chemotherapy is the major cause of therapy failure. Hence, the evaluation of cellular defense mechanisms is essential in the establishment of new chemotherapeutics. Archazolid B, a novel vacuolar H(+)-ATPase inhibitor, displayed cytotoxicity in the low nanomolar range on a panel of different tumor cell lines. First, we investigated tumor-specific cytotoxicity of archazolid B by comparing cancer to non-cancer cells. Breast, liver and colon cancer cells displayed higher drug sensitivity than corresponding non-tumorous cells, whereas leukemia cell lines were as sensitive as peripheral mononuclear blood cells. Investigating classical drug resistance mechanisms, archazolid B was identified as a possible substrate of the ABC transporters ABCB1 (P-glycoprotein) and ABCG2 (BCRP), whereas collateral sensitivity was observed in ABCB5-expressing cells. Our results pointed to a possible binding competition of archazolid B with verapamil on P-glycoprotein. However, archazolid B did not reverse resistance towards doxorubicin indicating that it might be a substrate but not an inhibitor of P-glycoprotein mediated transport. Furthermore, the cytotoxicity of archazolid B was independent of the p53 status of the cell. Mechanisms of aquired resistance were investigated establishing an archazolid B-resistant MCF-7 cell line. Interestingly, drug resistance was not conferred by aberrant expression or DNA mutations of the gene encoding vacuolar H(+)-ATPase subunit c, the direct target of archazolids. Instead, long-term treatment with archazolid B led to a slight overexpression of ABCB1 and a significant overexpression of the epidermal growth factor receptor and reduced cell growth, all of which can be assumed to contribute to archazolid B resistance.

Paenilarvins: Iturin family lipopeptides from the honey bee pathogen Paenibacillus larvae.

The bacterium Paenibacillus larvae has been extensively studied as it is an appalling honey bee pathogen. In the present work, we screened crude extracts derived from fermentations of P. larvae genotypes ERIC I and II for antimicrobial activity, following the detection of four putative secondary metabolite gene clusters that show high sequence homology to known biosynthetic gene clusters for the biosynthesis of antibiotics. Low molecular weight metabolites produced by P. larvae have recently been shown to have toxic effects on honey bee larvae. Moreover, a novel tripeptide, sevadicin, was recently characterized from laboratory cultures of P. larvae. In this study, paenilarvins, which are iturinic lipopeptides exhibiting strong antifungal activities, were obtained by bioassay-guided fractionation from cultures of P. larvae, genotype ERIC II. Their molecular structures were determined by extensive 2D NMR spectroscopy, high resolution mass spectrometry, and other methods. Paenilarvins are the first antifungal secondary metabolites to be identified from P. larvae. In preliminary experiments, these lipopeptides also affected honey bee larvae and might thus play a role in P. larvae survival and pathogenesis. However, further studies are needed to investigate their function.

Identification of myxobacteria-derived HIV inhibitors by a high-throughput two-step infectivity assay.

BACKGROUND: Drug-resistance and therapy failure due to drug-drug interactions are the main challenges in current treatment against Human Immunodeficiency Virus (HIV) infection. As such, there is a continuous need for the development of new and more potent anti-HIV drugs. Here we established a high-throughput screen based on the highly permissive TZM-bl cell line to identify novel HIV inhibitors. The assay allows discriminating compounds acting on early and/or late steps of the HIV replication cycle. RESULTS: The platform was used to screen a unique library of secondary metabolites derived from myxobacteria. Several hits with good anti-HIV profiles were identified. Five of the initial hits were tested for their antiviral potency. Four myxobacterial compounds, sulfangolid C, soraphen F, epothilon D and spirangien B, showed EC50 values in the nM range with SI > 15. Interestingly, we found a high amount of overlapping hits compared with a previous screen for Hepatitis C Virus (HCV) using the same library. CONCLUSION: The unique structures and mode-of-actions of these natural compounds make myxobacteria an attractive source of chemicals for the development of broad-spectrum antivirals. Further biological and structural studies of our initial hits might help recognize smaller drug-like derivatives that in turn could be synthesized and further optimized.

Screening of small molecules affecting mammalian P-body assembly uncovers links with diverse intracellular processes and organelle physiology.

Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. The mechanism of P-body assembly has been typically addressed by depleting P-body components. Here we apply a complementary approach and establish an automated cell-based assay platform to screen for molecules affecting P-body assembly. From a unique library of compounds derived from myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA), a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also inhibited, under stress conditions, phosphorylation of eIF2α and stress granule formation. Other hits uncovered interesting novel links between P-body assembly, lipid metabolism, and internal organelle physiology. The obtained results provide a chemical toolbox to manipulate P-body assembly and function.

Indiacens A and B: prenyl indoles from the myxobacterium Sandaracinus amylolyticus.

The gliding bacterium Sandaracinus amylolyticus, strain NOSO-4T, was recently characterized as the first representative of a new myxobacterial genus. A screening of the culture broth for antibiotically active metabolites followed by isolation and characterization revealed two unique 3-formylindol derivatives, indiacen A (1) and its chloro derivative indiacen B (2). Both are active against Gram-positive and Gram-negative bacteria as well as the fungus Mucor hiemalis. The biosynthetic origin of the isoprene-like side chain in 1 and 2 was studied by in vivo feeding experiments with ¹³C-labeled precursors.

Mutation in elongation factor G confers resistance to the antibiotic argyrin in the opportunistic pathogen Pseudomonas aeruginosa.

The natural myxobacterial product argyrin is a cyclic peptide exhibiting immunosuppressive activity as well as antibacterial activity directed against the highly intrinsically resistant opportunistic pathogen Pseudomonas aeruginosa. In this study, we used whole-genome sequencing technology as a powerful tool to determine the mode of action of argyrin. Sequencing of argyrin-resistant P. aeruginosa isolates selected in vitro uncovered six point mutations that distinguished the resistant mutants from their susceptible parental strain. All six mutations were localized within one gene: fusA1, which encodes for the elongation factor EF-G. After the reintroduction of selected mutations into the susceptible wild type, the strain became resistant to argyrin. Surface plasmon resonance experiments confirmed the interaction of argyrin A with FusA1. Interestingly, EF-G has been previously shown to be the target of the anti-Staphylococcus antibiotic fusidic acid. Mapping of the mutations onto a structural model of EF-G revealed that the mutations conveying resistance against argyrin were clustered within domain III on the side opposite to that involved in fusidic acid binding, thus indicating that argyrin exhibits a new mode of protein synthesis inhibition. Although no mutations causing argyrin resistance have been found in other genes of P. aeruginosa, analysis of the sequence identity in EF-G and its correlation with argyrin resistance in different bacteria imply that additional factors such as uptake of argyrin play a role in the argyrin resistance of other organisms.

Precursor-directed syntheses and biological evaluation of new elansolid derivatives.

The antibiotic elansolid C1 (8) was isolated from Chitinophaga sancti strain FxGBF13 after fermentation in the presence of anthranilic acid. Remarkably, 8 was also obtained by addition of anthranilic acid to a crude fermentation extract containing the macrolide elansolid A2 (1*). This Michael-type conjugate addition allowed us to generate 21 new derivatives of elansolid C1 (9-29) by using various nucleophiles. Biological activities of all derivatives were evaluated against Staphylococcus aureus, Micrococcus luteus, and the mouse cell line L929.

Sulfangolids, macrolide sulfate esters from Sorangium cellulosum.

Sulfangolids are the first sulfate ester containing secondary metabolites from myxobacteria. The metabolites 1-4 and the structurally related kulkenon (5) were isolated from different strains of the species Sorangium cellulosum. In the course of isolation all metabolites proved to be rather sensitive due to their conjugated double bond systems and the strong acidic nature of the sulfate ester in sulfangolids. The relative configuration of sulfangolid C (3) was assigned by extensive 1D and 2D NMR analysis and molecular modelling. In addition, the biosynthesis of 3 was studied by feeding experiments.

Hyaladione, an S-methyl cyclohexadiene-dione from Hyalangium minutum.

A bioassay-guided fractionation of the crude methanol extract of the myxobacterium Hyalangium minutum, strain NOCB-2(T) (DSM 14724(T)), led to the isolation of hyaladione (1), a novel S-methyl cyclohexadiene-dione. The structure of 1 was established by HRESIMS, NMR, and IR spectroscopy as well as X-ray crystallography. Compound 1 was active against growing mammalian cell lines, with IC(50) values ranging from 1.23 to 3.93 µM, in addition to a broad spectrum of antibacterial and antifungal activities, including inhibition of pathogenic methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa with an MIC of 0.83 and 8.5 µg mL(-1), respectively.