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.

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Antibiotic discovery and development is challenging as chemical scaffolds of synthetic origin often lack the required pharmaceutical properties, and the discovery of novel ones from natural sources is tedious. Herein, we report the discovery of new cystobactamids with a significantly improved antibacterial profile in a detailed screening of myxobacterial producer strains. Some of these new derivatives display antibacterial activities in the low-μg mL range against Gram-negative pathogens, including clinical isolates of Klebsiella oxytoca, Pseudomonas aeruginosa, and fluoroquinolone-resistant Enterobacteriaceae, which were not observed for previously reported cystobactamids. Our findings provide structure-activity relationships and show how pathogen resistance can be overcome by natural scaffold diversity. The most promising derivative 861-2 was prepared by total synthesis, enabling further chemical optimization of this privileged scaffold.

Tandem mass spectrometry is a widely applied and highly sensitive technique for the discovery and characterization of microbial natural products such as secondary metabolites from myxobacteria. Here, a data mining workflow based on MS/MS precursor lists targeting only signals related to bacterial metabolism is established using LC-MS data of crude extracts from shaking flask fermentations. The devised method is not biased toward specific compound classes or structural features and is capable of increasing the information content of LC-MS/MS analyses by directing fragmentation events to signals of interest. The approach is thus contrary to typical auto-MS(2) setups where precursor ions are usually selected according to signal intensity, which is regarded as a drawback for metabolite discovery applications when samples contain many overlapping signals and the most intense signals do not necessarily represent compounds of interest. In line with this, the method described here achieves improved MS/MS scan coverage for low-abundance precursor ions not captured by auto-MS(2) experiments and thereby facilitates the search for new secondary metabolites in complex biological samples. To underpin the effectiveness of the approach, the identification and structure elucidation of two new myxobacterial secondary metabolite classes is reported.

Vioprolides are a promising class of anticancer and antifungal lead compounds produced by the myxobacterium Cystobacter violaceus Cb vi35. Previously nothing had been reported about their biosynthesis, including the origin of the unusual 4-methylazetidinecarboxylic acid (MAZ) moiety. We describe the vioprolide biosynthetic gene cluster and solve the production obstacle by expression in three heterologous hosts. Starting from unstable production in the wild type at the single-digit mg L scale, we developed a stable host that eventually allowed for yields of up to half a gram per liter in fermenters. Gene inactivations coupled with isotope feeding studies identified an S-adenosylmethionine (SAM)-dependent enzyme and a methyltransferase as being responsible for the generation of the MAZ building block by a proposed mechanism unprecedented in bacteria. Furthermore, nonnatural vioprolide derivatives were generated via rational genetic engineering.

Amycolatopsis balhimycina DSM 5908 (formerly described as Amycolatopsis mediterranei) was first isolated from a soil sample originating from the Himalayas (20). It belongs to the order Actinomycetales and synthesizes the glycopeptide antibiotic balhimycin. Balhimycin differs only in its glycosylation pattern from vancomycin, which is used as an antibiotic of last resort against multidrug-resistant Gram-positive bacteria. Since the activities of balhimycin are comparable to those of vancomycin both in vitro and in vivo and A. balhimycina is accessible to genetic manipulation, this strain has become a model organism for analyzing glycopeptide synthesis and resistance (32). Glycopeptide antibiotics inhibit cell wall biosynthesis in Grampositive bacteria by binding the D-alanyl-D-alanine (D-Ala-DAla) terminus of peptidoglycan precursors on the outside surface of the cytoplasmic membrane (24). However, when the usage of vancomycin steadily increased in the 1980s (14), the first vancomycin-resistant enterococci (VRE) were isolated in hospitals (17). In these pathogens, cell wall biosynthesis was reprogrammed in such a way that the pentapeptide of the peptidoglycan precursor terminated in D-alanyl-D-lactate (DAla-D-Lac) rather than D-Ala-D-Ala, thereby causing an ϳ1,000-fold lower binding affinity of the glycopeptide (5). This alteration of the cell wall precursor requires the following three genes: vanH, coding for a dehydrogenase which converts pyruvate to D-Lac (1); vanA, which codes for a D-Ala-D-Lac ligase (4); and vanX, which codes for a DD-dipeptidase that cleaves the D-Ala-D-Ala dipeptide to ensure that only altered peptidoglycan precursors terminating in D-Ala-D-Lac are built up (25). van-like genes which have similarity to the resistance genes from VRE have been found in several glycopeptide producers, such as various Amycolatopsis spp. (16).We report here the rare case of an antibiotic producer carrying a biosynthetic gene cluster without essential resistance genes. The essential vanHAX resistance genes, located elsewhere in the chromosome, are expressed constitutively and are not controlled by the vanRS-like two component system vnlRS, resulting in constitutive production of peptidoglycan precursors terminating in D-Ala-D-Lac. MATERIALS AND METHODSBacterial strains and plasmids. The strains and plasmids used for this study are listed in Table 1.Media and culture conditions. Escherichia coli strains were grown in LB (26) at 37°C. Actinomycetes strains were grown in R5 medium (13) at 30°C. Media were supplemented with antibiotics when necessary to maintain plasmids.DNA preparation and manipulation. The methods used for the isolation and manipulation of DNA for E. coli and actinomycetes were described by Kieser et al. (13). PCR fragments were isolated from agarose gels by use of a QIAquick kit (Qiagen). Restriction endonucleases were obtained from various suppliers and were used according to their specifications.

Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal–adult-worm killing–treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4–5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.

Aetheramides are structurally distinctive cyclic peptides isolated from a novel myxobacterial genus proposed to be termed "Aetherobacter". The structures were solved by a combination of NMR analyses, quantum mechanical calculations, and chemical derivatizations. Aetheramides which contain a unique polyketide moiety and two amino acid residues potently inhibited HIV-1 infection with IC(50) values of ~0.015 μM. Furthermore aetheramides showed cytostatic activity against human colon carcinoma (HCT-116) cells with IC(50) values of 0.11 μM.