Mutasynthetic Production and Antimicrobial Characterization of Darobactin Analogs

There is great need for therapeutics against multidrug-resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that selectively kills Gram-negative bacteria by targeting the outer membrane protein BamA, was discovered. Its efficacy was proven in animal infection models of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, thus promoting darobactin A as a promising lead compound. Originally discovered from members of the nematode-symbiotic genus; Photorhabdus; , the biosynthetic gene cluster (BGC) encoding the synthesis of darobactin A can also be found in other members of the class; Gammaproteobacteria; . Therein, the precursor peptides DarB to -F, which differ in their core sequence from darobactin A, were identified; in silico; . Even though production of these analogs was not observed in the putative producer strains, we were able to generate them by mutasynthetic derivatization of a heterologous expression system. The analogs generated were isolated and tested for their bioactivity. The most potent compound, darobactin B, was used for cocrystallization with the target BamA, revealing a binding site identical to that of darobactin A. Despite its potency, darobactin B did not exhibit cytotoxicity, and it was slightly more active against Acinetobacter baumannii isolates than darobactin A. Furthermore, we evaluated the plasma protein binding of darobactin A and B, indicating their different pharmacokinetic properties. This is the first report on new members of this new antibiotic class, which is likely to expand to several promising therapeutic candidates.; IMPORTANCE; Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties. In combination with the structural data of the target BamA, this approach may contribute to structure-activity relationship (SAR) data to optimize inhibitors of this essential outer membrane protein of Gram-negative pathogens

Antimicrobial Potential of Bacteria Associated with Marine Sea Slugs from North Sulawesi, Indonesia

Nudibranchia, marine soft-bodied organisms, developed, due to the absence of a protective shell, different strategies to protect themselves against putative predators and fouling organisms. One strategy is to use chemical weapons to distract predators, as well as pathogenic microorganisms. Hence, these gastropods take advantage of the incorporation of chemical molecules. Thereby the original source of these natural products varies; it might be the food source, de novo synthesis from the sea slug, or biosynthesis by associated bacteria. These bioactive molecules applied by the slugs can become important drug leads for future medicinal drugs. To test the potential of the associated bacteria, the latter were isolated from their hosts, brought into culture and extracts were prepared and tested for antimicrobial activities. From 49 isolated bacterial strains 35 showed antibiotic activity. The most promising extracts were chosen for further testing against relevant pathogens. In that way three strains showing activity against methicillin resistant Staphylococcus aureus and one strain with activity against enterohemorrhagic Escherichia coli, respectively, were identified. The obtained results indicate that the sea slug associated microbiome is a promising source for bacterial strains, which hold the potential for the biotechnological production of antibiotics

Heterologous Expression of Pseudouridimycin and Description of the Corresponding Minimal Biosynthetic Gene Cluster

Pseudouridimycin (PUM) was recently discovered from Streptomyces sp. DSM26212 as a novel bacterial nucleoside analog that competes with UTP for access to the RNA polymerase (RNAP) active site, thereby inhibiting bacterial RNAP by blocking transcription. This represents a novel antibacterial mode of action and it is known that PUM inhibits bacterial RNAP in vitro, inhibits bacterial growth in vitro, and was active in vivo in a mouse infection model of Streptococcus pyogenes peritonitis. The biosynthetic gene cluster (BGC) was previously identified and characterized by knockout experiments. However, the minimal set of genes necessary for PUM production was not proposed. To identify the minimal BGC and to create a plug-and-play production platform for PUM and its biosynthetic precursors, several versions of a redesigned PUM BGC were generated and expressed in the heterologous host Streptomyces coelicolor M1146 under control of strong promotors. Heterologous expression allowed identification of the putative serine/threonine kinase PumF as an enzyme essential for heterologous PUM production and thus corroboration of the PUM minimal BGC

The Potential of Indonesian Heterobranchs Found around Bunaken Island for the Production of Bioactive Compounds

The species diversity of marine heterobranch sea slugs found on field trips around Bunaken Island (North Sulawesi, Indonesia) and adjacent islands of the Bunaken National Marine Park forms the basis of this review. In a survey performed in 2015, 80 species from 23 families were collected, including 17 new species. Only three of these have been investigated previously in studies from Indonesia. Combining species diversity with a former study from 2003 reveals in total 140 species from this locality. The diversity of bioactive compounds known and yet to be discovered from these organisms is summarized and related to the producer if known or suspected (might it be down the food chain, de novo synthesised from the slug or an associated bacterium). Additionally, the collection of microorganisms for the discovery of natural products of pharmacological interest from this hotspot of biodiversity that is presented here contains more than 50 species that have never been investigated before in regard to bioactive secondary metabolites. This highlights the great potential of the sea slugs and the associated microorganisms for the discovery of natural products of pharmacological interest from this hotspot of biodiversity

Characterization of a Radical SAM Oxygenase for the Ether Crosslinking in Darobactin Biosynthesis

International audienceDarobactin A is a ribosomally synthesized, post-translationally modified peptide (RiPP) with potent and broad-spectrum anti-Gram-negative antibiotic activity. The structure of darobactin A is characterized by an ether and C-C crosslinking. However, the specific mechanism of the crosslink formation, especially the ether crosslink, remains elusive. Here, using in vitro enzyme assays, we demonstrate that both crosslinks are formed by the DarE radical S-adenosylmethionine (SAM) enzyme in an O2-dependent manner. The relevance of the observed activity to darobactin A biosynthesis was demonstrated by proteolytic transformation of the DarE product into darobactin A. Furthermore, DarE assays in the presence of 18O2 or [18O]water demonstrated that the oxygen of the ether crosslink originates from O2 and not from water. These results demonstrate that DarE is a radical SAM enzyme that uses oxygen as a co-substrate in its physiologically relevant function. Since radical SAM enzymes are generally considered to function under anaerobic environments, the discovery of a radical SAM oxygenase represents a significant change in the paradigm and suggests that these radical SAM enzymes function in aerobic cells. Also, the study revealed that DarE catalyzes the formation of three distinct modifications on DarA; ether and C-C crosslinks and α,β-desaturation. Based on these observations, possible mechanisms of the DarE-catalyzed reactions are discussed

Description of a new Moridilla species from North Sulawesi, Indonesia (Mollusca: Nudibranchia: Aeolidioidea) - based on MicroCT, histological and molecular analyses

Schillo, Dorothee, Wipfler, Benjamin, Undap, Nani, Papu, Adelfia, Böhringer, Nils, Eisenbarth, Jan-Hendrik, Kaligis, Fontje, Bara, Robert, Schäberle, Till F., König, Gabriele M., Wägele, Heike (2019): Description of a new Moridilla species from North Sulawesi, Indonesia (Mollusca: Nudibranchia: Aeolidioidea) - based on MicroCT, histological and molecular analyses. Zootaxa 4652 (2): 265-295, DOI: https://doi.org/10.11646/zootaxa.4652.2.

Darobactin B stabilises a lateral-closed conformation of the BAM complex in <i>E. coli</i> cells

C.P. acknowledges the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/S018069/1; BB/W019795/1) for funding. cwEPR data were collected at the Pliotas Laboratory (BB/S018069/1) and PELDOR data were collected at the University of St Andrews, supported by equipment funding from the Wellcome Trust (WT) (099149/Z/12/Z) and BBSRC (BB/R013780/1). CryoEM data were collected at the Astbury Biostructure Laboratory, funded by the University of Leeds and the WT (108466/Z/15/Z; 221524/Z/20/Z). J.E.H. and K.F. acknowledge funding from the Medical Research Council (MRC) (MR/P018491/1). S.F.H. is funded by the White Rose BBSRC DTP (BB/M011151/1). J.M. is funded by the WT (222373/Z/21/Z). SER holds a Royal Society Professorial Fellowship (RSRP/R1/211057). T.F.S and N.B. acknowledge funding from the German Federal Ministry of Education and Research (BMBF, via grant GBi2S and German Centrefor Infection Research (DZIF) 09.918)The Β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.Publisher PDFPeer reviewe

Genome- and metabolome-guided discovery of marine BamA inhibitors revealed a dedicated darobactin halogenase

Darobactins represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics featuring a rare bicyclic structure. They target the Bam-complex of Gram-negative bacteria and exhibit in vivo activity against drug-resistant pathogens. First isolated from Photorhabdus species, the corresponding biosynthetic gene clusters (BGCs) are widespread among γ-proteobacteria, including the genera Vibrio, Yersinia, and Pseudoalteromonas (P.). While the organization of the BGC core is highly conserved, a small subset of Pseudoalteromonas carries an extended BGC with additional genes. Here, we report the identification of brominated and dehydrated darobactin derivatives from P. luteoviolacea strains. The marine derivatives are active against multidrug-resistant (MDR) Gram-negative bacteria and showed solubility and plasma protein binding ability different from darobactin A, rendering it more active than darobactin A. The halogenation reaction is catalyzed by DarH, a new class of flavin-dependent halogenases with a novel fold