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Abstract Various microbes isolated from healthy plants are detrimental under laboratory conditions, indicating the existence of molecular mechanisms preventing disease in nature. Here, we demonstrated that application of sodium chloride (NaCl) in natural and gnotobiotic soil systems is sufficient to induce plant disease caused by an otherwise non-pathogenic root-derived Pseudomonas brassicacearum isolate (R401). Disease caused by combinatorial treatment of NaCl and R401 triggered extensive, root-specific transcriptional reprogramming that did not involve down-regulation of host innate immune genes, nor dampening of ROS-mediated immunity. Instead, we identified and structurally characterized the R401 lipopeptide brassicapeptin A as necessary and sufficient to promote disease on salt-treated plants. Brassicapeptin A production is salt-inducible, promotes root colonization and transitions R401 from being beneficial to being detrimental on salt-treated plants by disturbing host ion homeostasis, thereby bolstering susceptibility to osmolytes. We conclude that the interaction between a global change stressor and a single exometabolite from a member of the root microbiome promotes plant disease in complex soil systems.

Summary: Arthropod venoms contain bioactive molecules attractive for biomedical applications. However, few of these have been isolated, and only a tiny number has been characterized. Pseudoscorpions are small arachnids whose venom has been largely overlooked. Here, we present the first structural and functional assessment of the checacin toxin family, discovered in the venom of the house pseudoscorpion (Chelifer cancroides). We combined in silico and in vitro analyses to establish their bioactivity profile against microbes and various cell lines. This revealed inhibitory effects against bacteria and fungi. We observed cytotoxicity against specific cell types and effects involving second messengers. Our work provides insight into the biomedical potential and evolution of pseudoscorpion venoms. We propose that plesiotypic checacins evolved to defend the venom gland against infection, whereas apotypic descendants evolved additional functions. Our work highlights the importance of considering small and neglected species in biodiscovery programs.

Summary High‐throughput platforms facilitating screening campaigns of environmental samples are needed to discover new products of natural origin counteracting the spreading of antimicrobial resistances constantly threatening human and agricultural health. We applied a combination of droplet microfluidics and fluorescence‐activated cell sorting (FACS)‐based technologies to access and assess a microbial environmental sample. The cultivation performance of our microfluidics workflow was evaluated in respect to the utilized cultivation media by Illumina amplicon sequencing of a pool of millions of droplets, respectively. This enabled the rational selection of a growth medium supporting the isolation of microbial diversity from soil (five phyla affiliated to 57 genera) including a member of the acidobacterial subgroup 1 (genus Edaphobacter). In a second phase, the entire diversity covered by 1071 cultures was used for an arrayed bioprospecting campaign, resulting in > 6000 extracts tested against human pathogens and agricultural pests. After redundancy curation by using a combinatorial chemical and genomic fingerprinting approach, we assigned the causative agents present in the extracts. Utilizing UHPLC‐QTOF‐MS/MS‐guided fractionation and microplate‐based screening assays in combination with molecular networking the production of bioactive ionophorous macrotetrolides, phospholipids, the cyclic lipopetides massetolides E, F, H and serratamolide A and many derivatives thereof was shown.

ABSTRACT The emergence and spread of antimicrobial resistance (AMR) in Gram-negative pathogens, such as carbapenem-resistant Pseudomonas aeruginosa, pose an increasing threat to health care. Patients with immunodeficiencies or chronic pulmonary disease, like cystic fibrosis (CF), are particularly vulnerable to Pseudomonas infections and depend heavily on antibiotic therapy. To broaden limited treatment options, this study evaluated the potency of the recently licensed drugs ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), and cefiderocol (FDC) as well as two novel preclinical antibiotics, darobactins B (DAR B) and B9 (DAR B9), against clinical P. aeruginosa isolates derived from respiratory samples of CF patients. We observed high levels of resistance to all three newly licensed drugs, with cefiderocol exhibiting the best activity. From the 66 investigated P. aeruginosa isolates, a total of 53% were resistant to CZA, 49% to C/T, and 30% to FDC. Strikingly, 52 of the evaluated isolates were obtained from CF patients prior to market introduction of the drugs. Thus, our results suggest that resistance to CZA, C/T, and FDC may be due to preexisting resistance mechanisms. On the other hand, our two novel preclinical compounds performed better than (CZA and C/T) or close to (FDC) the licensed drugs—most likely due to the novel mode of action. Thus, our results highlight the necessity of global consistency in the area of antibiotic stewardship to prevent AMR from further impairing the potency of antibiotics in clinical practice. Ultimately, this study demonstrates the urgency to support the development of novel antimicrobials, preferably with a new mode of action such as darobactins B and B9, two very promising antimicrobial compounds for the treatment of critically ill patients suffering from multidrug-resistant Gram-negative (MRGN) infections. IMPORTANCE Antimicrobial resistance (AMR) represents an ever increasing threat to the health care system. Even recently licensed drugs are often not efficient for the treatment of infections caused by Gram-negative bacteria, like Pseudomonas aeruginosa, a causative agent of lung infections. To address this unmet medical need, innovative antibiotics, which possess a new mode of action, need to be developed. Here, the antibiogram of clinical isolates derived from cystic fibrosis patients was generated and new bicyclic heptapeptides, which inhibit the outer membrane protein BamA, exhibited strong activity, also against multidrug-resistant isolates.

Honey bees coexist with fungi that colonize hive surfaces and pollen. Some of these fungi are opportunistic pathogens, but many are beneficial species that produce antimicrobial compounds for pollen conservation and the regulation of pathogen populations. In this study, we tested the in vitro antimicrobial activity of Talaromyces purpureogenus strains isolated from bee bread against Paenibacillus alvei (associated with European foulbrood disease) and three Aspergillus species that cause stonebrood disease. We found that methanol extracts of T. purpureogenus strains B18 and B195 inhibited the growth of P. alvei at a concentration of 0.39 mg/mL. Bioactivity-guided dereplication revealed that the activity of the crude extracts correlated with the presence of diketopiperazines, a siderophore, and three unknown compounds. We propose that non-pathogenic fungi such as Talaromyces spp. and their metabolites in bee bread could be an important requirement to prevent disease. Agricultural practices involving the use of fungicides can disrupt the fungal community and thus negatively affect the health of bee colonies.

ABSTRACT With progress in genome sequencing and data sharing, 1,000s of bacterial genomes are publicly available. Genome mining—using bioinformatics tools in terms of biosynthetic gene cluster (BGC) identification, analysis, and rating—has become a key technology to explore the capabilities for natural product (NP) biosynthesis. Comprehensively, analyzing the genetic potential of the phylum Bacteroidetes revealed Chitinophaga as the most talented genus in terms of BGC abundance and diversity. Guided by the computational predictions, we conducted a metabolomics and bioactivity driven NP discovery program on 25 Chitinophaga strains. High numbers of strain-specific metabolite buckets confirmed the upfront predicted biosynthetic potential and revealed a tremendous uncharted chemical space. Mining this data set, we isolated the new iron chelating nonribosomally synthesized cyclic tetradeca- and pentadecalipodepsipeptide antibiotics chitinopeptins with activity against Candida, produced by C. eiseniae DSM 22224 and C. flava KCTC 62435, respectively. IMPORTANCE The development of pipelines for anti-infectives to be applied in plant, animal, and human health management are dried up. However, the resistance development against compounds in use calls for new lead structures. To fill this gap and to enhance the probability of success for the discovery of new bioactive natural products, microbial taxa currently underinvestigated must be mined. This study investigates the potential within the bacterial phylum Bacteroidetes. A combination of omics-technologies revealed taxonomical hot spots for specialized metabolites. Genome- and metabolome-based analyses showed that the phylum covers a new chemical space compared with classic natural product producers. Members of the Bacteroidetes may thus present a promising bioresource for future screening and isolation campaigns.

ABSTRACT 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.

Marine flavobacterium Tenacibaculum discolor sv11 has been proven to be a promising producer of bioactive nitrogen-containing heterocycles. A chemical investigation of T. discolor sv11 revealed seven new heterocycles, including the six new imidazolium-containing alkaloids discolins C-H (1–6) and one pyridinium-containing alkaloid dispyridine A (7). The molecular structure of each compound was elucidated by analysis of NMR and HR-ESI-MS data. Furthermore, enzymatic decarboxylation of tryptophan and tyrosine to tryptamine and tyramine catalyzed by the decarboxylase DisA was investigated using in vivo and in vitro experiments. The antimicrobial activity of the isolated compounds (1–7) was evaluated. Discolin C and E (1 and 3) exhibited moderate activity against Gram-positive Bacillus subtilis DSM10, Mycobacterium smegmatis ATCC607, Listeria monocytogenes DSM20600 and Staphylococcus aureus ATCC25923, with MIC values ranging from 4 μg/mL to 32 μg/mL.

Linear cationic venom peptides are antimicrobial peptides (AMPs) that exert their effects by damaging cell membranes. These peptides can be highly specific, and for some, a significant therapeutic value was proposed, in particular for treatment of bacterial infections. A prolific source of novel AMPs are arthropod venoms, especially those of hitherto neglected groups such as pseudoscorpions. In this study, we describe for the first time pharmacological effects of AMPs discovered in pseudoscorpion venom. We examined the antimicrobial, cytotoxic, and insecticidal activity of full-length Checacin1, a major component of the Chelifer cancroides venom, and three truncated forms of this peptide. The antimicrobial tests revealed a potent inhibitory activity of Checacin1 against several bacteria and fungi, including methicillin resistant Staphylococcus aureus (MRSA) and even Gram-negative pathogens. All peptides reduced survival rates of aphids, with Checacin1 and the C-terminally truncated Checacin11−21 exhibiting effects comparable to Spinosad, a commercially used pesticide. Cytotoxic effects on mammalian cells were observed mainly for the full-length Checacin1. All tested peptides might be potential candidates for developing lead structures for aphid pest treatment. However, as these peptides were not yet tested on other insects, aphid specificity has not been proven. The N- and C-terminal fragments of Checacin1 are less potent against aphids but exhibit no cytotoxicity on mammalian cells at the tested concentration of 100 µM.

The ‘core’ metabolome of the Bacteroidetes genus Chitinophaga was recently discovered to consist of only seven metabolites. A structural relationship in terms of shared lipid moieties among four of them was postulated. Here, structure elucidation and characterization via ultra-high resolution mass spectrometry (UHR-MS) and nuclear magnetic resonance (NMR) spectroscopy of those four lipids (two lipoamino acids (LAAs), two lysophosphatidylethanolamines (LPEs)), as well as several other undescribed LAAs and N-acyl amino acids (NAAAs), identified during isolation were carried out. The LAAs represent closely related analogs of the literature-known LAAs, such as the glycine-serine dipeptide lipids 430 (2) and 654. Most of the here characterized LAAs (1, 5–11) are members of a so far undescribed glycine-serine-ornithine tripeptide lipid family. Moreover, this study reports three novel NAAAs (N-(5-methyl)hexanoyl tyrosine (14) and N-(7-methyl)octanoyl tyrosine (15) or phenylalanine (16)) from Olivibacter sp. FHG000416, another Bacteroidetes strain initially selected as best in-house producer for isolation of lipid 430. Antimicrobial profiling revealed most isolated LAAs (1–3) and the two LPE ‘core’ metabolites (12, 13) active against the Gram-negative pathogen M. catarrhalis ATCC 25238 and the Gram-positive bacterium M. luteus DSM 20030. For LAA 1, additional growth inhibition activity against B. subtilis DSM 10 was observed.

The discovery of novel natural products (NPs) that will serve as lead structures has to be an ongoing effort to fill the respective development pipelines. However, identification of NPs, which possess a potential for application in e.g., the pharma or agro sector, must be as cost effective and fast as possible. Furthermore, the amount of sample available for initial testing is usually very limited, not least because of the fact that the impact on the environment, i.e., the sampled biosystem, should be kept minimal. Here, our pipeline SeaPEPR is described, in which a primary bioactivity screening of crude extracts is combined with the analysis of their metabolic fingerprint. This enabled prioritization of samples for subsequent microfractionation and dereplication of the active compounds early in the workflow. As a case study, 76 marine sponge-derived extracts were screened against a microbial screening panel. Thereunder, human pathogenic bacteria (Escherichia coli ATCC35218 and Staphylococcus aureus ATCC33592) and yeast (Candida albicans FH2173), as well as the phytopathogenic fungus Septoria tritici MUCL45407. Overall, nine extracts revealed activity against at least one test organism. Metabolic fingerprinting enabled assigning four active extracts into one metabolic group; therefore, one representative was selected for subsequent microfractionation. Dereplication of the active fractions showed a new dibrominated aplysinopsin and a hypothetical chromazonarol stereoisomer derivative. Furthermore, inhibitory activity against the common plant pest Septoria tritici was discovered for NPs of marine origin.

Sponge-associated fungi are attractive targets for the isolation of bioactive natural products with different pharmaceutical purposes. In this investigation, 20 fungi were isolated from 10 different sponge specimens. One isolate, the fungus Penicillium citrinum strain WK-P9, showed activity against Bacillus subtilis JH642 when cultivated in malt extract medium. One new and three known citrinin derivatives were isolated from the extract of this fungus. The structures were elucidated by 1D and 2D NMR spectroscopy, as well as LC-HRMS. Their antibacterial activity against a set of common human pathogenic bacteria and fungi was tested. Compound 2 showed moderate activity against Mycobacterium smegmatis ATCC607 with a minimum inhibitory concentration (MIC) of 32 µg/mL. Compound 4 exhibited moderate growth inhibition against Bacillus subtilis JH642, B. megaterium DSM32, and M. smegmatis ATCC607 with MICs of 16, 16, and 32 µg/mL, respectively. Furthermore, weak activities of 64 µg/mL against B. subtilis DSM10 and S. aureus ATCC25923 were observed for compound 4.

Zostera marina (eelgrass) is a marine foundation species with key ecological roles in coastal habitats. Its bacterial microbiota has been well studied, but very little is known about its mycobiome. In this study, we have isolated and identified 13 fungal strains, dominated by Penicillium species (10 strains), from the leaf and the root rhizosphere of Baltic Z. marina. The organic extracts of the fungi that were cultured by an OSMAC (One-Strain−Many-Compounds) regime using five liquid culture media under both static and shaking conditions were investigated for their chemical and bioactivity profiles. All extracts showed strong anti-quorum sensing activity, and the majority of the Penicillium extracts displayed antimicrobial or anti-biofilm activity against Gram-negative environmental marine and human pathogens. HPLC-DAD-MS-based rapid metabolome analyses of the extracts indicated the high influence of culture conditions on the secondary metabolite (SM) profiles. Among 69 compounds detected in all Penicillium sp. extracts, 46 were successfully dereplicated. Analysis of SM relatedness in culture conditions by Hierarchical Cluster Analysis (HCA) revealed generally low similarity and showed a strong effect of medium selection on chemical profiles of Penicillium sp. This is the first study assessing both the metabolite and bioactivity profile of the fungi associated with Baltic eelgrass Z. marina.