Harzianic acid exerts antimicrobial activity against Gram-positive bacteria and targets the cell membrane.

The thermophilic fungus Oidiodendron flavum is a saprobe that is commonly isolated from soil. Here, we identified a Gram-positive bacteria-selective antimicrobial secondary metabolite from this fungal species, harzianic acid (HA). Using Bacillus subtilis strain 168 combined with dynamic bacterial morphology imaging, we found that HA targeted the cell membrane. To further study the antimicrobial activity of HA, we isolated an HA-resistant strain, Bacillus subtilis strain M9015, and discovered that the mutant had more translucent colonies than the wild type strain, showed cross resistance to rifampin, and harbored five mutations in the coding region of four distinct genes. Further analysis of these genes indicated that the mutation in atpE might be responsible for the translucency of the colonies, and mutation in mdtR for resistance to both HA and rifampin. We conclude that HA is an antimicrobial agent against Gram-positive bacteria that targets the cell membrane.

Loss of Shp1 impairs myeloid cell function and causes lethal inflammation in zebrafish larvae.

PTPN6 encodes SHP1, a protein tyrosine phosphatase with an essential role in immune cell function. SHP1 mutations are associated with neutrophilic dermatoses and emphysema in humans, which resembles the phenotype seen in motheaten mice that lack functional SHP1. To investigate the function of Shp1 in developing zebrafish embryos, we generated a ptpn6 knockout zebrafish line lacking functional Shp1. Shp1 knockout caused severe inflammation and lethality around 17 days post fertilization (dpf). During early development, the myeloid lineage was affected, resulting in a decrease in the number of neutrophils and a concomitant increase in the number of macrophages. The number of emerging hematopoietic stem and progenitor cells (HSPCs) was decreased, but due to hyperproliferation, the number of HSPCs was higher in ptpn6 mutants than in siblings at 5 dpf. Finally, the directional migration of neutrophils and macrophages was decreased in response to wounding, and fewer macrophages were recruited to the wound site. Yet, regeneration of the caudal fin fold was normal. We conclude that loss of Shp1 impaired neutrophil and macrophage function, and caused severe inflammation and lethality at the larval stage.

Paecilomycone Inhibits Quorum Sensing in Gram-Negative Bacteria.

Pseudomonas aeruginosa is an opportunistic pathogen that causes major health care concerns due to its virulence and high intrinsic resistance to antimicrobial agents. Therefore, new treatments are greatly needed. An interesting approach is to target quorum sensing (QS). QS regulates the production of a wide variety of virulence factors and biofilm formation in P. aeruginosa. This study describes the identification of paecilomycone as an inhibitor of QS in both Chromobacterium violaceum and P. aeruginosa. Paecilomycone strongly inhibited the production of virulence factors in P. aeruginosa, including various phenazines, and biofilm formation. In search of the working mechanism, we found that paecilomycone inhibited the production of 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), but not 2'-aminoacetophenone (2-AA). Therefore, we suggest that paecilomycone affects parts of QS in P. aeruginosa by targeting the PqsBC complex and alternative targets or alters processes that influence the enzymatic activity of the PqsBC complex. The toxicity of paecilomycone toward eukaryotic cells and organisms was low, making it an interesting lead for further clinical research. IMPORTANCE Antibiotics are becoming less effective against bacterial infections due to the evolution of resistance among bacteria. Pseudomonas aeruginosa is a Gram-negative pathogen that causes major health care concerns and is difficult to treat due to its high intrinsic resistance to antimicrobial agents. Therefore, new targets are needed, and an interesting approach is to target quorum sensing (QS). QS is the communication system in bacteria that regulates multiple pathways, including the production of virulence factors and biofilm formation, which leads to high toxicity in the host and low sensitivity to antibiotics, respectively. We found a compound, named paecilomycone, that inhibited biofilm formation and the production of various virulence factors in P. aeruginosa. The toxicity of paecilomycone toward eukaryotic cells and organisms was low, making it an interesting lead for further clinical research.

Gregatins, a Group of Related Fungal Secondary Metabolites, Inhibit Aspects of Quorum Sensing in Gram-Negative Bacteria.

Quorum sensing (QS) is a process that regulates gene expression based on cell density. In bacteria, QS facilitates collaboration and controls a large number of pathways, including biofilm formation and virulence factor production, which lead to lower sensitivity to antibiotics and higher toxicity in the host, respectively. Inhibition of QS is a promising strategy to combat bacterial infections. In this study, we tested the potential of secondary metabolites from fungi to inhibit bacterial QS using a library derived from more than ten thousand different fungal strains. We used the reporter bacterium, Chromobacterium violaceum, and identified 39 fungal strains that produced QS inhibitor activity. These strains expressed two QS inhibitors that had been described before and eight QS inhibitors that had not been described before. Further testing for QS inhibitor activity against the opportunistic pathogen Pseudomonas aeruginosa led to the identification of gregatins as an interesting family of compounds with QS inhibitor activity. Although various gregatins inhibited QS in P. aeruginosa, these gregatins did not inhibit virulence factor production and biofilm formation. We conclude that gregatins inhibit some, but not all aspects of QS.

Evolution-Informed Discovery of the Naphthalenone Biosynthetic Pathway in Fungi.

Fungi produce a wide diversity of secondary metabolites with interesting biological activities for the health, industrial, and agricultural sectors. While fungal genomes have revealed an unexpectedly high number of biosynthetic pathways that far exceeds the number of known molecules, accessing and characterizing this hidden diversity remain highly challenging. Here, we applied a combined phylogenetic dereplication and comparative genomics strategy to explore eight lichenizing fungi. The determination of the evolutionary relationships of aromatic polyketide pathways resulted in the identification of an uncharacterized biosynthetic pathway that is conserved in distant fungal lineages. The heterologous expression of the homologue from Aspergillus parvulus linked this pathway to naphthalenone compounds, which were detected in cultures when the pathway was expressed. Our unbiased and rational strategy generated evolutionary knowledge that ultimately linked biosynthetic genes to naphthalenone polyketides. Applied to many more genomes, this approach can unlock the full exploitation of the fungal kingdom for molecule discovery. IMPORTANCE Fungi have provided us with life-changing small bioactive molecules, with the best-known examples being the first broad-spectrum antibiotic penicillin, immunosuppressive cyclosporine, and cholesterol-lowering statins. Since the 1980s, exploration of chemical diversity in nature has been highly reduced. However, the genomic era has revealed that fungal genomes are concealing an unexpected and largely unexplored chemical diversity. So far, fungal genomes have been exploited to predict the production potential of bioactive compounds or to find genes that control the production of known molecules of interest. But accessing and characterizing the full fungal chemical diversity require rational and, thus, efficient strategies. Our approach is to first determine the evolutionary relationships of fungal biosynthetic pathways in order to identify those that are already characterized and those that show a different evolutionary origin. This knowledge allows prioritizing the choice of the pathway to functionally characterize in a second stage using synthetic-biology tools like heterologous expression. A particular strength of this strategy is that it is always successful: it generates knowledge about the evolution of bioactive-molecule biosynthesis in fungi, it either yields novel molecules or links the studied pathway to already known molecules, and it reveals the chemical diversity within a given pathway, all at once. The strategy is very powerful to avoid studying the same pathway again and can be used with any fungal genome. Functional characterization using heterologous expression is particularly suitable for fungi that are difficult to grow or not genetically tractable. Thanks to the decreasing cost of gene synthesis, ultimately, only the genome sequence is needed to identify novel pathways and characterize the molecules that they produce. Such an evolution-informed strategy allows the efficient exploitation of the chemical diversity hidden in fungal genomes and is very promising for molecule discovery.

Classification of antimicrobial mechanism of action using dynamic bacterial morphology imaging.

Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.

Berkchaetoazaphilone B has antimicrobial activity and affects energy metabolism.

Antimicrobial resistance has become one of the major threats to human health. Therefore, there is a strong need for novel antimicrobials with new mechanisms of action. The kingdom of fungi is an excellent source of antimicrobials for this purpose because it encompasses countless fungal species that harbor unusual metabolic pathways. Previously, we have established a library of secondary metabolites from 10,207 strains of fungi. Here, we screened for antimicrobial activity of the library against seven pathogenic bacterial strains and investigated the identity of the active compounds using ethyl acetate extraction, activity-directed purification using HPLC fractionation and chemical analyses. We initially found 280 antimicrobial strains and subsequently identified 17 structurally distinct compounds from 26 strains upon further analysis. All but one of these compounds, berkchaetoazaphilone B (BAB), were known to have antimicrobial activity. Here, we studied the antimicrobial properties of BAB, and found that BAB affected energy metabolism in both prokaryotic and eukaryotic cells. We conclude that fungi are a rich source of chemically diverse secondary metabolites with antimicrobial activity.

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein-Protein Interactions.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2.

Cercosporamide inhibits bone morphogenetic protein receptor type I kinase activity in zebrafish.

Zebrafish models are well-established tools for investigating the underlying mechanisms of diseases. Here, we identified cercosporamide, a metabolite from the fungus Ascochyta aquiliqiae, as a potent bone morphogenetic protein receptor (BMPR) type I kinase inhibitor through a zebrafish embryo phenotypic screen. The developmental defects in zebrafish, including lack of the ventral fin, induced by cercosporamide were strikingly similar to the phenotypes caused by renowned small-molecule BMPR type I kinase inhibitors and inactivating mutations in zebrafish BMPRs. In mammalian cell-based assays, cercosporamide blocked BMP/SMAD-dependent transcriptional reporter activity and BMP-induced SMAD1/5-phosphorylation. Biochemical assays with a panel of purified recombinant kinases demonstrated that cercosporamide directly inhibited kinase activity of type I BMPRs [also called activin receptor-like kinases (ALKs)]. In mammalian cells, cercosporamide selectively inhibited constitutively active BMPR type I-induced SMAD1/5 phosphorylation. Importantly, cercosporamide rescued the developmental defects caused by constitutively active Alk2 in zebrafish embryos. We believe that cercosporamide could be the first of a new class of molecules with potential to be developed further for clinical use against diseases that are causally linked to overactivation of BMPR signaling, including fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma.This article has an associated First Person interview with the first author of the paper.

A new perspective on fungal metabolites: identification of bioactive compounds from fungi using zebrafish embryogenesis as read-out.

There is a constant need for new therapeutic compounds. Fungi have proven to be an excellent, but underexplored source for biologically active compounds with therapeutic potential. Here, we combine mycology, embryology and chemistry by testing secondary metabolites from more than 10,000 species of fungi for biological activity using developing zebrafish (Danio rerio) embryos. Zebrafish development is an excellent model for high-throughput screening. Development is rapid, multiple cell types are assessed simultaneously and embryos are available in high numbers. We found that 1,526 fungal strains produced secondary metabolites with biological activity in the zebrafish bioassay. The active compounds from 39 selected fungi were purified by liquid-liquid extraction and preparative HPLC. 34 compounds were identified by a combination of chemical analyses, including LCMS, UV-Vis spectroscopy/ spectrophotometry, high resolution mass spectrometry and NMR. Our results demonstrate that fungi express a wide variety of biologically active compounds, consisting of both known therapeutic compounds as well as relatively unexplored compounds. Understanding their biological activity in zebrafish may provide insight into underlying biological processes as well as mode of action. Together, this information may provide the first step towards lead compound development for therapeutic drug development.