Genomic and metabolomic diversity within a familial population of Aspergillus flavus.

Aspergillus flavus is an agriculturally significant micro-fungus having potential to contaminate food and feed crops with toxic secondary metabolites such as aflatoxin (AF) and cyclopiazonic acid (CPA). Research has shown A. flavus strains can overcome heterokaryon incompatibility and undergo meiotic recombination as teleomorphs. Although evidence of recombination in the AF gene cluster has been reported, the impacts of recombination on genotype and metabolomic phenotype in a single generation are lacking. In previous studies, we paired an aflatoxigenic MAT1-1 A. flavus strain with a non-aflatoxigenic MAT1-2 A. flavus strain that had been tagged with green fluorescent protein and then 10 F1 progenies (a mix of fluorescent and non-fluorescent) were randomly selected from single-ascospore colonies and broadly examined for evidence of recombination. In this study, we determined four of those 10 F1 progenies were recombinants because they were not vegetatively compatible with either parent or their siblings, and they exhibited other distinctive traits that could only result from meiotic recombination. The other six progenies examined shared genomic identity with the non-aflatoxigenic, fluorescent, and MAT1-2 parent, but were metabolically distinct. This study highlights phenotypic and genomic changes that may occur in a single generation from the outcrossing of sexually compatible strains of A. flavus.

The fungal natural product fusidic acid demonstrates potent activity against Mycoplasma genitalium.

Antimicrobial resistance is extremely common in Mycoplasma genitalium, a frequent cause of urethritis in men and cervicitis, vaginitis, and pelvic inflammatory disease in women. Treatment of M. genitalium infections is difficult due to intrinsic and acquired resistance to many antibiotic classes. We undertook a program to identify novel antimicrobials with activity against M. genitalium from fungal natural products. Extracts of Ramularia coccinea contained a molecule with potent activity that was subsequently identified as fusidic acid, a fusidane-type antibiotic that has been in clinical use for decades outside the United States. We found that minimum inhibitory concentrations of fusidic acid ranged from 0.31 to 4 µg/mL among 17 M. genitalium strains including laboratory-passaged and low-passage clinical isolates. Time-kill data indicate that bactericidal killing occurs when M. genitalium is exposed to ≥10 µg/mL for 48 h, comparing favorably to serum concentrations obtained from typical loading dose regimens. Resistance to fusidic acid was associated with mutations in fusA consistent with the known mechanism of action in which fusidic acid inhibits protein synthesis by binding to elongation factor G. Interestingly, no mutants resistant to >10 µg/mL fusidic acid were obtained and a resistant strain containing a F435Y mutation in FusA was impaired for growth in vitro. These data suggest that fusidic acid may be a promising option for the treatment of M. genitalium infections.

Iron(III) Binding Properties of PF1140, a Fungal N-Hydroxypyridone, and Activity against Mycoplasma genitalium.

Mycoplasma genitalium is a sexually transmitted bacterium associated with urogenital disease syndromes in the US and worldwide. The global rise in drug resistance in M. genitalium necessitates the development of novel drugs to treat this pathogen. To address this need, we have screened extracts from a library of fungal isolates assembled through the University of Oklahoma Citizen Science Soil Collection Program. Analysis of one of the bioactive extracts using bioassay-guided fractionation led to the purification of the compound PF1140 (1) along with a new and several other known pyridones. The N-hydroxy pyridones are generally regarded as siderophores with high binding affinity for iron(III) under physiological conditions. Results from UV-vis absorption spectroscopy-based titration experiments revealed that 1 complexes with Fe3+. As M. genitalium does not utilize iron, we propose that the PF1140-iron complex induces cytotoxicity by facilitating the cellular uptake of iron, which reacts with endogenous hydrogen peroxide to produce toxic hydroxyl radicals.

Chemoreactive 2,5-Diketopiperazines from a Penicillium sp., Structure Revision of Reported Analogues and Proposed Facile Transformation Pathways.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous cancer. Two new prenylated indole 2,5-diketopiperazine alkaloids, brevianamides E1 (1) and E2 (2), were isolated from a Penicillium fungus. Both compounds showed moderate cytotoxic activity against select MCC cell lines (i.e., MCC13, MKL-1, UISO, and WaGa) in the low micromolar range. The relative and absolute configurations of 1 and 2 were determined by combined approaches, including NOESY spectroscopy, DFT ECD and DP4 plus calculations, and Marfey's reaction. Literature research and the comparison of NMR and ECD data led to the structure revision of three previously reported natural analogues, notoamides K and P and asperversiamide L. The structurally unstable 1 and 2 underwent steady interconversion under neutral aqueous conditions. Investigation of the degradation of 2 in acidic methanol solutions led to the identification of a new methoxylated derivative (6) and two new ring-opened products (7 and 8) with the rearranged, elongated, 4-methylpent-3-ene side chain. The facile transformation of 2 to 7 and 8 was promoted by the intrinsic impurity (i.e., formaldehyde) of HPLC-grade methanol through the aza-Cope rearrangement.

Resolving a Natural Product Cold Case: Elucidation of Fusapyrone Structure and Absolute Configuration and Demonstration of Their Fungal Biofilm Disrupting Properties.

Fusapyrones are fungal metabolites, which have been reported to have broad-spectrum antibacterial and antifungal properties. Despite the first members of this chemical class being described three decades prior, many aspects of their structures have remained unresolved, thereby constraining efforts to fully understand structure-activity relationships within this metabolite family and impeding the design of streamlined syntheses. Among the main challenges posed by fusapyrones is the incorporation of several single and groups of stereocenters separated by atoms with freely rotating bonds, which have proven unyielding to spectroscopic analyses. In this study, we obtained a series of new (2-5 and 7-9) and previously reported fusapyrones (1 and 6), which were subjected to a combination of spectroscopic, chemical, and computational techniques enabling us to offer proposals for their full structures, as well as provide a pathway to reinterpreting the absolute configurations of other published fusapyrone metabolites. Biological testing of the fusapyrones revealed their abilities to inhibit and disrupt biofilms made by the human fungal pathogen, Candida albicans. These results show that fusapyrones reduce hyphae formation in C. albicans, as well as decrease the surface adherence capabilities of planktonic cells and cells transitioning into early-stage biofilm formation.

Persephacin Is a Broad-Spectrum Antifungal Aureobasidin Metabolite That Overcomes Intrinsic Resistance in Aspergillus fumigatus.

Fungi pose a persistent threat to humankind with worrying indications that emerging and re-emerging pathogens (e.g., Candida auris, Coccidioides spp., drug-resistant Aspergilli, and more) exhibit resistance to the limited number of approved antifungals. To address this problem, our team is exploring endophytic fungi as a resource for the discovery of new antifungal natural products. The rationale behind this decision is based on evidence that endophytes engage with plants in mutualistic relationships wherein some fungi actively participate by producing chemical defense measures that suppress pathogenic microorganisms. To improve the odds of bioactive metabolite discovery, we developed a new hands-free laser-cutting system capable of generating >50 plant samples per minute that, in turn, enabled our team to prepare and screen large numbers of endophytic fungi. One of the fungal isolates obtained in this way was identified as an Elsinoë sp. that produced a unique aureobasidin analogue, persephacin (1). Some distinctive features of 1 are the absence of both phenylalanine residues combined with the incorporation of a novel amino acid residue, persephanine (9). Compound 1 exhibits potent antifungal effects against a large number of pathogenic yeast (including several clinical C. auris strains), as well as phylogenetically diverse filamentous fungi (e.g., Aspergillus fumigatus). In an ex vivo eye infection model, compound 1 outperformed standard-of-care treatments demonstrating the ability to suppress fluconazole-resistant Candida albicans and A. fumigatus at a concentration (0.1% solution) well below the clinically recommended levels used for fluconazole and natamycin (2% and 5% solutions, respectively). In 3D tissue models for acute dermal and ocular safety, 1 was found to be nontoxic and nonirritating at concentrations required to elicit antifungal activity. Natural product 1 appears to be a promising candidate for further investigation as a broad-spectrum antifungal capable of controlling a range of pathogens that negatively impact human, animal, and plant health.

Appraisal of Fungus-Derived Xanthoquinodins as Broad-Spectrum Anti-Infectives Targeting Phylogenetically Diverse Human Pathogens.

Xanthoquinodins make up a distinctive class of xanthone-anthraquinone heterodimers reported as secondary metabolites from several fungal species. Through a collaborative multi-institutional screening program, a fungal extract prepared from a Trichocladium sp. was identified that exhibited strong inhibitory effects against several human pathogens (Mycoplasma genitalium, Plasmodium falciparum, Cryptosporidium parvum, and Trichomonas vaginalis). This report focuses on one of the unique samples that exhibited a desirable combination of biological effects: namely, it inhibited all four test pathogens and demonstrated low levels of toxicity toward HepG2 (human liver) cells. Fractionation and purification of the bioactive components and their congeners led to the identification of six new compounds [xanthoquinodins NPDG A1-A5 (1-5) and B1 (6)] as well as several previously reported natural products (7-14). The chemical structures of 1-14 were determined based on interpretation of their 1D and 2D NMR, HRESIMS, and electronic circular dichroism (ECD) data. Biological testing of the purified metabolites revealed that they possessed widely varying levels of inhibitory activity against a panel of human pathogens. Xanthoquinodins A1 (7) and A2 (8) exhibited the most promising broad-spectrum inhibitory effects against M. genitalium (EC50 values: 0.13 and 0.12 µM, respectively), C. parvum (EC50 values: 5.2 and 3.5 µM, respectively), T. vaginalis (EC50 values: 3.9 and 6.8 µM, respectively), and P. falciparum (EC50 values: 0.29 and 0.50 µM, respectively) with no cytotoxicity detected at the highest concentration tested (HepG2 EC50 > 25 µM).

Assessing Microbial Metabolic and Biological Diversity to Inform Natural Product Library Assembly.

The pressing need for novel chemical matter to support bioactive compound discovery has led natural product researchers to explore a wide range of source organisms and environments. One of the implicit guiding principles behind those efforts is the notion that sampling different environments is critical to accessing unique natural products. This idea was tested by comparing fungi from disparate biomes: aquatic sediments from Lake Michigan (USA) and terrestrial samples taken from the surrounding soils. Matched sets of Penicillium brevicompactum, Penicillium expansum, and Penicillium oxalicum from the two source environments were compared, revealing modest differences in physiological performance and chemical output. Analysis of LC-MS/MS-derived molecular feature data showed no source-dependent differences in chemical richness. High levels of scaffold homogeneity were also observed with 78-83% of scaffolds shared among the terrestrial and aquatic Penicillium spp. isolates. A comparison of the culturable fungi from the two biomes indicated that certain genera were more strongly associated with aquatic sediments (e.g., Trichoderma, Pseudeurotium, Cladosporium, and Preussia) versus the surrounding terrestrial environment (e.g., Fusarium, Pseudogymnoascus, Humicola, and Acremonium). Taken together, these results suggest that focusing efforts on sampling the microbial resources that are unique to an environment may have a more pronounced effect on enhancing the sought-after natural product diversity needed for chemical discovery and screening collections.

Tolypocladamides A-G: Cytotoxic Peptaibols from Tolypocladium inflatum.

Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.

Leveraging Peptaibol Biosynthetic Promiscuity for Next-Generation Antiplasmodial Therapeutics.

Malaria remains a worldwide threat, afflicting over 200 million people each year. The emergence of drug resistance against existing therapeutics threatens to destabilize global efforts aimed at controlling Plasmodium spp. parasites, which is expected to leave vast portions of humanity unprotected against the disease. To address this need, systematic testing of a fungal natural product extract library assembled through the University of Oklahoma Citizen Science Soil Collection Program has generated an initial set of bioactive extracts that exhibit potent antiplasmodial activity (EC50 < 0.30 µg/mL) and low levels of toxicity against human cells (less than 50% reduction in HepG2 growth at 25 µg/mL). Analysis of the two top-performing extracts from Trichoderma sp. and Hypocrea sp. isolates revealed both contained chemically diverse assemblages of putative peptaibol-like compounds that were responsible for their antiplasmodial actions. Purification and structure determination efforts yielded 30 new peptaibols and lipopeptaibols (1-14 and 28-43), along with 22 known metabolites (15-27 and 44-52). While several compounds displayed promising activity profiles, one of the new metabolites, harzianin NPDG I (14), stood out from the others due to its noteworthy potency (EC50 = 0.10 µM against multi-drug-resistant P. falciparum line Dd2) and absence of gross toxicity toward HepG2 at the highest concentrations tested (HepG2 EC50 > 25 µM, selectivity index > 250). The unique chemodiversity afforded by these fungal isolates serves to unlock new opportunities for translating peptaibols into a bioactive scaffold worthy of further development.