Nature Communications
Nature Communications
Coniontins, lipopetaibiotics active against Candida auris identified from a microbial natural product fractionation library
The rise of drug-resistant fungal pathogens, including Candida auris, highlights the urgent need for innovative antifungal therapies. We have developed a cost-effective platform combining microbial extract prefractionation with rapid mass spectrometry-bioinformatics-based dereplication to efficiently prioritize previously uncharacterized antifungal scaffolds. Screening C. auris and Candida albicans reveals coniotins, lipopeptaibiotics isolated from Coniochaeta hoffmannii, which are undetectable in crude extracts. Coniotins exhibit potent activity against critical priority fungal pathogens listed by the World Health Organization, including C. albicans, Cryptococcus neoformans, multidrug-resistant Candida auris, and Aspergillus fumigatus, with high selectivity and low resistance potential. Coniotin A targets beta-glucan, compromising fungal cell wall integrity, remodelling, and sensitizing C. auris to caspofungin. Identification of its hybrid polyketide synthase-nonribosomal peptide synthetase biosynthetic gene cluster facilitates discovering structurally diverse lipopeptaibiotics. Here, we show that natural product prefractionation enables the discovery of previously hidden bioactive scaffolds and introduces coniotins as candidates for combating multidrug-resistant fungal pathogens.
Fungal diseases represent a significant threat to public health, affecting over a billion people globally and resulting in more than two point five million deaths annually, surpassing mortality rates from tuberculosis and malaria. Developing antifungal therapies is particularly challenging due to the overlapping cell components, as well as conserved metabolic and biochemical pathways between fungi and their human hosts, leading to a limited repertoire of available treatments for invasive fungal infections. The emergence of drug-resistant fungal pathogens, such as Candida auris, which has caused recent outbreaks in healthcare settings, further exacerbates this issue. C. auris is recognized as a critical priority pathogen by the World Health Organization and has been classified as an urgent threat by the US Centers for Disease Control and Prevention. C. auris isolates resistant to all existing drugs are increasingly common. Unlike other Candida species, C. auris efficiently colonizes the skin, leading to rapid nosocomial transmission and systemic infections with mortality rates of forty to sixty percent. The urgent need for next-generation antifungal drugs is critical to prevent further failures in controlling fungal infections within hospitals and healthcare facilities.
Natural products and their derivatives have been an invaluable source of therapeutic agents, ranging from antibiotics to anticancer agents, thanks to their structural novelty, chemical complexity, and intrinsic bioactivity; consequently, natural products hold promise as leads for antifungal drug discovery. The traditional compound-first discovery strategy using phenotypic cell growth inhibition screens of crude extracts of bacteria and fungi contributed to over half of the antibiotics and antifungal drugs in everyday use today. However, the rediscovery of well-known chemical scaffolds, including antifungal classes such as the polyenes, is a challenge given the phenotypic dominance of highly expressed common scaffolds in natural product extracts.
Owing to the rapid advancement of DNA sequencing technology, genome sequences of natural product producers have revealed large numbers of untapped biosynthetic gene clusters of metabolites, predicting that traditional extract screens vastly under-sample the available chemical space of natural products. A genes-first genome mining strategy, coupled with advanced molecular technologies, is leading to the discovery of novel chemical entities. However, predicting the biological activities of the natural products discovered based on bioinformatic analyses is difficult, even with known compound classes, which limits their application in drug development.
An orthogonal approach to access untapped natural product chemical space entails prefractionating crude natural product extracts before biological testing, thereby uncovering bioactive compounds that are often low in abundance or masked by other activities within complex mixtures. This approach typically improves the hit rate in phenotypic screens and streamlines dereplication by concentrating minor components, reducing sample viscosity for automated platforms, and separating ubiquitous nuisance compounds from rare metabolites. To further broaden accessible chemical diversity prior to fractionation, the one strain, many compounds approach was integrated, which systematically modulates cultivation parameters to enhance microbial metabolite diversity. By altering media composition, aeration, and culture formats, OSMAC enables the activation of silent or poorly expressed biosynthetic gene clusters, facilitating the discovery of cryptic metabolites. Although semipreparative high-performance liquid chromatography-based fractionation approaches remain widespread, their scalability in academic settings is challenging due to the substantial resources required for the dedicated equipment and personnel to prepare the libraries. In contrast, reversed-phase flash chromatography offers a scalable alternative compatible with resource-constrained laboratories.
Building on these insights, we developed a cost-effective prefractionation library using medium-pressure reverse-phase separation, optimized for broad deployment in academic labs. Here, we report a pilot application of this platform for antifungal discovery targeting C. auris and Candida albicans, coupling high-resolution mass spectrometry and tandem mass spectrometry with genome-mining of the relevant biosynthetic gene clusters for rapid dereplication. This workflow efficiently identified and triaged known antifungal families (e.g., enniatins, surfactins, and tunicamycins) and, critically, enabled the prioritization and discovery of a lipopeptaibiotic antifungal coniotin, which exhibits broad activity against multidrug-resistant fungal pathogens and is phenotypically undetectable in the crude extract. Unlike channel-forming lipopeptides, coniotin targets the fungal cell wall by binding beta-glucan, disrupting cell wall remodeling, and sensitizing resistant C. auris to caspofungin, with promising selectivity and a low potential for resistance development. The identification of the linear nonribosomal peptide synthetase-polyketide synthase hybrid gene cluster and the proposed coniotin biosynthetic pathway facilitate the exploration of structurally diverse lipopeptaibiotics from related clusters. This work highlights the utility of the prefractionation library platform coupled with rapid high-resolution mass spectrometry and tandem mass spectrometry dereplication for the discovery of previously undetectable antifungal scaffolds and identifies coniotins as structurally unique candidates that target fungal cell wall integrity in important fungal pathogens.
Results
Results
Antifungal screening of a fractionated natural product library
The PFL was derived from the medium-pressure reverse phase separation of fermentation methanolic extracts of our in-house collection of bacteria and fungi, resulting in eight fractions of metabolites sorted by hydrophilicity for each strain. A total of three thousand forty-eight fractions and the corresponding three hundred eighty-one crude extracts were screened against Candida auris CBS one zero nine one three in duplicate, identifying forty-three hits that showed growth inhibition from fractions, while only twelve hits were from crude extracts. Similarly, a parallel screen against Candida albicans ATCC nine zero zero two eight yielded twenty-eight active fractions and nine crude hits. To identify broad-spectrum antifungal agents, nine hits shared across the two PFL screens were selected for further validation. Among these, antifungal bioactivity from WAC one one zero eight four, WAC one one one one three, and WAC one one one six one was exclusively observed in the fractionated samples, whereas their corresponding crude extracts showed minimal activity and were not identified during the cross-species hit screening. These results highlight the advantage of screening fractionated extracts to improve hit detection.