A microbial natural product fractionation library screen with HRMS/MS dereplication identifies new lipopeptaibiotics against Candida auris

Abstract

The rise of drug-resistant fungal pathogens, including Candida auris, highlights the urgent need for novel antifungal therapies. We developed a cost-effective platform combining microbial extract prefractionation with rapid MS/MS-bioinformatics-based dereplication to efficiently prioritize new antifungal scaffolds. Screening C. auris and C. albicans revealed novel lipopeptaibiotics, coniotins, from Coniochaeta hoffmannii WAC11161, which were undetectable in crude extracts. Coniotins exhibited potent activity against critical fungal pathogens on the WHO Fungal Priority Pathogens List, including C. albicans, C. neoformans, multidrug-resistant C. auris, and Aspergillus fumigatus, with high selectivity and low resistance potential. Coniotin A targets β-glucan, compromising fungal cell wall integrity, remodelling, and sensitizing C. auris to caspofungin. Identification of a PKS-NRPS biosynthetic gene cluster further enables the discovery of related clusters encoding potential novel lipopeptaibiotics. This study demonstrates the power of natural product prefractionation in uncovering bioactive scaffolds and introduces coniotins as promising candidates for combating multidrug-resistant fungal pathogens.

Figure 1. A scheme for the discovery of derisked novel antifungal natural products

a, Overview of a rapid and risk-minimized approach for discovering novel antifungal natural products. The flowchart outlines a streamlined pipeline, starting from the construction of a prefractionation library (PFL), followed by high-throughput screening against target organisms (Candida albicans and Candida auris), rapid dereplication using tandem mass spectrometry (MS²) fingerprinting and bioinformatics analysis, structure determination, off-target assessment in mammalian cells (HEK: Human Embryonic Kidney cells + RBC: Red Blood Cell), broad-spectrum bioactivity evaluation, rapid therapeutic assessment using high-throughput animal models, and characterization of the mechanism of action (MOA). b, Scatter plots illustrating high-throughput screening results of crude methanolic extracts and PFL against C. albicans (y-axis) and C. auris (x-axis). Colored circles within the red box represent active hits: crude extracts (blue) or fractions (red) that inhibit Candida growth by at least 75% compared to the untreated control. Active hits with their WAC and fraction identities are shown in Extended Data Fig. 1a, with validation results in Extended Data Fig. 1b.

Figure 2. Rapid identification of known chemical scaffolds using high-resolution (HR) mass spectrometry coupled with bioinformatics analysis.

a, Identification of enniatin from active fractions of WAC11175 through Global Natural Products Social Molecular Networking (GNPS) based on HRMS and MS/MS data. (i) The enniatin molecular network, with tandem MS fragment ion matches between the sample entity (blue) and reference enniatin B1 (black), is shown in the inset panel. (ii) HR-LCMS analysis for enniatins identified in active fractions of WAC11175, including the structures and representative HRMS spectra of enniatin A1 (iii), enniatin B (v), and enniatin B1 (vii). Accurate MS/MS spectra for enniatins A1, B, and B1, acquired at a single collision energy of 20 eV, along with fragmentation analyses, are shown in (iv), (vi), and (viii). N-Me-Val, N-Me-Ile, and Hiv represent N-Methyl-L-valine, N-Methyl-L-isoleucine, and 2-hydroxyisovaleric acid, respectively. b, Identification of the enniatin biosynthetic gene cluster (BGC) in the genome of WAC11175 and depiction of its biosynthesis. Esyn1, the enniatin synthetase, contains the following domains: C (condensation), A (adenylation), P (phosphopantetheine attachment site), and nMT (N-methyltransferase). The precursors, L-valine and D-hydroxycarboxylic acids, are activated at the A domain, and the building blocks are transferred between modules via P-domains. The final condensation, cyclization, and release from the enzyme are catalyzed by the C-domains. c, Identification of surfactin from active fractions of WAC11084. (i) GNPS molecular networking, constructed from HR-MS/MS data, showing tandem MS fragment ion matches (inset panel) between the identified entity (blue) and reference surfactin C (black). MS/MS fragmentation patterns and structures of surfactins are shown below: (ii) Surfactin A ([M + H]⁺, m/z 1008.6565) fragments into main ions at 667.4, 568.3, and 455.3; (iii) Surfactin B ([M + H]⁺, m/z 1022.6731) into 909.5, 681.4, and 582.4; (iv) Surfactin C ([M + H]⁺, m/z 1036.6907) into 923.6, 695.4, and 596.4. Additional fragment ions [M + H]⁺ confirm specific amino acid residue sequences, including m/z 227.1750 [Leu+Leu+H]⁺, 229.1145 [Asp+Leu+H]⁺, and 441.2699 [Leu+Asp+Val+Leu+H]⁺. d, Genetic organization of the surfactin BGC in WAC11084 and proposed biosynthesis. The surfactin synthetase complex consists of three modular units: SrfA, SrfB, mono-modular SrfC, and SrfD, responsible for synthesizing the seven amino acids of surfactin. Key domains include C (condensation), A (adenylation), T (thiolation), E (epimerization), and TE (thioesterase). The TE domain facilitates the release and cyclization of surfactin.

Figure 3. Characterization of novel antifungal lipopeptaibiotics from the Coniochaeta fungus WAC11161.

a, High-resolution mass spectrum of compound 1 obtained with QToF mass spectrometry, showing the [M+H]⁺ ion at m/z 2057.2609. In-source fragmentation produced ions at m/z 506.36, 602.31, 690.48, 843.45, 931.62, 1126.65, and 1215.81. b, MS/MS analysis of compound 1 using collision-induced dissociation (CID) combined with a product ion scan (MS/MS) of nominal m/z 2057.26. Precursor ion indicated with a blue square. The structure of the lipopeptide 1 (termed coniotin A) is displayed above, along with its collision-induced fragmentation pattern, which corresponds to the detected b ions in the MS/MS spectrum. The b ions are labeled in blue, and the y ions are labeled in red. c, The structures of antifungal lipopeptaibiotics analogues 1, 2, 3, and 4, identified from the Coniochaeta fungus WAC11161, termed coniotin A, B, C, and D. d, High-resolution mass spectrum of coniotin B obtained with QToF mass spectrometry, showing the [M+H]⁺ ion at m/z 2056.2781 (calculated for C98H171N22O25, 2056.2780). e, High-resolution mass spectrum of coniotin C obtained with QToF mass spectrometry, showing the [M+H]⁺ ion at m/z 2043.2464 (calculated for C97H168N21O26⁺, 2043.2464) and the [M+Na]⁺ ion at m/z 2065.2255. f, High-resolution mass spectrum of coniotin D obtained using QToF mass spectrometry, showing the [M+H]⁺ ion at m/z 2042.2689 (calculated for C97H169N22O25⁺, 2042.2624), the [M+Na]⁺ ion at m/z 2064.2536 and the [M+K]⁺ ion at m/z 2080.2316.

Figure 4. Antifungal activity of coniotin against C. albicans and multidrug-resistant Candidaauris

a, Coniotin A (CAN) synergizes with caspofungin (CAP) in Candida species. Checkerboard assays depicted as heatmaps show the average growth of biological duplicates, normalized to controls without compounds. The potentiation of coniotin A and caspofungin was evaluated against C. auris CBS12775 and C. albicans ATCC90028. Relative growth is depicted by colour, as indicated by the scale bar in the bottom right. Fractional Inhibitory Concentration Index (FICI) values, calculated as described in the Methods, are shown in the top right corner of each checkerboard. FICI values below 0.5 denote synergistic interactions. b, Rapid assessment of the therapeutic potential of coniotin A using high-throughput phenotypic screening in a Caenorhabditis elegans-Candida albicans infection model. C. elegans were infected with C. albicans ATCC90028 and treated with various concentrations of coniotin A. Representative images show worms treated with DMSO (i, negative control), 1 µg/ml coniotin A (ii), and 8 µg/ml coniotin A (iii). Scale bar = 0.2 mm. c, Survival of C. elegans infected with C. auris CBS 12775 and treated with amphotericin B (AMB), coniotin A (CNA), or vehicle dimethyl sulfoxide (DMSO). Twenty-five worms per condition were observed over a 48-hour period in three independent trials. Survival was analyzed using Kaplan-Meier survival curves, and statistical significance was determined by the Log-rank (Mantel-Cox) test, comparing CNA (1× MIC) treatment to the DMSO control group, with p-values reported as **** for p < 0.0001.

Figure 5. Coniotin A targets β-glucan impairing cell wall integrity.

a, Intracellular accumulation of coniotin A (CNA), caspofungin (CAP) and iturin A (ITA) in C. auris CBS10913 (Cau) and C. albicans ATCC90028 (Cal) were quantified after 10 minutes of treatment. Surface-bound compound was removed using silicone oil prior to analysis. Results are expressed as the mean ± SD from three independent biological replicates. b, Increased cell wall chitin levels following coniotin A (CNA) and caspofungin (CAP) treatment. Quantification of calcofluor white (CFW) staining was performed to assess total cell wall chitin levels in log-phase cells of C. albicans ATCC90028, C. auris CBS10913, and C. neoformans H99. Cells were treated for four hours with half the MIC of CNA, CAP, or vehicle control (DMSO), stained with CFW, and imaged using a Nikon Eclipse Ti inverted microscope. Fluorescence intensity per cell was quantified using ImageJ and CellProfiler, analyzing 100 cells from at least three images per condition. Data are presented as the log mean edge fluorescence intensity ± SEM. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test, with each treatment group compared to its respective DMSO control (*P < 0.05; ** P < 0.01; ***P < 0.001). c, Representative images of CFW staining in treated fungal cells related to Fig. 6b. Representative images showing C. albicans ATCC90028, C. auris CBS10913, and C. neoformans H99 cells treated with half the MIC of coniotin A (CNA), caspofungin (CAP), or vehicle control (DMSO), followed by CFW staining to assess chitin content. In CNA- and CAP-treated cells, bright, thickened septa were observed, forming proximal to the normal location at the mother-bud neck region. Scale bar = 10 µm. d, Structural organization and composition of Candida yeast cell wall. The outer cell wall of Candida yeasts is enriched with highly mannosylated proteins, predominantly anchored to the β-glucan and chitin core via glycosylphosphatidylinositol (GPI) remnants. Echinocandins target glucan synthase Fks1, a key enzyme displayed in the cell membrane, which is essential for the synthesis and integrity of the cell wall. e, The mannoprotein component of the fungal cell wall was fluorescently labelled with ConA-Alex647. C. albicans ATCC90028 cultures were grown to the mid-log phase in the presence of coniotin A (CNA), caspofungin (CAP), or vehicle control (DMSO) and stained with ConA-Alex647 for 10 minutes. Z-stack images were acquired using a Zeiss LSM980 Inverted Confocal Microscope using a 63×/1.4 oil-immersion objective. 3D projections of the yeast cells were generated in ImageJ. Blue arrows indicate cell wall damage. Scale bars = 5 μm. f, Perimeter of mid-log phase C. albicans ATCC90028 cells grown in SDB medium at 30°C in the presence of coniotin A (CNA), caspofungin (CAP), or vehicle control (DMSO). The cell periphery was visualized by staining with ConA-Alex647, and the perimeter and diameter were measured and analyzed using ImageJ by examining ~150 cells. Statistical significance was evaluated using two-tailed pairwise Student’s t-tests (**P < 0.01; ***P < 0.001). g, Quantitative analysis of coniotin A (CNA) binding in the pull-down assay. β−1,3-glucan (1 mg/mL) or chitin (1 mg/mL) was incubated with 32 µg/mL coniotin A (CNA) in PBS for 1 hour. Following incubation, the polysaccharides were collected, washed, and extracted with DMSO for analysis. CNA bound to β−1,3-glucan (Glu-B) or chitin (Chi-B), or remaining in the supernatant of β−1,3-glucan (Glu-S) or chitin (Chi-S) solutions, was quantified using high-resolution mass spectrometry. The Y-axis shows the relative abundance of CNA based on MS peak area, and the X-axis indicates the sample groups. Data are presented as mean ± SD from three independent biological replicates. h, Inhibition of β−1,3-glucan (Glu) digestion by coniotin A (CNA). Relative abundance of glucanase (GCase) digestion products (β−1,3-linked oligosaccharides: hexa-glucose) from 125 µg/mL laminarin (a β−1,3-glucan), incubated with glucanase for 0.5 h in the absence or presence of different concentrations of coniotin A (64 µg/mL, ×0.5; 128 µg/mL, ×1; 256 µg/mL, ×2). Presence is indicated as “+” and absence as Data were acquired using high-resolution mass spectrometry and presented as mean ± SD from triplicate runs. Statistical significance was determined using an unpaired t-test with Welch’s correction, comparing each coniotin A treatment to untreated controls. p-values: *** < 0.001, **** < 0.0001. i Kinetic curves of β-glucan activation of limulus coagulation factor G. The kinetic chromogenic reaction specific to (1,3)-β-D-glucan (Glu) using Glucatell^® kits: 100 pg/mL β−1,3-glucan was preincubated with or without coniotin A (CNA) at concentrations of 0.625 µg/mL (1×), 5 µg/mL (8×), and 40 µg/mL (64×). The samples were mixed with 100 µL reconstituted Glucatell reagent containing limulus coagulation factor G, and analyzed in a preheated plate reader at 37°C for 1 hour. The rate of change (mAbs/30s) was measured at 405 nm to determine intact (1,3)-β-D-glucan abundance. j, Transmission Electron Microscopy (TEM) images of C. auris CBS12766 and C. neoformans H99 showing abnormal cell wall structures following coniotin A (CNA) treatment. Cells were cultured with (+) and without (−) half MIC of CNA, fixed, and visualized via TEM. Vehicle (DMSO)-treated C. auris cells are shown in (i, ii), CNA-treated cells in (iii, iv), similarly, vehicle (DMSO)-treated C. neoformans cells in (v, vi), and CNA-treated C. neoformans (2 µg/mL) in (vii, viii). Observed cell wall defects in CNA-treated cells include detached membranes (blue arrowheads), compromised cell wall integrity (orange arrowheads), and abnormally increased thickness. Brackets denote distinct cell wall layers: G+C, β-glucan and chitin; M, mannoproteins. Additional cellular structures are labeled: nucleus (N) and mitochondria (m). Scale bars are depicted in each image, with units in nanometers (nm).

Figure 6. The biosynthetic gene cluster and proposed biosynthetic pathway of coniotin A.

a, Organization of the coniotin biosynthetic gene cluster and proposed pathway in Coniochaeta hoffmannii WAC11161. Open reading frames (ORFs) involved in coniotin biosynthesis are color-coded as follows: pink for the polyketide synthase (PKS) gene (conA), blue for the non-ribosomal peptide synthetase (NRPS) genes (conB-D), dark blue for the acyl-CoA ligase gene (conE), light purple for the transferase gene (conF), light yellow for the ABC transporter, and green for potential functional genes. For ORF annotations, see also Supplementary Table 5. The PKS domains in ConA are labeled as follows: AT (acyltransferase), KS (keto synthase), ACP (acyl carrier protein), KR (ketoreductase), DH (dehydratase), ER (enoylreductase), and MT (methyltransferase). The NRPS domains in ConB-D are labeled as follows: C (condensation domain), A (adenylation domain), T (thiolation domain), and TD (terminal domain). b, A phylogenetic tree of ConA PKS analogs was constructed using the Jukes-Cantor genetic distance model and the neighbor-joining method, based on amino acid sequences of 22 ConA homologues (Left panel). To ensure statistical robustness, bootstrap resampling with 1,000 replicates and a random seed of 996327 was performed. Bootstrap values are displayed next to the nodes, and the ConA homologues are labeled with their respective protein IDs. The gene organization of biosynthetic gene clusters containing the corresponding ConA PKS analogs is shown on the right, compared and aligned with the phylogenetic tree. Protein-coding genes adjacent to the PKSs within these clusters are depicted as colored arrows, indicating transcriptional orientation. Genes with similar functions are color-coded for clarity: purple arrows denote ConA PKS analogs, with connecting linkers indicating sequence alignment identities exceeding 30%; pink arrows highlight NRPS genes.