Fig. 5: Coniotin A targets β-glucan and impairs 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) after 10 min treatment. Data represent mean ± SD from four biological replicates. b Chitin levels in fungal cells after 4 h treatment with ½ MIC of CNA, CAP, or vehicle control (DMSO). Calcofluor white (CFW)-stained cells were analyzed by epifluorescence microscopy; bars represent the mean, with individual cell values (from three independent biological replicates) overlaid as dots. Statistical significance was determined using unpaired two-tailed t-tests with Welch’s correction; all comparisons between CNA and DMSO yielded P < 0.000001. c Representative CFW-stained images corresponding to (b). Scale bars, 10 µm. d Schematic of the Candida cell wall. Glycosylphosphatidylinositol-anchored mannoproteins form the outer layer linked to a β-glucan–chitin core. Echinocandins target the glucan synthase Fks1, compromising wall integrity. Created with BioRender.com. e Mannoprotein staining in C. albicans (ConA–Alexa Fluor 647) after treatment. Confocal 3D projections showing cell wall disruption (blue arrows). Scale bars, 5 µm. f Quantification of cell perimeter from treatments as in (e), based on ~150 cells (ImageJ). Significance was determined by a two-tailed unpaired Student’s t-test: exact P values are shown in the figure. g CNA binding to β−1,3-glucan and chitin assessed by mass spectrometry after pull-down assay. Relative abundance shown for bound (Glu-B, Chi-B) and unbound (Glu-S, Chi-S) fractions. Data represent mean ± SD of three biological replicates; each dot corresponds to one replicate. h CNA inhibits β−1,3-glucanase (GCase) digestion. Hexa-glucose oligosaccharides released from laminarin (a β−1,3-glucan) after 0.5 h hydrolysis with or without CNA (64, 128, or 256 µg/mL) were quantified by mass spectrometry. Data from three independent replicates are shown as dots; values are mean ± SD. Significance was determined using two-tailed unpaired t-tests with Welch’s correction. Exact P values are shown in the figure. i CNA inhibits β−1,3-glucan activation of Limulus factor G in a dose-dependent manner. β−1, 3-glucan (Glu) was incubated without/with CNA (0.625, 5, or 40 µg/mL; 1×, 8×, and 64×) and assayed using Glucatell®. Reactions were monitored at 405 nm for 1 h. Data represent mean ± SD from n = 3 independent biological replicates. j TEM of C. auris and C. neoformans ± CNA (½ MIC). CNA induces membrane detachment (blue arrowheads), loss of integrity (orange), and cell wall thickening. G + C β-glucan/chitin, M mannoprotein, N nucleus, m mitochondria. Scale bars, nm. Figures 5b, f–i, Source data and full statistical results are provided as a Source Data file.