Raman Imaging of an Amphotericin B Potentiator in Single-Cell <i>Saccharomyces cerevisiae</i>

Nectriatide, a fungal cyclotetrapeptide, was previously found to enhance the antifungal activity of amphotericin B (AmB), with nectriatide linear derivatives (NLDs) exhibiting even stronger potentiation. Although we showed that NLDs have an affinity for ergosterol <i>in vitro</i>, the precise mechanism underlying these interactions within living fungal cells remained elusive. Here, we demonstrate for the first time a label-free, single-cell Raman microspectroscopic imaging approach to visualize how NLD-1 colocalizes with ergosterol in <i>Saccharomyces cerevisiae</i>. Our results revealed clear evidence of NLD-1 localization in ergosterol-rich regions, indicating direct interaction. Furthermore, antifungal-enhancing effect of NLD-1 was markedly reduced in <i>ERG4</i>-deficient <i>S. cerevisiae</i>─supporting its specific recognition of ergosterol. This unprecedented real-time, <i>in situ</i> visualization of an AmB potentiator in living yeast cells not only provides new insights into the mechanism of AmB enhancement but also sets the stage for broader applications in understanding other antifungal synergies at the molecular level.