Fingerprint SRS Imaging Unveils Ergosteryl Ester as a Metabolic Signature of Azole-ResistantCandida albicans

Abstract

AbstractCandida albicans(C. albicans), a major fungal pathogen, causes life-threatening infections in immunocompromised individuals. Fluconazole (FLC) is recommended as first-line therapy for treatment of invasive fungal infections. Yet, the widespread use of FLC has resulted in increased antifungal resistance among different strains ofCandida, especiallyC. albicans, which is a leading source of hospital-acquired infections. Here, by hyperspectral stimulated Raman scattering (hSRS) imaging of single fungal cells in the fingerprint window and pixel-wise spectral unmixing, we report aberrant ergosteryl ester accumulation in azole-resistantC. albicanscompared to azole-susceptible species. This accumulation was a consequence ofde novolipogenesis. Lipid profiling by mass spectroscopy identified ergosterol oleate to be the major species stored in azole-resistantC. albicans. Blocking ergosterol esterification by oleate and suppressing sterol synthesis by FLC synergistically suppressed the viability ofC. albicans in vitroand limited the growth of biofilm on mouse skinin vivo. Our findings highlight a metabolic marker and a new therapeutic strategy for targeting azole-resistantC. albicansby interrupting the esterified ergosterol biosynthetic pathway.Significance StatementInvasive fungal infections and increasing antifungal resistance are emerging threats to public health with high morbidity and mortality. Despite the advances in azole resistance mechanisms, it remains unclear why some fungal species are intrinsically resistant to or easily acquire resistance to multiple antifungal drugs. Here, using fingerprint SRS microscopy, we uncovered a molecular signature, aberrant ergosteryl ester accumulation, linked to the azole resistance ofCandidaspecies. An antifungal treatment strategy combining oleate (inhibitor of ersgosteryl esterification) and azole significantly attenuates the azole resistance and the viability ofC. albicans in vitroandin vivo. Our work opens a new way to detect and treat azole-resistant fungal infections by targeting ergosterol metabolism.