Elucidating the Eradication Mechanism of Perillyl Alcohol against Candida glabrata Biofilms: Insights into the Synergistic Effect with Azole Drugs

[Image: see text] Increased incidences of fungal infections and associated mortality have accelerated the need for effective and alternative therapeutics. Perillyl alcohol (PA) is a terpene produced by the hydroxylation of limonene via the mevalonate pathway. In pursuit of an alternative antifungal agent, we studied the effect of PA on the biofilm community of Candida glabrata and on different cellular pathways to decipher its mode of action. PA efficiently inhibited growth and eradicated biofilms by reducing carbohydrate and eDNA content in the extracellular matrix. PA reduced the activity of hydrolytic enzymes in the ECM of C. glabrata biofilm. The chemical profiling study has given insights into the overall mode of action of PA in C. glabrata and the marked involvement of the cell wall and membrane, ergosterol biosynthesis, oxidative stress, and DNA replication. The spectroscopic and RT-PCR studies suggested a strong interaction of PA with chitin, β-glucan, ergosterol, and efflux pump, thus indicating increased membrane fluidity in C. glabrata. Furthermore, the microscopic and flow cytometry analysis emphasized that PA facilitated the change in mitochondrial activity, increased Ca(2+) influx via overexpression of voltage-gated Ca(2+) channels, and enhanced cytochrome C release from mitochondria. In addition, PA interferes with DNA replication and thus hinders the cell cycle progression at the S-phase. All these studies together established that PA mitigates the C. glabrata biofilms by targeting multiple cellular pathways. Interestingly, PA also potentiated the efficacy of azole drugs, particularly miconazole, against C. glabrata and its clinical isolates. Conclusively, the study demonstrated the use of PA as an effective antifungal agent alone or in combination with FDA-approved conventional drugs for fungal biofilm eradication.