Rosemary essential oil and its components 1,8-cineole and α-pinene induce ROS-dependent lethality and ROS-independent virulence inhibition in Candida albicans

Abstract

The essential oil from Rosmarinus officinalis L., a composite mixture of plant-derived secondary metabolites, exhibits antifungal activity against virulent candidal species. Here we report the impact of rosemary oil and two of its components, the monoterpene α-pinene and the monoterpenoid 1,8-cineole, against Candida albicans, which induce ROS-dependent cell death at high concentrations and inhibit hyphal morphogenesis and biofilm formation at lower concentrations. The minimum inhibitory concentrations (100% inhibition) for both rosemary oil and 1,8-cineole were 4500 μg/ml and 3125 μg/ml for α-pinene, with the two components exhibiting partial synergy (FICI = 0.55 ± 0.07). At MIC and 1/2 MIC, rosemary oil and its components induced a generalized cell wall stress response, causing damage to cellular and organelle membranes, along with elevated chitin production and increased cell surface adhesion and elasticity, leading to complete vacuolar segregation, mitochondrial depolarization, elevated reactive oxygen species, microtubule dysfunction, and cell cycle arrest mainly at the G1/S phase, consequently triggering cell death. Interestingly, the same oils at lower fractional MIC (1/8-1/4) inhibited virulence traits, including reduction of mycelium (up to 2-fold) and biofilm (up to 4-fold) formation, through a ROS-independent mechanism.

Candida albicans Reactive Oxygen Species (ROS)-Dependent Lethality and ROS-Independent Hyphal and Biofilm Inhibition by Eugenol and Citral

“…Chitin content was elevated ( Fig. 2b ) only for eugenol and the positive control (amphotericin B [Amp B]) at MIC ( P < 0.05 and P < 0.01, respectively), consistent with our previous data ( 41 ).…”

“…5 ). Citral disrupts MTs in plant and animal cells, with no damage to the cell membrane nor the actin cytoskeleton, but plant cells exposed to limonene and (+)-citronellal have membrane damage associated with MT disruption ( 62 , 63 ), consistent with this study and similar to the impact of other EO(C)s ( 41 , 64 ).…”

“…4 ). Similar findings have been reported for C. albicans exposed to known membrane disruptors such as Amp B ( 41 ), cationic peptides ( 54 ), clove, thyme ( 55 ) rosemary and its major components ( 41 ).…”

“…The cell wall and cell membrane are essential not only for Candida cell viability and morphology, but also virulence through several signaling pathways ( 44 ). Therefore, relatively slight changes to the structural integrity of the cell wall ( 41 ) or membrane can result in cell death. Eugenol and citral are both lethal for C. albicans at relatively high concentrations compared to commercial antifungals ( 45 ) and, together, their anticandidal activity is additive ( Fig.…”

“…Vacuolar defects were assessed using the method described previously ( 41 ). Briefly, C. albicans RSY150 overnight cultures were suspended in freshly prepared YPD medium to a cell density of ~10 7 CFU/mL, treated with EOCs at MIC and 1/2 MIC, or the two at their FICI for 4 h. Samples were then washed three times with PBS and transferred (5 μL) onto glass slides, sealed (nail polish) with clean coverslips, and imaged (Axio Observer Z1 inverted epifluorescence microscope; Oberkochen, Germany) using a 63× objective in bright field.…”