Fig 1.
Overview of PPI Network of C. albicans genes for strains resistant and sensitive to EOCs.
(a,b) Comparison of genes showing resistance and sensitivity to 1,8-cineole and α-pinene, including protein-protein interaction networks. (a) There were 5 genes conferring resistance to both 1,8-cineole and α-pinene and (b) 8 conferring sensitivity to both 1,8-cineole and α-pinene. Thick lines represent very high confidence, yellow nodes represent highly interconnected subnetworks. (c) Heatmap of gene product localization to cellular components.
Fig 2.
Effects of RM oil, 1,8-cineole, and α-pinene on C. albicans RSY150 cell separation, chitin production and surface remodelling.
(a) Representative epifluorescence images of C. albicans with our without exposure to oils for 4h, followed by calcofluor white staining. Cells in media served as a negative control. Scale bar for the control is 5 μm and represents the scale for all images. (b) Bar graphs of total fluorescence intensity. AFM QI™ data for control and RM oil treated C. albicans at (c, left) low (10 μm, 128 × 128 pixel) (c, right) and high (1 μm, 128 × 128 pixel) resolution was used to generate representative topographic images of the cell surface (boxed area images), revealing no change in surface roughness (visually and quantitatively), but the data indicate a significant increase in (d) adhesion and (e) elasticity for treated cells. Data are presented as the mean ± SEM of three biological replicates, with (b) 300 representative cells per replicate, or (d and e) 17 cells per replicate, for which statistical significance (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05) was analysed by an unpaired Student’s t-test of treated compared to control.
Fig 3.
Effects of RM oil, 1,8-cineole and α-pinene on C. albicans RSY150 cell membrane polarization and on C. albicans RBY1132 cell membrane integrity.
(a) Bar graphs show a significant (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05) increase in C. albicans RSY150 membrane depolarization with exposure to EO(C)s at MIC, 1/2 MIC and the two components at FICI, compared to the controls. Pearson correlation indicates a positive association (r = 0.96–0.99, p < 0.001–0.01) between the oil concentration (1/16, 1/8, 1/4, 1/2 MIC and MIC) and membrane depolarization. (b) LSCM images show increased dye content in treated cells compared to control. Scale bar for control is 2 μm and represents that for all images. (c) Bar graphs show significantly (***, p < 0.001; **, p < 0.01 *, p < 0.05) higher PI uptake in oil treated C. albicans RBY1132 compared to control, with 100 cells per replicate. (d) Representative merged (bright-field/fluorescence) (left) and fluorescence (right) images (λex = 493 nm; λem = 636 nm) of treated Candida show PI uptake compared to controls. Scale bars are 5 μm, applicable to all images. Data in bar graphs are presented as the mean ± SEM of three biological replicates, as evaluated by (a) a one-way ANOVA, followed by Dunnett’s multiple comparison of each condition versus control or (c) by an unpaired Student’s t-test.
Fig 4.
Impact of RM oil and its components on C. albicans organelles.
(a) RM oil, 1,8-cineole, and α-pinene at MIC, and the two components at FICI, induced vacuolar fragmentation (pink arrows) as compared to control (orange arrow). The positive control, Amp B, at MIC showed partial (yellow arrow) segregation compared to EO(C)-treated cells. Scale bar for control is 2 μm and represents the scale for all images. (b) Bar graphs show significant differences in % segregated vacuoles for control versus treated cells. (c) Bar graphs show C. albicans exposed to EO(C)s have a greater number of damaged mitochondria compared to control. (d) Representative bright field (left) and fluorescence (right; λex = 644 nm; λem = 665 nm) images of Candida exposed to RM oil, 1,8-cineole, α-pinene at 1/2 MIC and MIC, and the two components at 1/2 and full FICI showed poor uptake of the Mitotracker deep red as compared to control. Scale bars of control images are 5 μm and applicable to all images. Data are presented as the mean ± SEM of three biological replicates, with 300 and 100 cells per replicate for vacuole and mitochondria, respectively, for which statistical significance (***, p < 0.001; **, p < 0.01) was analysed by an unpaired Student’s t-test.
Fig 5.
Impact of RM oil and its components on C. albicans intracellular ROS accumulation.
Bar graphs of intracellular ROS in untreated C. albicans RBY1132, and those treated with RM oil, 1,8-cineole, α-pinene and H2O2 (25 mM). Fluorescence intensity was measured in a plate reader (λex = 485 nm; λem = 528 nm, gain 35) and data are presented as the mean ± SEM of three biological replicates for which statistical significance (****, p < 0.0001; *, p < 0.05) was evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison of each condition versus control.
Fig 6.
Effects of RM oil, 1,8-cineole, and α-pinene on C. albicans RSY150 microtubule formation and cell cycle phases.
(a) The majority of cells (90–95%) treated with RM oil and its components at 1/2 MIC and MIC show diffuse tubulin (purple arrows), compared to untreated controls with normal spindle formation during mitosis (long MTs (white arrows) and short (blue arrows) MTs). Cells exposed to the positive control, nocodazole at 1/2 MIC and MIC had concentrated spots of green fluorescence (yellow arrow) similar to EO(C)-treated cells at 1/4 MIC. Images were collected by LSCM (Tub2-GFP λex = 488 nm; λem = 512 nm and Htb-RFP λex = 543 nm; λem = 605 nm). Scale bar for control is 2 μm and represents the scale for all images. (b‒d) Bar graphs of β-tubulin arrangement (long MT, short MT, diffuse). (e) Cell cycle (S/G1, G2, or M phase) frequency (%) following exposure to RM oil, 1,8-cineole, α-pinene at 1/2 MIC and MIC and the two components at FICI show arrest at the G1/S phase. Data are presented as the mean ± SEM of three biological replicates, with 250 cells per replicate, for which statistical significance ((****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05), was evaluated by (b‒d) a one-way ANOVA, followed by Dunnett’s multiple comparison of each condition versus control for or (e) an unpaired Student’s t-test.
Fig 7.
RM oil and its components impact C. albicans RSY150 germ tube, mycelium formation and preformed biofilm.
(a) Representative fluorescence microscopy (λex = 365 nm; λem = 435 nm) images show that C. albicans treated with 10% FBS in YPD medium containing either RM oil, 1,8-cineole or α-pinene at 1/2 MIC or MIC, or the latter at their FICI (4 h exposure) form pseudohyphae (yellow arrows), while control cells mostly produced germ tubes (white arrows). Scale bar for the control is 10 μm and applies to all images. (b) Bar graphs show all EO(C)s inhibit the yeast to hyphal transition in C. albicans. (c) Bar diagram of the MTT assay shows a dose dependent (r = 0.95–0.99, p < 0.001 for all conditions, FICI: r = 0.86, p < 0.05) biofilm reduction after treatment with RM oil, 1,8-cineole, and α-pinene. Data are presented as the mean ± SEM of three biological replicates, with 100 cells per replicate, for which statistical significance (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05) was analysed by (b) an unpaired Student’s t-test and (c) a one-way ANOVA, followed by Dunnett’s multiple comparison of each condition versus control. (d) Representative stereoscopic bright-field images of control and treated RSY150 on spider media. Bar for control is 250 μm, and represents the scale for all images. (e) Results are summarized in tabular format for which ++++, +++, ++ and + indicate the relative amount of mycelial growth.
Table 1.
Summary of mutant strain sensitivity.
Fig 8.
Cartoon representation of potential C. albicans biological pathways and overall impact by RM oil and its major components.
(a) At higher concentrations (1/2 to full MIC), EOCs can pass through the C. albicans cell wall and gain access to (1) the cell membrane, wherein they cause membrane depolarization. (2) Once EOCs pass through the cell membrane they can enter the membrane of other organelles, such as vacuoles, mitochondria and the nucleus. (3) Mitochondria are the main source of cellular ROS, in which membrane damage would lead to cellular stress and accumulation of ROS, which subsequently (4) causes damage to macromolecules (e.g. MTs) and (5) eventually cell death. (b) At lower fractional MIC, EO(C)s also diffuse through the cell wall and (1) enter through the cell membrane, where they cause (2) membrane defects, proposed to retard major cell wall virulence proteins from passing through and reaching the cell wall surface. This would impact important cell wall virulence proteins, including Als1, Als3, Hwp1, which play an important role in adhesion, biofilm and hyphal formation. (3) Direct impacts of EO(C)s on vacuoles and MTs also play an important role in (4) inhibiting hyphal formation. Abnormal MT morphology can also contribute to the cell separation defects, which lead to pseudohyphal growth. (c) Heat map of major impacts to C. albicans RSY150 treated with RM oil and its components, 1,8-cineole and α–pinene. Colour scale indicates fold change in parameter with EO(C) exposure. The major C. albicans impacts associated with RM oil are attributable to 1,8-cineole, which accounts 53% of the essential oil.