Fig 1.
(a) Bar graph depicting the revival of untreated candidal cells (-Ger) and non revival of Ger treated (MIC80) cells in normal YPD media confirming the fungicidal nature of Ger. (b) Quadrant streaking on YPD plates supplemented with and without Ger depicting the revival of untreated candidal cells (-Ger) and non revival of Ger treated (MIC80) cells in YPD agar media reconfirming the fungicidal nature of Ger.
Fig 2.
R6G and NR intracellular accumulation.
(a) Left panel showing the fluorescence images of R6G stained AD1-8u-, AD-CDR1, AD-MDR1 strains in presence of Ger (135μg/ml). Right panel shows the bar graph of the fluorescence intensity measured by Image J software. (b) Left panel showing the fluorescence images of NR stained AD1-8u-, AD-CDR1, AD-MDR1 strains in presence of Ger (135μg/ml). Right panel shows the bar graph of the fluorescence intensity measured by Image J software.
Fig 3.
(a) Extracellular R6G concentrations in control cells (AD1-8u-), cells overexpressing CaCdr1p (AD-CDR1) and Wild-type strain SC5314 of C. albicans. The energy dependent R6G efflux was initiated by adding glucose (2%; indicated by an arrow) and quantified by measuring the absorbance of the supernatant at 527 nm. The values are the means and standard deviations (indicated by error bars) from three independent experiments. (b) Spot assays of AD1-8u- and AD-CDR1 in presence and absence of Ger, R6G and FLC. (c) Lineweaver-Burk plot of CaCdr1p-mediated R6G efflux in the presence of Ger. The x axis (1/S) represents the various concentrations (μM) of R6G used, and the y axis (1/V) shows the rate of release of R6G in the presence of Ger (135μg/ml). The rate of each reaction was calculated as nanomoles of R6G released/minute/5X106 cells.
Fig 4.
3D structure prediction and validation by I-TASSER.
(a) Three dimensional structure of C. albicans CaCdr1p predicted by I-TASSER. Alignment of query protein (pink) with structural analog (cyan) 5do7A in PDB library (right panel). (b) Validation of top score model by PROCHECK Ramachandran plot (left panel), MolProbity Ramachandran plot (right panel).
Fig 5.
Molecular docking of Ger with CaCdr1p.
(a) Cartoon model of Cdr1p protein with Ger (left panel), surface view of CaCdr1p with Ger (middle panel) and 2D schematic diagram showing interactions of Ger to the CaCdr1p. Residues involved in hydrogen bonding, Vander Waals interactions, Pi-sigma and Pi-alkyl are represented in different color indicated in inset (right panel). (b) Cartoon model of CaCdr1p with Farnesol (known inhibitor) (left panel), surface view of CaCdr1p with Farnesol (middle panel) and 2D schematic diagram showing interactions of Farnesol to the CaCdr1p. Residues involved in hydrogen bonding, Vander Waals interactions, Pi-sigma and Pi-alkyl are represented in different color indicated in inset (right panel).
Table 1.
Binding energy and specific interaction of CaCdr1p with Ger.
Fig 6.
Expression and localization of CaCdr1p in presence of Ger.
(a) RT-PCR of CDR1 in response to Ger. The left panel shows transcript levels of the gene in lanes control and Ger treated cells. The right panel shows the quantitation (density expressed as Intensity/mm2) of the respective transcript normalized with constitutively expressed ACT1 transcript. (b) Western blot analysis showing the CaCdr1p-GFP protein levels in presence of Ger (sub inhibitory concentration) and developed by anti-GFP antibody. (c) Confocal microscopy images showing the membrane mislocalization in the AD-CDR1-GFP tagged strain in presence of Ger.
Fig 7.
Synergistic effect of Ger with known antifungal drugs.
(a) Checkerboard assay showing the synergism of Ger with the FLC and FICI was calculated (FICI < 0.5). (b) Disk diffusion assay using the AD1-8u-, AD-CDR1 and wild type SC5314 by growing them in presence of Ger and FLC along with the combinations. (c) Ger enhances the antifungal efficiency of current therapeutic drugs Amp B and CAS.
Fig 8.
Effect of Ger on FLC resistant clinical isolates.
(a) Spot assay of Gu4/Gu5 and F2/F5 in presence of Ger (135 μg/mL) and FLC. (b) R6G extracellular efflux showing the extracellular R6G concentrations in overexpressing FLC resistant and sensitive match pairs Gu4/ Gu5. The energy dependent R6G efflux was initiated by adding glucose (2%; indicated by an arrow) and quantified by measuring the absorbance of the supernatant at 527 nm. The values are the mean and standard deviations (indicated by error bars) from three independent experiments. (c) UV spectrophotometric ergosterol profiles of Gu4/Gu5 strains scanned between 220 and 300 nm in presence of Ger (135 μg/mL) as depicted in left panel. The relative percentage of ergosterol content in the presence of Ger (135μg/mL) is presented in right panel.
Fig 9.
Effect of Ger on mitochondria.
(a) Fluorescent microscopy images showing Rhodamine B probe for monitoring mitochondrial membrane potential (mtΨm) in the presence of Ger. Scale is 20μm. (b) Spot assay demonstrating no growth defect of C. albicans lacking RTG3 in presence of Ger (135 μg/mL).
Fig 10.
Effect of Ger on virulence markers.
(a) Extracellular Phospholipase activity and calculation of the precipitation zone represented as the ratio of the diameter of the colony to the cloudy zone plus colony diameter. P <0.05 compared to the control. C. albicans (90028) with Ger (at SubMIC & MIC values), FLC (1 mg/mL) along with the combinations of Ger and FLC. All data are the averages of triplicate experiments. (b) Effect of Ger on cell adherence. The microscopic images have shown the adherence of Candida to human buccal epithelial cells in presence of Ger (135 μg/mL). The control cells shows adherence of cells to epithelial cells (depicted by arrow) and Ger treated panel shows non adherence of candidal cells to epithelial cells. (c) Effect of Ger on biofilm biomass formed on silicone sheets. Mean of dry weight ± SD of three independent sets of experiments are depicted on Y-axis and * depicts P < 0.05. Inset depicts fluorescence microscopy images of CFW stained biofilms.
Fig 11.
In-vivo studies of Ger on nematode model C. elegans.
(a) Toxicity test of Ger on C. elegans depicted by Kaplan–Meier curve showing % survival of C. elegans in the presence of Ger at sub MIC concentration. Microscopic images (magnification 4x) of nematodes in presence of Ger. Worm survival was determined based on movement (upper panel).The toxicity of Ger was studied on nematodes by determining survival rates after 3 days (lower panel). (b) Ger prolongs the survival of C. albicans infected C. elegans. Microscopic images showing the survival of infected C. elegans when treated with Ger on day 1, day2 and day 3. Kaplan–Meier curve showing % survival of C. albicans infected C. elegans in the presence of Ger (lower panel). (c) Intestinal persistence of C. albicans. Upper panel shows the Act1p-GFP C. albicans visualization confirming lower fungal burden in Ger treated worms contrary to untreated nematodes. Lower panel represents the CFW stained C. albicans visualized in proximal and distal intestine of C. elegans treated with Ger showing lesser fungal burden. (d). Hemolytic activity of Ger depicted as percentage on Y axis in comparison to the positive control Triton X 100.
Fig 12.
Schematic proposal of the multi-target activity of Ger.