Cellular energy status is indispensable for perillyl alcohol mediated abrogated membrane transport in Candida albicans

The prevalence of fungal infections and their resistance patterns in fungal isolates from large number of patients with impaired immunity still remains poorly monitored. In spite of significant advances being made in the improvement of antifungal drugs, only a limited number of antifungal drugs are currently available. The present study aimed to gain further mechanistic insights into the previously described anticandidal activity of natural monoterpenoid, perillyl alcohol (PA). We found that cellular transport across cell membrane was abrogated in presence of PA. This was demonstrated by dose and time dependent enhanced cellular leakage accompanied by inhibited sodium and potassium cellular transport. In addition, we found disrupted pH homeostasis which was depicted by enhanced extracellular pH. We further observed that mitochondrial energy status is highly integrated with the antifungal activity of PA. This was evident from inhibited propidium iodide (PI) uptake in presence of sodium azide and di-nitro phenol (DNP) which showed no fluorescence when treated with PA. Moreover, we observed that PA leads to disrupted mitochondrial membrane potential. Additional cell death hallmarks in response to PA such as nuclear fragmentation was also observed with 4\u27,6-diamidino-2-phenylindole (DAPI) staining. Taken together, PA is a novel candidate that deserves further attention to be exploited as effective antifungal agent of pharmacological interest

Sesamol: A Natural Phenolic Compound with Promising Anticandidal Potential

We investigated the antifungal effects of sesamol (Ses), a natural phenolic compound, and exemplified that it could be mediated through disruption of calcineurin signaling pathway in C. albicans, a human fungal pathogen. The repertoire of antifungal activity not only was limited to C. albicans and its six clinical isolates tested but also was against non-albicans species of Candida. Interestingly, the antifungal effect of Ses affects neither the MDR efflux transporter activity nor passive diffusion of drug. We found that C. albicans treated with Ses copies the phenotype displayed by cells having defect in calcineurin signaling leading to sensitivity against alkaline pH, ionic, membrane, salinity, endoplasmic reticulum, and serum stresses but remained resistant to thermal stress. Furthermore, the ergosterol levels were significantly decreased by 63% confirming membrane perturbations in response to Ses as also visualized through transmission electron micrographs. Despite the fact that Ses treatment mimics the phenotype of compromised calcineurin signaling, it was independent of cell wall integrity pathway as revealed by spot assays and the scanning electron micrographs. Taken together, the data procured from this study clearly ascertains that Ses is an effectual antifungal agent that could be competently employed in treating Candida infections

Susceptibility assays to reveal functional indispensability of calcineurin against PA in <i>C</i>. <i>albicans</i>.

<p><b>(a)</b> Spot assay with and without PA (175 μg ml^-1) as controls. <b>(b)</b> Spot assay with PA at alkaline pH10. <b>(c)</b> Spot assay with PA in serum (50% w/v). <b>(d)</b> Spot assay with PA under ER stress by DTT (20mM). <b>(e)</b> Spot assays with PA under various ionic stress conditions with LiCl (0.4M), CaCl₂ (0.3M), NaCl (1M). <b>(f)</b> Spot assay with PA in presence of membrane perturbing agent SDS (0.02% w/v). <b>(g)</b> Spot assay with PA at elevated temperatures of 37°C and 42°C. <b>(h)</b> Spot assay depicting loss of growth in <i>Δcnb1</i> mutant in the presence of PA (225μg ml ^-1) while the <i>Δcrz1</i> mutant and CNB1-1/CNB1 (calcineurin strain with constitutively expressed hyperactive allele of <i>CNB1</i>) were efficiently growing in presence of PA.</p

List of strains used.

<p>List of strains used.</p

Anticandidal Effect and Mechanisms of Monoterpenoid, Perillyl Alcohol against <i>Candida albicans</i> - Fig 2

<p><b>(a) Transcriptional profiling of <i>C</i>. <i>albicans</i> in response to PA.</b> Pie chart showing various gene categories differentially regulated in PA treated cells. Green color show downregulated genes and red color shows upregulated genes. <b>(b) RT-PCR of differentially regulated genes in response to PA.</b> The left panels show transcript levels of <i>CNB1</i>, <i>VCX1</i>, <i>NPC2</i>, <i>KRE62</i>, <i>SKO1</i>, <i>GLN3</i>, <i>TPK1</i>, <i>RFX2</i>, <i>HWP1</i>, <i>DOT5</i>, <i>RAD57</i>, <i>CSM3</i>, <i>SPC98</i>, <i>CLB4</i> in lanes (1) Control, (2) PA (175μg ml^-1). The right panel shows the quantitation (density expressed as Intensity/mm²) of the respective transcripts normalized with constitutively expressed <i>ACT1</i> transcripts.</p

Effect of PA on mitochondria activity.

<p><b>(a)</b> Spot assays in absence and presence of PA (175 μg ml^-1) in YEPD (fermentable carbon source) and YPG media (non-fermentable carbon source). <b>(b)</b> Effect of PA (175μg ml^-1) on mitochondrial activity of <i>C</i>. <i>albicans</i> depicted as bar graph and quantified by using MTT assay as described in material and methods. Mean of O.D₅₇₀ nm ± SD of three independent sets of experiments is depicted on Y-axis and * depicts P value <0.05.</p

Effect of PA on DNA repair and cell cycle.

<p>(a) Spot assay in absence (control) and presence of PA (175μg ml^-1) depicting loss of growth with DNA damaging agent EtBr (50μg ml^-1). (b) Cell cycle analysis of control and PA (175μg ml^-1) treated <i>C</i>. <i>albicans</i> by Flow Cytometry.</p

Effect of PA on cell membrane of <i>C</i>. <i>albicans</i>.

<p><b>(a)</b> Spot assay with FLC (1μg ml^-1), PA (175μg ml^-1) and in combination of both. <b>(b)</b> Left panel shows UV spectrophotometric ergosterol profiles of <i>C</i>. <i>albicans</i> scanned between 220 and 300 nm from overnight culture grown in absence (control) and presence of PA (175 μg ml^-1). Right panel shows relative percentages of ergosterol content in the absence (control) and presence of PA (175 μg ml^-1). Mean of % ergosterol levels is calculated as described in materials and methods normalized by considering the untreated control as 100 ± SD of three independent sets of experiments is depicted on Y-axis and * depicts <i>P</i> value <0.05. <b>(c)</b> Intracellular pH (pHi) in presence of PA (MIC₈₀) in <i>C</i>. <i>albicans</i> cells. Mean of pHi ± SD of three independent sets of experiments is depicted on Y-axis with respect to control & PA on X- axis and * depicts <i>P</i> value <0.05.</p

Effect of PA on the cell wall integrity of <i>C</i>. <i>albicans</i>.

<p><b>(a)</b> Susceptibility assay showing hypersensitivity to PA (175 μg ml^-1) in the presence of cell wall perturbing agents; CFW (10 μg ml^-1) and CR (10 μg ml^-1). <b>(b)</b> SEM images showing the smooth surface of untreated cell (control) and the crinkled cell wall with the leakage of its cell contents (marked with an arrow) because of the extensive damage caused due to PA. <b>(c)</b> Relative sedimentation of <i>C</i>. <i>albicans</i> cells. Left panel shows O.D₆₀₀ of untreated (control) and PA treated (175 μg/mL) cells depicted on <i>y</i>-axis with respect to time (minutes) on <i>x</i>-axis. Right panel shows sedimentation rates per min on y-axis for control and PA on <i>x</i>-axis, calculated by estimating the difference in OD₆₀₀ from 0 till 30 min per unit interval.</p