10 research outputs found
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<sup>-1</sup>) 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), CaCl<sub>2</sub> (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 <sup>-1</sup>) 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
Drug susceptibility assays against <i>C</i>. <i>albicans</i> and non-<i>albicans</i> species of <i>Candida</i> in the presence of PA.
<p><b>(a)</b> Broth microdilution assay to determine the MIC<sub>80</sub> of <i>C</i>. <i>albicans</i> reference strain SC5314, clinical isolates (D1, D2, D4, D7, D18 and D20) and <i>C</i>. <i>glabrata</i>, <i>C</i>. <i>krusei</i>, <i>C</i>. <i>parapsilosis</i> and <i>C</i>. <i>tropicalis</i> in presence of PA. Data was quantitatively displayed with color (see color bar), where each shade of color represents relative optical densities of the cell. The minimum drug concentration that inhibits growth by 80% relative to the drug-free growth control is indicated as MIC<sub>80</sub> for each strain. <b>(b)</b> Spot assay of <i>C</i>. <i>albicans</i> reference strain SC5314, clinical isolates (D1, D2, D4, D7, D18 and D20) and <i>C</i>. <i>glabrata</i>, <i>C</i>. <i>krusei</i>, <i>C</i>. <i>parapsilosis</i> and <i>C</i>. <i>tropicalis</i> in the absence (control) and presence of PA.</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<sup>-1</sup>). The right panel shows the quantitation (density expressed as Intensity/mm<sup>2</sup>) 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<sup>-1</sup>) in YEPD (fermentable carbon source) and YPG media (non-fermentable carbon source). <b>(b)</b> Effect of PA (175μg ml<sup>-1</sup>) 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.D<sub>570</sub> nm ± SD of three independent sets of experiments is depicted on Y-axis and * depicts P 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<sup>-1</sup>) in the presence of cell wall perturbing agents; CFW (10 μg ml<sup>-1</sup>) and CR (10 μg ml<sup>-1</sup>). <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.D<sub>600</sub> 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 OD<sub>600</sub> from 0 till 30 min per unit interval.</p
Anticandidal Effect and Mechanisms of Monoterpenoid, Perillyl Alcohol against <i>Candida albicans</i>
<div><p>This study explored the antifungal potential of perillyl alcohol (PA), a natural monoterpene alcohol, against most prevalent human fungal pathogen <i>C</i>. <i>albicans</i>, its clinical isolates and four non<i>-albicans</i> species of <i>Candida</i>. To resolve the potential mechanisms, we used whole genome transcriptome analyses of PA treated <i>Candida</i> cells to examine the affected cellular circuitry of this pathogen. The transcriptome data revealed a link between calcineurin signaling and PA as among the several categories of PA responsive genes the down regulation of calcineurin signaling gene <i>CNB1</i> was noteworthy which was also confirmed by both molecular docking and susceptibility assays. We observed that PA treated <i>Candida</i> phenocopied compromised calcineurin pathway stress responses and turned sensitive to alkaline pH, ionic, membrane, salinity, endoplasmic reticulum and serum stresses. Indispensability of functional calcineurin was further confirmed as calcineurin mutant was hypersensitive to PA while constitutively expressed calcineurin strain remained resistant. We explored that PA leads to perturbed membrane integrity as depicted through depleted ergosterol levels and disrupted pH homeostasis. Moreover, PA caused cell wall damage which was evident from hypersensitivity against cell wall perturbing agents (congo red, calcoflour white), SEM and enhanced rate of cell sedimentation. Furthermore, PA inhibited potential virulence traits including morphological transition, biofilm formation and displayed diminished capacity to adhere both to the polystyrene surface and buccal epithelial cells. The study also revealed that PA leads to cell cycle arrest and mitochondrial dysfunction in <i>C</i>. <i>albicans</i>. Together, the present study provides enough evidence for further work on PA so that better strategies could be employed to treat <i>Candida</i> infections.</p></div
Summary of PA mode of action in <i>C</i>. <i>albicans</i>.
<p>PA blocks calcineurin signaling, affects cell surface integrity (cell wall and cell membrane), yeast to hyphal transition, biofilm formation, cell cycle arrest at S phase and mitochondria dysfunction (shown by dark lines). Abrogated membrane homeostasis, cell wall integrity, yeast to hyphal transition could also be the result of compromised calcineurin signaling because of PA (denoted by red dashed lines). All these disrupted mechanisms due to PA are necessary to sustain drug tolerance and virulence in <i>C</i>. <i>albicans</i>.</p
Effect of PA on DNA repair and cell cycle.
<p>(a) Spot assay in absence (control) and presence of PA (175μg ml<sup>-1</sup>) depicting loss of growth with DNA damaging agent EtBr (50μg ml<sup>-1</sup>). (b) Cell cycle analysis of control and PA (175μg ml<sup>-1</sup>) 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<sup>-1</sup>), PA (175μg ml<sup>-1</sup>) 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<sup>-1</sup>). Right panel shows relative percentages of ergosterol content in the absence (control) and presence of PA (175 μg ml<sup>-1</sup>). 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 (MIC<sub>80</sub>) 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