Carvacrol-Induced Vacuole Dysfunction and Morphological Consequences in Nakaseomyces glabratus and Candida albicans.

With the prevalence of systemic fungal infections caused by Candida albicans and non-albicans species and their resistance to classical antifungals, there is an urgent need to explore alternatives. Herein, we evaluate the impact of the monoterpene carvacrol, a major component of oregano and thyme oils, on clinical and laboratory strains of C. albicans and Nakaseomyces glabratus. Carvacrol induces a wide range of antifungal effects, including the inhibition of growth and hyphal and biofilm formation. Using biochemical and microscopic approaches, we elucidate carvacrol-induced hyphal inhibition. The significantly reduced survival rates following exposure to carvacrol were accompanied by dose-dependent vacuolar acidification, disrupted membrane integrity, and aberrant morphology. Germ tube assays, used to elucidate the relationship between vacuolar dysfunction and hyphal inhibition, showed that carvacrol significantly reduced hyphal formation, which was accompanied by a defective C. albicans morphology. Thus, we show a link between vacuolar acidification/disrupted vacuole membrane integrity and compromised candidal morphology/morphogenesis, demonstrating that carvacrol exerts its anti-hyphal activity by altering vacuole integrity.

A Simple and Reproducible Stereomicroscopic Method to Assess Fungal Biofilms: Application to Antifungal Susceptibility Testing.

Candida albicans, a well-known opportunistic pathogen, is a major cause of human fungal infections. Biofilm formation is considered an important pathogenesis factor. Biofilms are less sensitive to antibiotics and immune responses, allowing them to colonize and persist in host niches. Biofilm screening is important in the identification of anti-biofilm drugs. However, developing nations, with limited financial resources, often do not have access to advanced scientific equipment. Here, we describe an in vitro, protocol using common materials and simple equipment to evaluate static microbial biofilms.

Key essential oil components delocalize Candida albicans Kar3p and impact microtubule structure.

BACKGROUND: Treatment of Candida albicans associated infections is often ineffective in the light of resistance, with an urgent need to discover novel antimicrobials. Fungicides require high specificity and can contribute to antifungal resistance, so inhibition of fungal virulence factors is a good strategy for developing new antifungals. OBJECTIVES: Examine the impact of four plant-derived essential oil components (1,8-cineole, α-pinene, eugenol, and citral) on C. albicans microtubules, kinesin motor protein Kar3 and morphology. METHODS: Microdilution assays were used to determine minimal inhibitory concentrations, microbiological assays assessed germ tube, hyphal and biofilm formation, confocal microscopy probed morphological changes and localization of tubulin and Kar3p, and computational modelling was used to examine the theoretical binding of essential oil components to tubulin and Kar3p. RESULTS: We show for the first time that essential oil components delocalize the Kar3p, ablate microtubules, and induce psuedohyphal formation with reduced biofilm formation. Single and double deletion mutants of kar3 were resistant to 1,8-cineole, sensitive to α-pinene and eugenol, but unimpacted by citral. Strains with homozygous and heterozygous Kar3p disruption had a gene-dosage effect for all essential oil components, resulting in enhanced resistance or susceptibility patterns that were identical to that of cik1 mutants. The link between microtubule (αß-tubulin) and Kar3p defects was further supported by computational modeling, showing preferential binding to αß-tubulin and Kar3p adjacent to their Mg2+-binding sites. CONCLUSION: This study highlights how essential oil components interfere with the localization of the kinesin motor protein complex Kar3/Cik1 and disrupt microtubules, leading to their destabilization which results in hyphal and biofilm defects.

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

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.

Candida albicans is part of the normal human flora but is most frequently isolated as the causative opportunistic pathogen of candidiasis. Plant-based essential oils and their components have been extensively studied as antimicrobials, but their antimicrobial impacts are poorly understood. Phenylpropenoids and monoterpenes, for example, eugenol from clove and citral from lemon grass, are potent antifungals against a wide range of pathogens. We report the cellular response of C. albicans to eugenol and citral, alone and combined, using biochemical and microscopic assays. The MICs of eugenol and citral were 1,000 and 256 µg/mL, respectively, with the two exhibiting additive effects based on a fractional inhibitory concentration index of 0.83 ± 0.14. High concentrations of eugenol caused membrane damage, oxidative stress, vacuole segregation, microtubule dysfunction and cell cycle arrest at the G1/S phase, and while citral had similar impacts, they were reactive oxygen species (ROS) independent. At sublethal concentrations (1/2 to 1/4 MIC), both oils disrupted microtubules and hyphal and biofilm formation in an ROS-independent manner. While both compounds disrupt the cell membrane, eugenol had a greater impact on membrane dysfunction. This study shows that eugenol and citral can induce vacuole and microtubule dysfunction, along with the inhibition of hyphal and biofilm formation. IMPORTANCE Candida albicans is a normal resident on and in the human body that can cause relatively benign infections. However, when our immune system is severely compromised (e.g., cancer chemotherapy patients) or underdeveloped (e.g., newborns), this fungus can become a deadly pathogen, infecting the bloodstream and organs. Since there are only a few effective antifungal agents that can be used to combat fungal infections, these fungi have been exposed to them over and over again, allowing the fungi to develop resistance. Instead of developing antifungal agents that kill the fungi, some of which have undesirable side effects on the human host, researchers have proposed to target the fungal traits that make the fungus more virulent. Here, we show how two components of plant-based essential oils, eugenol and citral, are effective inhibitors of C. albicans virulence traits.

Cinnamon Leaf and Clove Essential Oils Are Potent Inhibitors of Candida albicans Virulence Traits.

Plant-based essential oils are promising anti-virulence agents against the multidrug-resistant opportunistic pathogen Candida albicans. Gas chromatography-mass spectrometry of Cinnamomum zeylanicum (cinnamon) leaf and Eugenia caryophyllus (clove) flower bud essential oils revealed eugenol (73 and 75%, respectively) as their major component, with ß-caryophyllene, eugenyl acetate, and α-humulene as common minor components. Cinnamon leaf and clove essential oils had minimum inhibitory concentrations of 600 and 500 µg/mL, respectively against the C. albicans RSY150 reference strain and 1000 and 750 µg/mL, respectively for the clinical reference strain ATCC 10231. The combined oils are additive (FICI = 0.72 ± 0.16) and synergistic (0.5 ± 0.0) against RSY150 and the clinical reference strain, respectively. Mycelial growth was inhibited by sublethal concentrations of either essential oil, which abolished colony growth. At half of the lowest combined lethal concentration for the two oils, the yeast-to-hyphal transition and mycelial growth was potently inhibited. Mutant strains als1Δ/Δ, als3Δ/Δ, hwp1Δ/HWP1+, and efg1Δ/Δ were sensitive to either or both oils, especially efg1Δ/Δ. In conclusion, oils of cinnamon leaf and clove and their combination significantly impact C. albicans virulence by inhibiting hyphal and mycelial growth.

Identification of bioactive metabolites and evaluation of in vitro anti-inflammatory and in vivo antinociceptive and antiarthritic activities of endophyte fungi isolated from Elaeocarpus floribundus blume.

ETHNOPHARMACOLOGICAL RELEVANCE: Functional disability associated with rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease is a challenging concern in healthcare systems. Along with environmental factors and epigenetic disorders, multiple pathways are reported as prominent mechanism for the progression of RA symptoms including; pain, swelling and stiffness of joints. Elaeocarpus floribundus Blume has been used as a folklore medicine for RA from ancient times. This plant harbours a suite of endophytic fungi that produce a range of metabolites of potential interest. Thus, for the establishment of a scientific basis for this folklore use, it is essential to find out the involvement, if any, of the endophytic fungi living in this plant and the metabolites they elaborate, for the management of RA. AIM OF THE STUDY: This study was designed to isolate, identify and evaluate the in vitro anti-inflammatory and in vivo antinociceptive and antiarthritic activities of the compounds produced by the endophytic fungi living in different parts of Elaeocarpus floribundus Blume. MATERIALS AND METHODS: Endophytic fungi from different parts of the plant were isolated and cultured for the production of secondary metabolites. Chromatographically fractionated fungal extracts were assessed for anti-inflammatory and antinociceptive activities. For the evaluation of anti-inflammatory activity, in vitro cyclooxygenase (COX1/COX2) and 5-lipoxygenase (5-LOX) inhibitory assays were performed. For the evaluation of in vivo antinociceptive activity, hot plate acetic acid induced writhing, and formalin induced paw licking methods were adopted, whereas complete Freund's adjuvant (CFA) induced poly-arthritic method was adopted for the evaluation of antiarthritic activity. The most effective fraction was analyzed by liquid chromatography-mass spectroscopy (LC-MS) in search of the bioactive extracellular metabolites. RESULTS: Five endophytic fungi viz. Aspergillus fumigatus, Aspergillus niger, Rhizoctonia oryzae, Rhizopus oryzae, and Syncephalastrum racemosum were isolated. COX1/COX2 and 5-LOX inhibitory assays state that the Aspergillus niger fraction possesses the greatest activity against these enzymes of inflammatory process. In vivo antinociceptive showed significant (***P<0.001) reduction of pain in a dose dependent manner. As well, significant (***P<0.001) reduction of paw volume was observed in CFA induce poly-arthritic test. LC/MS analysis of the Aspergillus niger fraction revealed the presence of bioactive compounds including tensyuic acid, hexylitaconic acid, chlorogenic acid, nigragillin, TMC-256C1, asnipyrone B, asperenone, fumaric acid and fusarubin, all having reported pharmacological activities. CONCLUSION: The present study demonstrates that secondary metabolites produced by endophytic fungi living in various parts of Elaeocarpus floribundus Blume had potential to relief pain and inflammation. The endophytes were found to contain multiple biomolecules effective in rheumatoid arthritis. These findings provide a rationale for the folklore use of the plant in the management of rheumatoid arthritis.

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time.

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

Cinnamomum zeylanicum bark essential oil induces cell wall remodelling and spindle defects in Candida albicans.

BACKGROUND: Cinnamon (Cinnamomum zeylanicum) bark extract exhibits potent inhibitory activity against Candida albicans but the antifungal mechanisms of this essential oil remain largely unexplored. RESULTS: We analyzed the impact of cinnamon bark oil on C. albicans RSY150, and clinical strains isolated from patients with candidemia and candidiasis. The viability of RSY150 was significantly compromised in a dose dependent manner when exposed to cinnamon bark oil, with extensive cell surface remodelling at sub inhibitory levels (62.5 µg/mL). Atomic force microscopy revealed cell surface exfoliation, altered ultrastructure and reduced cell wall integrity for both RSY150 and clinical isolates exposed to cinnamon bark oil. Cell wall damage induced by cinnamon bark oil was confirmed by exposure to stressors and the sensitivity of cell wall mutants involved in cell wall organization, biogenesis, and morphogenesis. The essential oil triggered cell cycle arrest by disrupting beta tubulin distribution, which led to mitotic spindle defects, ultimately compromising the cell membrane and allowing leakage of cellular components. The multiple targets of cinnamon bark oil can be attributed to its components, including cinnamaldehyde (74%), and minor components (< 6%) such as linalool (3.9%), cinamyl acetate (3.8%), α-caryophyllene (5.3%) and limonene (2%). Complete inhibition of the mitotic spindle assembly was observed in C. albicans treated with cinnamaldehyde at MIC (112 µg/mL). CONCLUSIONS: Since cinnamaldehyde disrupts both the cell wall and tubulin polymerization, it may serve as an effective antifungal, either by chemical modification to improve its specificity and efficacy or in combination with other antifungal drugs.

Exposure to Sub-lethal 2,4-Dichlorophenoxyacetic Acid Arrests Cell Division and Alters Cell Surface Properties in Escherichia coli.

Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force - laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage.