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.
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.
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 β-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.
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 G
/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.
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.
, 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.
With the prevalence of systemic fungal infections caused by
and non-
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
and
. 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
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.
is a robust, easily adaptable and culturable bacterium
, 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
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
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.
Cinnamon (
bark extract exhibits potent inhibitory activity against
but the antifungal mechanisms of this essential oil remain largely unexplored.
We analyzed the impact of cinnamon bark oil on
...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
treated with cinnamaldehyde at MIC (112 μg/mL).
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.
Candida albicans is one of the most common fungi associated with lifethreateninginfections. Treatment of the associated infections is often ineffective inthe light of resistance, and so there is an ...urgent need to discover novel antifungals.Rather than killing the fungal cells, which requires quite high specificity and canlead to the emergence of further resistance, inhibiting growth and virulence factorsin fungal cells represents a good alternative for the development of new antifungaldrugs. Recently, there has been a resurged interest in essential oils and theiractive components, in relation to their pharmacological properties. The primaryobjective of this research was to evaluate the antifungal activity of cinnamon barkand rosemary essential oils, along with its major components cinnamadehyde, and1,8-cineole, α-pinene, respectively, and two other common essential oilcomponents, namely eugenol and citral, against C. albicans. Despite many reportson the antimicrobial activities of essential oils, the results have been diverse andthe evaluation methods inconsistent.At higher essential oil concentrations, reactive oxygen species weregenerated, impacting a wide range of processes including cell membranedepolarization, vacuolar segregation, mitochondrial dysfunction, cell-cyclecheckpoint deficiency, and mitotic catastrophe, which resulted in C. albicans death.At lower fractional MICs, essential oils had a ROS-independent response thatinhibited mycelial growth and biofilm formation, which may be attributable todefects at the cell membrane. However, microtubule inhibition also plays a role inlimiting hyphal growth. I explored the microtubule defects and how they relate toKar3p, a member of the kinesin-14 family shown to be linked to microtubulestability. Interestingly, both tubulin and Kar3 protein was delocalized with essentialoil components exposure at levels for which endogenous ROS levels were normal.I further demonstrate that the level of Kar3 is associated with resistance andsusceptibility of C. albicans to essential oil components in both liquid and on solidgrowth media. All mutant strains grown on hyphae-inducing media in the presenceof essential oil components exhibited an enhanced tendency to form pseudohyphalcells, a common phenotype for the homozygous and heterozygous deletionstrains. Towards these ends, microtubule defects were linked with theoreticalbinding between essential oil components and α-tubulin and Kar3p adjacent tocofactor binding sites, consistent with experimentally observed hyphal defects andbiofilm inhibition. This study for the first time uncovers a new mode of essential oilmediatedmicrotubule defects, which does not follow the known mechanisms ofconventional microtubule inhibition. The essential oil components appear to impactC. albicans eventually giving rise to psuedohyphal formation, microtubule loss,hyphal and biofilm reduction.Since those essential oil components impact multiple Candida targets, theyshould be less susceptible to resistance. Further, many components showed antivirulence,and may represent an effective approach for inhibiting Candida,especially in the context of physically preventing Candida from entering its hostthrough catheters and prosthetics.
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