Efficacy of oregano oils and phenolic derivatives against S. cerevisiae.
A first step in our study was to determine whether the susceptibility of S. cerevisiae
to terpenoid phenols recapitulated the results of published studies in various pathogenic fungi. The strength of oregano oil was analyzed by looking at the dose dependence of halo formation on a lawn of yeast. Oregano oil effectively prevented yeast growth up to a dilution of 1:8 (Fig. ), and no colonies appeared even after prolonged incubation, consistent with potent fungicidal activity. By comparison, another essential oil with antifungal activity, tea tree (Melaleuca
) oil, only worked well at full strength, and small colonies appeared around the filters after 2 weeks (data not shown), suggesting a more fungistatic mechanism of action. As a control, we showed that mineral oil had no effect on yeast growth. In metabolically active cells, the vital stain FUN-1 is converted to a red intravacuolar spindle-like structure, whereas the loss of metabolic activity is associated with bright greenish-yellow fluorescence (19
). In yeast cells treated briefly (15 min) with oregano oil but not in yeast cells treated with mineral oil, FUN-1 green fluorescence was indicative of a loss of metabolic activity (Fig. ).
Next, we evaluated the relative efficacies of purified components of oregano oil and related compounds. The monoterpenoid phenol carvacrol and its structural isomer thymol together constitute 72 to 83% of extracts from Origanum vulgarum
species, although they vary reciprocally as the predominant components in distinct chemotypes (23
). Out of a panel of structurally related phenolic compounds (Fig. , bottom), carvacrol was found to be the most potent in inhibiting yeast growth, with a MIC of 0.008% (or 79.8 μg/ml), which was 1,500 times more effective than oregano oil (Fig. ). Thymol was only slightly less efficacious, whereas eugenol, a major phenolic component of clove oil (Eugenia
sp.), was significantly less effective than carvacrol in the yeast growth assay (Fig. ). In contrast, the monoterpene hydrocarbon γ-terpinene, which is the biosynthetic precursor of carvacrol and also a component of oregano oil, was ineffective as a fungicide in the same concentration range as the terpenoid phenols. Other phenolic compounds, vanillin and guaiacol, were also ineffective as fungicides (Fig. ). These results parallel the work of Tampieri et al. (27
), showing that out of a panel of terpenoid phenols, carvacrol was the most effective in killing C. albicans
at a MIC of 100 ppm. Eugenol was slightly less active (MIC = 250 ppm) than carvacrol, and methyl eugenol was even weaker (MIC = 1,000 ppm). Unlike carvacrol, which has a free hydroxyl group, eugenol has a methylated hydroxyl group, and methyl eugenol has two methyl groups. Similarly, hydroxymethyl derivatives of carvacrol, thymol, and eugenol had significantly lower antifungal activities relative to those of the parent compounds (18
), although interestingly, the derivatives had superior free radical-scavenging activities and protective effects as antioxidants. This suggests that the antifungal activity depends on the structure and makeup of the terpenoid phenols, specifically, the presence of a free hydroxyl group and an aromatic ring.
Carvacrol exerted dose-dependent inhibition on the yeast growth rate, with complete inhibition of growth at 0.01%, as shown by the results in Fig. . Quantification of FUN-1 fluorescence showed that carvacrol elicited dose-dependent loss of metabolic activity in yeast (Fig. ) with more potency than the parent essential oil mixture (not shown), confirming that it was the major active ingredient of oregano oil.
Carvacrol disrupts ion homeostasis in yeast.
We have previously shown that an unrelated membrane-active compound, amiodarone, elicits cytosolic Ca2+
bursts and downstream Ca2+
-related stress responses in yeast (15
). Therefore, we examined the effect of carvacrol on cytosolic Ca2+
levels in yeast expressing the protein aequorin, after reconstitution with its cofactor coelenterazine. The aequorin-coelenterazine complex emits light upon binding to Ca2+
, and the luminescence intensity quantitatively correlates to the Ca2+
concentration. Upon the addition of carvacrol to final concentrations ranging from 0.0125 to 0.05%, we observed immediate dose-dependent Ca2+
elevations, followed by a decrease to baseline within 1 to 2 min (Fig. ). These characteristic spikes have been described before (15
) and are consistent with rapid influx of Ca2+
from the extracellular medium and from the vacuole and other intracellular stores, followed by sequestration into stores or efflux from cells and concomitant desensitization of channels. Similar Ca2+
bursts were observed with the structural isomer thymol, whereas eugenol showed smaller amplitudes of burst, and the remaining compounds tested (vanillin, guaiacol, γ-terpinene, and p
-cymene) failed to elicit any change in luminescence (Fig. ). Overall, the ability of phenolic compounds to elicit Ca2+
bursts correlated well with their antifungal activity (Fig. ).
levels are tightly controlled within a narrow range that is compatible with cellular viability by an array of ion pumps and transporters. To distinguish whether the Ca2+
burst was directly in the pathway leading to cell death or a mere bystander effect, we evaluated carvacrol toxicity in a yeast vma2Δ
mutant lacking a functional vacuolar H+
pump. In the absence of vacuolar acidification, which provides the driving force for H+
exchangers, the clearance of cytosolic Ca2+
is severely impaired (12
). We show that vma2Δ
mutants are clearly more sensitive to growth inhibition by carvacrol than the isogenic wild type, consistent with toxicity of the Ca2+
burst (Fig. ).
Since the vacuolar H+
pump is also critical for pH homeostasis (17
), we monitored the effect of carvacrol on both vacuolar and cytosolic pH. Cells were loaded with the acetoxymethyl derivative of the pH-sensitive fluorescent dye, BCECF, which has been shown to stably accumulate in yeast vacuoles (3
). The addition of carvacrol elicited a 0.5-unit increase in the vacuolar pH and persistent alkalinization (Fig. ), suggesting a loss of protons out of the vacuolar lumen. Concurrently, carvacrol was able to induce immediate acidification of the yeast cytosol (Fig. ), which was monitored by the pH-dependent fluorescence of the green fluorescent protein derivative pHluorin (8
). Acidification was dose dependent: whereas 0.01% carvacrol elicited a modest drop in pH, 0.05% carvacrol resulted in an immediate drop in pH of ~0.5 pH unit, followed by a precipitous decrease beginning around 30 min after exposure to the phenolic compound. This second phase of cytosolic acidification induced by 0.05% carvacrol correlated with a reciprocal increase in vacuolar pH (Fig. ) and was well downstream of the Ca2+
burst (Fig. ). We conclude that carvacrol disrupts both Ca2+
homeostasis in yeast and that these disruptions likely lead to loss of cell viability.
Transcriptional profiling in carvacrol resembles the Ca2+ stress response.
As an independent approach toward elucidating the antifungal mechanism of terpenoid phenols, we analyzed the transcriptional response to carvacrol in yeast. Because disruption of Ca2+ and H+ homeostasis and loss of metabolic activity occur within minutes of carvacrol treatment, we reasoned that 15 min following drug exposure would be the ideal time to capture the transcriptional effect of carvacrol. Exponentially growing cells were treated for 15 min with 0.005% and 0.01% carvacrol. These concentrations were shown to cause about half-maximal and maximal inhibition of growth rates relative to the growth of the control (Fig. ).
As summarized in Table , the number of gene transcripts showing 2-fold or greater upregulation increased from 492 to 800 in 0.005% and 0.01% carvacrol, respectively, with 91 genes showing robust (≥2-fold) dose-dependent increases at higher carvacrol concentrations. A smaller number of gene transcripts were downregulated with exposure to carvacrol (Table ).
Summary of differential regulation of genes in response to carvacrol
The functional categories of genes, according to the MIPS classification, that were differentially regulated by at least 2-fold in response to 0.01% carvacrol are shown in Fig. . The inner circle depicts the functional distribution of the yeast proteome, whereas the outer circle represents categories that were significantly overrepresented in the carvacrol data sets (P
≤ 5 × 10−3
). Prominent among the upregulated genes (Fig. ) were functions associated with alternative metabolic or energy-handling pathways, including glucogen and trehalose biosynthesis, polyamine degradation, and fatty acid transport and oxidation. Next in abundance were pathways associated with stress response/signaling and cell rescue, including sporulation, oxygen and free radical detoxificaton, heat shock proteins and chaperones, autophagy, and vacuolar degradation mechanisms (Fig. and Table ). A robust induction of drug efflux mechanisms was observed, including many members of the ABC drug transporter family, such as SNQ2, YOR1, PDR5, PDR10, and PDR15 (Table ). In contrast to this broad array of cellular functions affected by carvacrol, the pathways represented by repressed (Fig. ) genes were overwhelmingly associated with nucleic acid metabolism and RNA synthesis, processing, and modification. In dividing cells, most of the gene transcription (>80% of nucleotides used) is dedicated to synthesis of ribosomes and tRNA (29
). Carvacrol rapidly shut down these pathways, consistent with cessation of growth (Table and Fig. ).
FIG. 3. Functional distribution of genes differentially regulated by carvacrol. Genes that showed transcriptional alteration by ≥2-fold in response to 0.01% carvacrol were categorized according to the MIPS classification system (see Materials (more ...)
Representative examples of genes that are differentially regulated in response to 0.01% carvacrol
Yeast transcriptional profiles have been documented in response to a wide array of antifungal drugs (5-fluorocytosine, amphotericin B, caspofungin, and ketoconozole), metabolic conditions (diauxic shift, nitrogen depletion, rapamycin treatment, and YPD) and agents known to induce ionic stress (amiodarone and Ca2+
). Clustering analysis of microarray data revealed that the transcriptional response to carvacrol most closely resembled the Ca2+
stress response (Fig. ). We had previously observed that the transcriptional response to the antifungal agent amiodarone also clusters closely with Ca2+
stress, consistent with the ability of amiodarone to evoke bursts of cellular Ca2+
. Here, we show a similarity in overall gene regulation by carvacrol and amiodarone (Fig. ). These observations corroborate our hypothesis that carvacrol elicits Ca2+
-mediated cell death. Interestingly, the carvacrol response also closely resembles the effect of rapamycin, an inhibitor of the TOR signaling pathway that controls cell growth in response to nutrients and stress by regulating mRNA transcription and stability, protein translation, ribosome biogenesis and autophagy, and nutrient transport. To examine this further, we evaluated the overlap in transcriptional profiles in response to carvacrol, rapamycin, and Ca2+
stress (Fig. ). As seen in the Venn depictions, the overall transcriptional response to carvacrol was 58% identical to that in response to rapamycin and 63% identical to that in response to Ca2+
stress. Of the genes upregulated by carvacrol, a third (166) were also induced by the other two conditions and chiefly included genes involved in metabolic pathways of carbohydrate, protein, or energy. An even greater overlap existed among downregulated genes, wherein 60% of genes repressed by carvacrol (257) were common to downregulation by Ca2+
stress and rapamycin. Shared genes belonged largely to categories of RNA metabolism (60.7%) or ribosome biogenesis (55.6%). As these transcriptional responses are characteristic of inhibition of the TOR pathway, these findings raise the intriguing possibility that carvacrol may affect the TOR pathway, either independently or through calcium signaling. A recent screen of >3,500 compounds identified amiodarone as an inhibitor of mTORC1, based on the induction of autophagy in nutrient-rich medium (5
); a similar effect may be predicted with carvacrol, given the robust induction of autophagy genes observed (Table ).
FIG. 4. Carvacrol elicits transcriptional response similar to calcium stress and rapamycin. (A) Hierarchical clustering of transcriptional response to carvacrol (Carv) (15 min [15′]), CaCl2 (Ca; 5 and 30 min), rapamycin (30 min), thymol (90 min), time (more ...)
Recently, Bi et al. (7
) described the transcriptional response of S. cerevisiae
to thymol, the structural isomer of carvacrol. As shown by hierarchical clustering analysis (Fig. ), the transcriptional responses to carvacrol and thymol were similar. As both compounds induce a rapid and robust cytosolic calcium surge (Fig. ), the mechanisms by which these two phenolic isomers kill fungal cells are likely to be substantially similar. We also noted differences in the transcriptional response to the two isomers. Thymol repressed multiple genes implicated in thiamine (vitamin B1) biosynthesis (THI4
, and PET18
) and sulfur metabolism, while this response was not observed after carvacrol treatment. It is possible that these differences represent distinct cellular responses to the two isomers. However, given the different times of assessment of the transcriptional responses to thymol (90 min; 7
) and carvacrol (15 min, this study), it may well be that the induction of sulfur metabolism and the repression of thiamine biosynthesis are both late-stage transcriptional responses to these compounds. Furthermore, the size of the transcriptional response appears to diminish substantially with time, with differential regulation of 922 genes by at least 2-fold after a 15-min exposure to carvacrol (0.005%) (Table ) and 305 genes after a 90-min treatment with thymol (7
) at equivalent drug concentrations. A similar transient transcriptional response has been previously observed for the membrane-active drug amiodarone, which also elicits a rapid transcriptional response peaking at between 10 and 15 min, followed by significant decay at 30 min (13
). Thus, we speculate that the study of Bi et al. provides insight into a later window of cellular response that is distinct from the early observations described in this work.
Mechanism of action of terpenoid phenols.
The hydrophobic nature of terpenoid phenols ensures their preferential partition into the lipid membrane; thus, carvacrol has a log P
value of 3.26 for partition into phosphatidylethanolamine membranes relative to that of buffer (28
). However, hydrophobicity alone does not ensure toxicity, since p
-cymene, a precursor of carvacrol, has a higher partition coefficient for lipid membranes but is nontoxic. The presence of the hydroxyl group is critical for toxicity, as seen by the lack of microbicidal effects of p
-cymene and carvacrol methylesters (6
). It has been proposed that the delocalized electron system in carvacrol facilitates the dissociation of H+
from the −OH group. This, in turn, would allow carvacrol to shuttle H+
and monovalent cations, such as K+
, across membranes, dissipating pH and K+
gradients across cell membranes (28
). Consistent with this mechanism, carvacrol was also shown to depolarize bacterial cell membranes and decrease the accumulation of the fluorescent dye 5(6)-carboxyfluorescein diacetate, suggestive of an increase in membrane permeability (30
). Such a mechanism, however, does not explain the transient Ca2+
bursts associated with cellular interaction with carvacrol. It may be that effects on membrane expansion and fluidity (28
) cause the opening of ion channels, followed by their rapid desensitization.
The distinct phases of Ca2+
and pH transients argue against a simple mechanism involving catastrophic membrane lesion, as has been previously proposed based on the uptake of propidium iodide (21
). Propidium iodide staining of cells at MIC confirms cell death but does not elucidate the mechanisms leading up to cell death and loss of membrane integrity. The transient nature of the cytosolic Ca2+
surge upon exposure to carvacrol indicates that cells maintain their ability to regulate ion flux (Fig. ). In addition, the robust transcriptional responses that largely overlap Ca2+
stress and nutrient starvation point to the activation of specific signaling pathways downstream of membrane interaction with terpenoid phenols. These signaling cascades reveal additional fungal targets that can be used in combination with carvacrol and similar essential oil components. Recent studies have shown that azole drugs inhibit vacuolar acidification and exacerbate the Ca2+
transients elicited by amiodarone, consistent with synergic effects of these drugs (33
). Future studies could examine potential interactions between essential oils and azoles. In addition, calcineurin inhibitors (cyclosporine A and FK506) that enhance Ca2+
dysregulation and rapamycin analogs that block TOR signaling could be tested for drug interactions with carvacrol against pathogenic fungi.