Abstract

The antifungal mode of action of thymol was investigated by a chemical-genetic profile analysis. Growth of each of ∼ 4700 haploid Saccharomyces cerevisiae gene deletion mutants was monitored on medium with a subinhibitory concentration (50 µg/ml) of thymol and compared to growth on non-thymol control medium. This analysis revealed that, of the 76 deletion mutants with the greatest degree of susceptibility to thymol, 29% had deletions in genes involved in telomere length maintenance. A telomere restriction fragment (TRF) length assay showed that yeast exposed to a subinhibitory concentration of thymol for 15 days had telomere size reductions of 13–20% compared to non-thymol controls. By accelerating telomere shortening, thymol may increase the rate of cell senescence and apoptosis. Furthermore, real-time RT-PCR analysis revealed approximately two-fold reductions in EST2 mRNA but no change in TLC1 RNA in thymol-treated S. cerevisiae relative to untreated cells. EST2 encodes the essential reverse transcriptase subunit of telomerase that uses TLC1 RNA as a template during addition of TG(1–3) repeats to maintain telomere ends. This study provides compelling evidence that a primary mode of thymol antifungal activity is through inhibition of transcription of EST2 and thus telomerase activity.

Introduction

New antifungal drugs with distinct modes of action need to be identified because of the increasing incidence of fungal resistance to existing antibiotics [1]. Plant secondary metabolites have great potential as sources of antifungal agents, and an advantage to using ethnobotanicals with antimicrobial activity is that many phytomedicines have been employed by traditional healers for thousands of years with few or no adverse effects. The genus Thymus L. comprises about 215 aromatic plant species that are native to the Mediterranean region, among which is the well-known culinary herb thyme [2] and its species are used extensively in traditional medicine. The leaves and flowering parts are widely used as tonic and herbal tea, antiseptic, antitussive and carminative as well as for treating colds [3,4]. Oils and extracts from Thymus spp. are commonly employed in pharmaceutical, cosmetic and perfume industries, and for flavoring and preservation of food products [5]. Several studies have also shown that thyme oils have strong antimicrobial and antioxidant activities [6,7]. Many of these biological activities are believed to be due to volatile compounds that may include thymol, carvacrol, ρ-cymene, and β-caryophyllene [8–11].

Despite many reports on the antimicrobial properties of essential oils and of the main monoterpenes (thymol and carvacrol) found in most Thymus species, there are few investigations on the antifungal mode of action of these compounds. Previous studies primarily focus on the pronounced fungicidal activity of thymol against azole-resistant Candida strains. In vitro experiments showed that thymol inhibits H(+)-ATPase in the cytoplasmic membrane [12], disrupts membrane integrity [13] and drug efflux pumps [14]. Thus thymol may have several targets in the fungal cell.

We reasoned that cell-based screens, rather than in vitro experiments, would provide us with the main target site(s) of thymol. We employed a high throughput thymol-sensitivity screen using ∼ 4700 haploid gene deletion strains of Saccharomyces cerevisiae. The dominant group of most sensitive mutants, indicative of a primary thymol target, had deletions of genes that are involved in telomere length maintenance. Telomeres comprise specific DNA sequences at the ends of chromosomes in most eukaryotes that protect the chromosome from end-to-end fusion and help to maintain chromosome integrity [15]. Yeast telomeres are about 350–500 bp in length and are made up of tandem TG(1–3) repeats [16]. Telomeres are prone to shortening at each replication event and this sequence loss is normally prevented by the action of the ribonucleoprotein enzyme telomerase, which reverse-transcribes telomeric repeats onto telomeric ends [17]. Nevertheless, telomere shortening is implicated in cell senescence, aging, and entry into apoptosis and thus offers an interesting chemotherapeutic target.

Material and methods

Growth media and compounds

Standard rich (YPD) and synthetic media were used for the experiments [18]. Yeast cells were grown at 30°C for 1–2 days. The YPD medium containing Geneticin (G418; 200 µg/ml) was used for the maintenance of deletion strains carrying the G418R selectable marker. G418 was purchased from Sigma-Aldrich (Oakville, ON, Canada) and thymol was obtained from Bioshop (Burlington, ON, Canada).

Antifungal activity

Saccharomyces cerevisiae (S288C) was used in antifungal activity assays. Minimum Inhibitory Concentration (MIC) for thymol was measured using the broth microdilution assay protocol [19]. A three-fold dilution of thymol (concentration range of 5.1−3.95 × 10−25 mg/ml) was added to sterile 96-well microtitre plates. Plates were incubated at 30°C for 1–2 days. Inhibition of growth was visually compared with control wells containing no thymol.

Gene deletion array (GDA) analysis

For high throughput phenotypic screenings, approximately 4700 MATa haploid gene deletion strains of S. cerevisiae derived from BY4741 (MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0) were maintained in an ordered array of approximately 384 individual strains in 16 plates [20]. YPD agar plates without (control), and with a sub-inhibitory concentration of thymol (50 µg/ml) (experimental), were inoculated by hand-pinning sets of 384 mutant strains per plate using a floating pin replicator as previously described [21]. After 1–2 days incubation at 30°C, digital images of the plates were captured and colony sizes were analyzed using Growth Detector software [22]. The relative size of colonies was used as a measure for growth rates under experimental (+ thymol) versus control (− thymol) conditions. Each experiment was repeated three times. Colonies that demonstrated 70% or more reduction in size in at least two replicates were classified as supersensitive (i.e., highly susceptible mutants). Gene ontology annotation analysis was performed with online software (gprofiler; http://biit.cs.ut.ee/gprofiler/, Profcom; http://webclu.bio.wzw.tum.de/profcom/, GeneMANIA; http://www.genemania.org/) and Saccharomyces Genome Database [23] was used for functional profiling of highly susceptible mutants in our large-scale experiment.

Spot test analysis

Sensitivity of selected mutant strains identified in the GDA screens were confirmed by spot test analyses. Yeast cells were grown in YPD liquid medium to mid-log phase and 10-fold serially diluted. From each dilution, 15 µl was spotted on medium containing sub-inhibitory concentrations of thymol (50 µg/ml), or without thymol (control). The growth patterns were compared after 2 days at 30°C. Each experiment was repeated a minimum of three times.

Telomere restriction fragment (TRF) length analysis

Genomic DNA was prepared from the haploid yeast strain BY4741 grown in YPD medium according to the protocol of Hoffman et al. [24]. For Southern blots, DNA was digested overnight with XhoI and subjected to agarose gel electrophoresis. DNA fragments were transferred to Hybond-N+ membrane (Amersham, Baied'Urfe, PQ, Canada) and cross-linked using UV light. Hybridization was with biotinylated telomeric probe (5′-/BioTEG/ TGTGGGTGTGGTGTGTGGGTGTGGTG/BioTEG/-3′) for 20 h at 55°C and bands were visualized using streptavidin-HRP (Sigma) – chemiluminescence (Amersham), and exposure to X-ray film [25]. Average telomere sizes were calculated using internal control fragments containing S. cerevisiae telomeric repeats generated by Bsm AI and TaqI digests of the plasmid pYt103 [26].

β-galactosidase expression assay

This assay used an inducible β-galactosidase reporter gene in p416 as described previously [27]. Briefly, cells of BY4741 harboring p416GAL1-lacZ were incubated in SC-ura medium containing 2% galactose with subinhibitory amounts (60 and 75 µg/ml) of thymol and without thymol (negative control). Cycloheximide (1 µg/ml, Sigma) was used as a positive control for translation inhibition. After 20 h at 30°C, yeast cells were harvested by centrifugation, cell density was measured at OD600 and β-galactosidase activity was measured as described in Miller et al. [28].

Real-Time RT-PCR quantification of EST2 and TLC1

The yeast strain BY4741 was grown in YPD medium containing either a subinhibitory concentration (60 µg/ml) of thymol or without thymol for 24 h and total RNA was prepared using an RNA extraction kit (Bio-Rad, Mississauga, ON, Canada). RNA samples were DNase treated (Ambion, Burlington, ON, Canada). Total RNA concentrations were determined using a NanoDrop-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and cDNA was synthesized using 1 µg of total extracted RNA and M-MulV reverse transcriptase (New England Biolabs, Pickering, ON, Canada) as described previously [25]. Oligonucleotide primers for real-time PCR were designed for coding sequences from the Saccharomyces Genome Database (http://www.yeastgenome.org) using the on-line IDT SciTools software PrimerQuest (http://www.idtdna.com/scitools/applications/primerquest/). Primers used were as follows:

PGK1f = 5′-CAGACCATTCTTGGCCATCT-3′, PGK1r = 5′-CGAAGATGGAGTCACCGATT-3′;

EST2f = 5′-GTTACTGAAAGGCGCTGCTT GGAA-3′, EST2r = 5′-AGCAGTTGCGGATGATGAGG ATGA-3′;

TLC1f = 5′-TTAAGCATCGGTTAGGTTTGCGGG-3′, TLC1r = 5′-AAATGAACACACGGTTCCTTCCGC-3′.

Real-time PCR was conducted using a Rotor Gene RG-300 from Corbett Research (Montreal Biotech, Dorval, PQ, Canada) and SYBR Green Supermix (Bio-Rad) as per manufacturer's recommendations. Reactions were initiated with a 5 min incubation at 94°C followed by 40 cycles at 94°C for 60 s (denaturation), 60°C for 60 s (primer annealing), and 72°C for 60 s (elongation). Three biological replicates were done for each treatment. The PCR quantification and melting curves were generated using the Rotor Gene 6 software and threshold was set to 0.025 relative fluorescence units. Relative values of gene expression were calculated with untreated samples as control, and normalized to levels of PGK1 (phosphoglycerate kinase), according to the method of Pfaffl et al. [29]. Finally, to ensure that there were no false positives in PCR reactions, the PCR products were examined by electrophoresis in 1.2% agarose gels.

Statistical analysis

Statistical significance in the data sets was assessed by Student's t-test using Microsoft Excel 2007 (Microsoft Corporation, USA). The difference was considered to be statistically significant when P-value < 0.05.

Results and discussion

Antifungal activity of thymol

We determined that the minimum inhibitory concentration (MIC100) for thymol was 80 µg/ml. This MIC100 was defined as the lowest concentration that resulted in complete inhibition of visible growth of S. cerevisiae strain S288C using a broth microdilution assay [19].

Gene deletion array (GDA) analysis

The haploid yeast gene deletion array (yGDA) was screened for sensitivity to a subinhibitory concentration of thymol (50 µg/ml) so that only strains with increased sensitivity to thymol would exhibit a significant growth reduction. Colony size measurements revealed 76 deletion mutants were super-sensitive to thymol. These represent genes that are not normally required for growth under laboratory conditions, and slow growth by the corresponding deletion strains is likely a result of a chemical-genetic interaction. The genes deleted in super-sensitive strains were clustered based on the cellular processes in which they participate (Fig. 1A). The profile obtained reveals an interesting set of genes with diverse cellular functions falling into at least five main categories: approximately 29% of strains supersensitive to thymol are involved in telomere length maintenance (P-value: 8.3 × 10−25), whereas 13%, 11%, 8% and 9% are associated with ribosome assembly and translation, vesicular transport, transcription and carbohydrate metabolism, respectively. Of the dominant cluster, most of the genes involved in telomere length maintenance cause telomere shortening when deleted (Table 1) [23,30–32]. The smaller clusters could represent additional target sites (side-effects) of thymol or could also represent novel secondary functions for certain genes, some of which may link telomere length maintenance to other cellular processes. There are considerable overlaps between the dominant cluster and the three smaller clusters of ribosome assembly and translation, vesicular transport and transcription (Fig. 1B). For instance, five of 22 supersensitive mutants in the main cluster, SRB5, SRB8, SSN3, MFT1 and GTR1, not only affect telomere length but also play important roles in Pol II-associated transcriptional processes in yeast [23]. This could represent cross-talk between telomere length maintenance and the other cellular processes such as transcription in S. cerevisiae.

Fig. 1

Clustering of thymol-sensitive gene deletion mutants. The haploid non-essential yeast gene deletion array was subjected to a subinhibitory concentration of thymol. Colony size reduction was used to detect sensitivity. (A) The mutants most sensitive to thymol were clustered according to the cellular processes in which their deleted genes participated. (B) Overlap between the dominant cluster (telomere length maintenance) and three smaller clusters indicate cross-talk between telomere length maintenance and other cellular processes. This Figure is reproduced in color in the online version of Medical Mycology.

Fig. 1

Clustering of thymol-sensitive gene deletion mutants. The haploid non-essential yeast gene deletion array was subjected to a subinhibitory concentration of thymol. Colony size reduction was used to detect sensitivity. (A) The mutants most sensitive to thymol were clustered according to the cellular processes in which their deleted genes participated. (B) Overlap between the dominant cluster (telomere length maintenance) and three smaller clusters indicate cross-talk between telomere length maintenance and other cellular processes. This Figure is reproduced in color in the online version of Medical Mycology.

Table 1

List of thymol-sensitive gene deletion mutants involved in telomere length maintenance.

Systematic name Standard name Gene function % of colony size reduction Telomere length1 
YLR318W EST2 Telomerase reverse transcriptase 86.2 VS 
YIL009C-A EST3 Telomerase holoenzyme complex 73.1 VS 
YMR224C MRE11 DNA repair, MRX complex 74.6 VS 
YGL173C KEM1 Exonuclease, RNA degradation 93.1 VS 
YGR036C CAX4 ER, N-glycosylation, phosphatase 96.1 VS 
YEL033W MTC7 Maintenance of Telomere Capping 72.6 VS 
YGR104C SRB5 Transcription, Mediator complex component 85.2 
YMR186W HSC82 Heat shock protein 75.5 
YGL084C GUP1 Glycerol uptake 88.7 
YML121W GTR1 Phosphate transport 77.1 
YLR372W SUR4 Fatty acid synthesis, transport 94.3 
YCL061C MRC1 DNA damage response 89.9 
YDR418W RPL12B Large ribosomal subunit 75.3 
YDR532C KRE28 Spindle pole protein 91.6 
YML062C MFT1 Tho and Paf1 complexes 78.7 ss 
YCR081W SRB8 Transcription, Mediator complex component 81.0 
YPL042C SSN3 Transcription, Mediator complex component 82.1 
YOL109W ZEO1 Plasma membrane protein 71.3 
YCR047C BUD23 Bud site selection 72.1 sl 
YBL032W HEK2 RNA binding protein 71.5 Unknown 
YNL273W TOF1 Topoisomerase I-interacting Factor 79.7 Abnormal 
YPR018W RLF2 Chromatin Assembly Complex, Large Subunit 75.5 Unknown 
Systematic name Standard name Gene function % of colony size reduction Telomere length1 
YLR318W EST2 Telomerase reverse transcriptase 86.2 VS 
YIL009C-A EST3 Telomerase holoenzyme complex 73.1 VS 
YMR224C MRE11 DNA repair, MRX complex 74.6 VS 
YGL173C KEM1 Exonuclease, RNA degradation 93.1 VS 
YGR036C CAX4 ER, N-glycosylation, phosphatase 96.1 VS 
YEL033W MTC7 Maintenance of Telomere Capping 72.6 VS 
YGR104C SRB5 Transcription, Mediator complex component 85.2 
YMR186W HSC82 Heat shock protein 75.5 
YGL084C GUP1 Glycerol uptake 88.7 
YML121W GTR1 Phosphate transport 77.1 
YLR372W SUR4 Fatty acid synthesis, transport 94.3 
YCL061C MRC1 DNA damage response 89.9 
YDR418W RPL12B Large ribosomal subunit 75.3 
YDR532C KRE28 Spindle pole protein 91.6 
YML062C MFT1 Tho and Paf1 complexes 78.7 ss 
YCR081W SRB8 Transcription, Mediator complex component 81.0 
YPL042C SSN3 Transcription, Mediator complex component 82.1 
YOL109W ZEO1 Plasma membrane protein 71.3 
YCR047C BUD23 Bud site selection 72.1 sl 
YBL032W HEK2 RNA binding protein 71.5 Unknown 
YNL273W TOF1 Topoisomerase I-interacting Factor 79.7 Abnormal 
YPR018W RLF2 Chromatin Assembly Complex, Large Subunit 75.5 Unknown 

1Telomere length classifications follow those in previous reports [23,30,32]: ss, slightly short (< 50 bp shorter than wild type); S, short (50–150 bp); VS, very short (> 150 bp shorter); sl, slightly long (< 50 bp longer than wild type); L, long (50–150 bp).

Based on previous systematic genome-wide studies in S. cerevisiae, it can be said that nearly 5% of the ∼ 6000 genes in yeast are involved in telomere length maintenance [30–32]. Some of these genes affect the telomeres or telomerase enzyme directly, but most lack obvious connections to telomere maintenance. The functions of these indirectly related genes include transcription, ribosome assembly and translation, vesicular transport and chromatin modification. These previous findings are completely consistent with the profile obtained from our GDA analysis.

Spot test analysis verifies GDA

To investigate the accuracy of our large-scale approach to detect thymol-sensitive mutants, nine deletion strains that were supersensitive to thymol based on the GDA assay were randomly selected and subjected to spot test analyses (Fig. 2). This analysis confirmed that deletion of these genes confers increased sensitivity to thymol, and confirmed the large-scale screen based on the GDA approach.

Fig. 2

Strain sensitivity to thymol. Wild type and nine randomly selected gene deletion mutant strains that were thymol-sensitive based on GDA analysis were 10-fold serially diluted and spotted on solid YPD medium with a subinhibitory concentration (50 µg/ml) of thymol or without thymol (control). The plates were incubated at 30°C for 1–2 days and then photographed. All deletion mutants selected exhibited increased sensitivity to thymol, providing verification of the GDA analysis. This Figure is reproduced in color in the online version of Medical Mycology.

Fig. 2

Strain sensitivity to thymol. Wild type and nine randomly selected gene deletion mutant strains that were thymol-sensitive based on GDA analysis were 10-fold serially diluted and spotted on solid YPD medium with a subinhibitory concentration (50 µg/ml) of thymol or without thymol (control). The plates were incubated at 30°C for 1–2 days and then photographed. All deletion mutants selected exhibited increased sensitivity to thymol, providing verification of the GDA analysis. This Figure is reproduced in color in the online version of Medical Mycology.

Thymol induces telomere shortening

According to our GDA analysis, the dominant cluster of mutants with increased sensitivity to thymol had deletions of genes with roles in telomere length maintenance. To clarify whether the antifungal activity of thymol in yeast was related to telomere shortening, we monitored changes in telomere length, if any, following thymol treatment using a telomere restriction fragment (TRF) assay. Yeast cultures were grown in YPD medium with a sub-inhibitory concentration of thymol (50 µg/ml) and telomere length was measured at days 4 and 15. Treatment for 4 days with thymol did not result in a significant reduction in telomere length compared to the untreated control (Fig. 3A, lanes 3 and 4). This may be explained by the relatively few rounds (32–48) of cell divisions occurring over a 4-day interval and that telomeres become incrementally shorter during successive cell divisions. At day 15, however, telomeres of yeast cells exposed to thymol were on average 67 bp shorter than those of the no-thymol control (Fig. 3A, lanes 1 and 2). This is equivalent to an average size reduction of 13–20%, given that yeast telomeres are about 350–500 bp in length [16]. This result was in accordance with our large-scale screens and demonstrated that thymol interferes with the biological pathways that are involved in telomere length maintenance and causes telomere shortening over time.

Fig. 3

Secondary assays testing thymol mode of action. (A) Telomere restriction fragment (TRF) length assay for telomere length measurement in yeast cells (strain BY4741) following 4 and 15 days exposure to a subinhibitory concentration of thymol (50 µg/ml) and without thymol (control). Internal size standards (m) are fragments of the plasmid pYt103 containing telomeric sequences. Arrow heads correspond to average size of fragments containing telomeres as follows from left to right: 883 bp (15d, + thymol), 950 bp, 983 bp and 983 bp (4d, − thymol). (B)β-galactosidase reporter gene expression decreases in S. cerevisiae with increasing concentrations of subinhibitory levels of thymol. Thymol exposure at 60 and 75 µg/ml limited the expression of β-galactosidase to 38% and 53% of that in the untreated control, respectively. The values are expressed as mean ± SD of triplicates and difference between treatment and control groups is indicated as P < 0.05 (*) and P < 0.01 (**). (C) Real-time RT-PCR was used to determine the expression levels of EST2 and TLC1 genes. Gene expression was normalized to that of the phosphoglycerate kinase (PGK1) gene. The results indicate approximately twofold reductions in relative contents of EST2 mRNAs in the presence of 60 µg/ml thymol relative to untreated control, while no significant difference in the amounts of TLC1 RNAs was observed. Asterisk shows significant differences at P < 0.05.

Fig. 3

Secondary assays testing thymol mode of action. (A) Telomere restriction fragment (TRF) length assay for telomere length measurement in yeast cells (strain BY4741) following 4 and 15 days exposure to a subinhibitory concentration of thymol (50 µg/ml) and without thymol (control). Internal size standards (m) are fragments of the plasmid pYt103 containing telomeric sequences. Arrow heads correspond to average size of fragments containing telomeres as follows from left to right: 883 bp (15d, + thymol), 950 bp, 983 bp and 983 bp (4d, − thymol). (B)β-galactosidase reporter gene expression decreases in S. cerevisiae with increasing concentrations of subinhibitory levels of thymol. Thymol exposure at 60 and 75 µg/ml limited the expression of β-galactosidase to 38% and 53% of that in the untreated control, respectively. The values are expressed as mean ± SD of triplicates and difference between treatment and control groups is indicated as P < 0.05 (*) and P < 0.01 (**). (C) Real-time RT-PCR was used to determine the expression levels of EST2 and TLC1 genes. Gene expression was normalized to that of the phosphoglycerate kinase (PGK1) gene. The results indicate approximately twofold reductions in relative contents of EST2 mRNAs in the presence of 60 µg/ml thymol relative to untreated control, while no significant difference in the amounts of TLC1 RNAs was observed. Asterisk shows significant differences at P < 0.05.

Telomere shortening can induce cell cycle arrest, apoptosis, and as a consequence, cell death in S. cerevisiae. In recent years, there has been increasing interest in antitumor properties of thymol and thymoquinone (TQ), a derivative of thymol. TQ has been reported to induce DNA fragmentation and kill cancer cells by a process that involves apoptosis and cell cycle arrest [33,34]. Gurung et al. [35] showed that TQ induces DNA damage, telomere shortening by inhibiting telomerase enzyme, and cell death in glioblastoma cells. It has been reported that the cytotoxic effect of thymol on HL-60 cells is associated with induction of cell cycle arrest and apoptotic cell death based on genomic DNA fragmentation [36]. These reports are consistent with our TRF assay and indicate that target pathways for thymol are probably conserved between yeast and human cells.

It is noteworthy that telomere length in lane 2, Figure 3A (control after 15 days) was about 33 bp shorter than in lane 4 (control after 4 days). Mozdy and Cech [37] found that the number of TLC1 (RNA component of telomerase enzyme) molecules in a haploid yeast is about 29, less than the 64 chromosome ends in late S-phase. Because the number of telomerase holoenzymes cannot exceed the number of TLC1 molecules, they concluded that chromosome ends out number telomerase holoenzymes in yeast. Limited telomerase may contribute to an explanation for why so few telomeres are extended during a single cell cycle in yeast [38] and observations of chronologic aging of yeast populations [39], and may also explain the differences in telomere length of untreated controls over time that was observed in the present study.

Gene expression analysis using β-galactosidase reporter

To investigate inhibitory effects of thymol on genes involved in transcription or translation in yeast (two smaller clusters in our GDA analysis), we used an inducible β-galactosidase reporter construct. As seen in Figure 3B, the addition of subinhibitory concentrations of thymol to yeast cells resulted in a reduction of β-galactosidase activity in a dose dependent manner. Compared to the untreated control, significant reductions in β-galactosidase activities were observed (P-value < 0.05, paired t-test) when thymol was at 60 µg/ml (38% reduction) and 75 µg/ml (53% reduction). These observations support GDA-based indications that thymol reduces the efficiency of transcription or translation in yeast cells. This is also consistent with the reported link between telomere length maintenance and gene expression pathway in S. cerevisiae [30,32].

Effect of thymol on expression levels of EST2 and TLC1 genes

According to our TRF and β-galactosidase assays, it can be hypothesized that thymol-mediated changes in telomere length may be related to thymol interference with transcriptional or translational processes in S. cerevisiae. To investigate the first possibility, we used real-time RT-PCR to measure the transcript abundance of EST2 (the catalytic component of yeast telomerase) and TLC1 (RNA template component of telomerase) after exposing yeast to a subinhibitory concentration of thymol (60 µg/ml) for 24 h. EST2 mRNA and TLC1 RNA levels were normalized to PGK1 mRNA levels with REST software [29]. The results indicated that the EST2 gene was down-regulated in the presence of thymol by a factor of 2.2 in comparison to untreated controls. This difference in EST2 mRNA abundance between control and experimental samples was significant (P-value = 0.01). However, we observed no significant difference in the amounts of TLC1 RNAs in treated and untreated samples (Fig. 3C). Electrophoresis of PCR products confirmed that a single amplicon of expected size was produced in each assay (data not shown).

Telomeres are nucleoprotein structures that protect the ends of eukaryotic linear chromosomes from degradation and chromosomal fusions [15]. Several conserved pathways have been identified that regulate telomere length in yeast and humans. Telomerase is a highly specialized enzyme that catalyzes extension of 5′-ends of the lagging DNA strand using an RNA template [17]. Yeast telomerase is a ribonucleoprotein containing a catalytic protein component, EST2 (telomerase reverse transcriptase) [40], and an associated RNA moiety, TLC1 [41], which serves as the template to extend telomeric DNA sequences. Telomerase activity can overcome telomere shortening that results from the end-replication problem [42]. Our real-time RT-PCR quantification of EST2 and TLC1, showed approximately two-fold reductions in contents of catalytic component of telomerase (EST2) transcripts in cells exposed to 60 µg/ml thymol relative to untreated cells. This observation suggests that thymol probably interferes with the biological pathways that are involved in transcription of the EST2 gene. Reduction in the content of EST2 mRNAs can reduce the telomerase activity during the cell cycle and finally cause telomere shortening over time in the yeast cell as indicated in our TRF assay. This result is in agreement with a recent study that demonstrated inhibitory effects of thymoquinone on telomerase enzyme, and as a result, telomere shortening and cell death in glioblastoma cells [35]. Diminishing RNA transcription or processing of telomerase core components or their regulators will have an influence on telomere length. For example, previous work showed that a subgroup of the telomere length maintenance genes influences telomere length by affecting the abundance of TLC1 transcripts [43]. However, no significant difference in the contents of TLC1 RNAs was observed in treated and untreated samples in the present study, leading to the proposal that thymol specifically interferes with expression of a catalytic component (EST2) rather than an RNA component (TLC1) of telomerase, and that different genes probably participate in transcription of these two telomerase core components in yeast cells.

This interesting mode of action of thymol in S. cerevisiae further suggests why it may be of value for inhibiting the growth of fungi that are resistance to existing antibiotics [13,14]. For example, little overlap would be expected in the thymol mode of action described here and that of the front-line commercial azole antifungals that target the ergosterol biosynthetic pathway. In addition, in recent years, there is increasing interest in natural plant products as potential chemotherapy agents because they are relatively non-toxic for the normal cellular system, inexpensive and available in an ingestible form. Thymol may exhibit high antifungal specificity given that most human somatic cells do not express telomerase [44]. Telomerase inhibition by thymol also presents an attractive target for cancer therapeutics, since in more than 90% of human cancers activation of telomerase prevents telomere shortening and thus allows for unlimited replicative capability [45]. Given the conservation of telomere maintenance mechanisms throughout evolution and the mode of action of thymol in S. cerevisiae as identified herein, thymol could also be useful as a potential chemotherapeutic drug for human cancers. However, detailed studies are required to profile the genome-wide effects of thymol in human cell lines to exploit its therapeutic potential more effectively. Finally, the study provides further evidence for the usefulness of the GDA approach in uncovering the mode of action of natural products.

Acknowledgments

This work was supported by Discovery Grants from Natural Sciences and Engineering Research Council (NSERC) of Canada to A.G. and M.L.S., and Ministry of Science, Research and Technology, Iran to E.D. We would like to thank Dr Janis Shampay (University of California) for generous gift of pYt103 plasmid. We also thank Md Alamgir, M. Jessulat, B. Samanfar, I. Galvan and D. Lafontaine for valuable technical advice.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.

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This paper was first published online on Early Online on 23 May 2013.