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Article

Augmenting the Activity of Monoterpenoid Phenols against Fungal Pathogens Using 2-Hydroxy-4-methoxybenzaldehyde that Target Cell Wall Integrity

by
Jong H. Kim
*,
Kathleen L. Chan
and
Noreen Mahoney
Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2015, 16(11), 26850-26870; https://doi.org/10.3390/ijms161125988
Submission received: 19 September 2015 / Revised: 27 October 2015 / Accepted: 2 November 2015 / Published: 10 November 2015
(This article belongs to the Special Issue Phenolics and Polyphenolics 2015)
Browse Figures
Versions Notes

Abstract

:
Disruption of cell wall integrity system should be an effective strategy for control of fungal pathogens. To augment the cell wall disruption efficacy of monoterpenoid phenols (carvacrol, thymol), antimycotic potency of benzaldehyde derivatives that can serve as chemosensitizing agents were evaluated against strains of Saccharomyces cerevisiae wild type (WT), slt2Δ and bck1Δ (mutants of the mitogen-activated protein kinase (MAPK) and MAPK kinase kinase, respectively, in the cell wall integrity pathway). Among fourteen compounds investigated, slt2Δ and bck1Δ showed higher susceptibility to nine benzaldehydes, compared to WT. Differential antimycotic activity of screened compounds indicated “structure-activity relationship” for targeting the cell wall integrity, where 2-hydroxy-4-methoxybenzaldehyde (2H4M) exhibited the highest antimycotic potency. The efficacy of 2H4M as an effective chemosensitizer to monoterpenoid phenols (viz., 2H4M + carvacrol or thymol) was assessed in yeasts or filamentous fungi (Aspergillus, Penicillium) according to European Committee on Antimicrobial Susceptibility Testing or Clinical Laboratory Standards Institute M38-A protocols, respectively. Synergistic chemosensitization greatly lowers minimum inhibitory or fungicidal concentrations of the co-administered compounds. 2H4M also overcame the tolerance of two MAPK mutants (sakAΔ, mpkCΔ) of Aspergillus fumigatus to fludioxonil (phenylpyrrole fungicide). Collectively, 2H4M possesses chemosensitizing capability to magnify the efficacy of monoterpenoid phenols, which improves target-based (viz., cell wall disruption) antifungal intervention.

Graphical Abstract

1. Introduction

Filamentous fungi in the genus Aspergillus are ubiquitous opportunistic pathogens, which are most notable as causative agents of highly enervating human diseases such as aspergillosis [1]. They form extremely invasive human infections, particularly in immunocompromised patients or in people suffering chronic granulomatosis [2,3,4]. Aspergillus flavus and Aspergillus parasiticus also produce highly (hepato)carcinogenic aflatoxins, which contaminate various agricultural/food commodities [5]. Filamentous fungi in the genus Penicillium also frequently cause food contamination or postharvest decay, where P. expansum is the main producer of the mycotoxin patulin that negatively affects human and animal health [6].
Mycotic diseases/infections are becoming a serious problem since effective antifungal drugs or fungicides, especially agents for treating drug/fungicide-resistant fungi, are very limited. Development of fungal resistance to conventional antimycotic agents not only triggers global public health issues, but also threatens the safety of food supplies, especially for the products susceptible to mycotoxin contamination [7,8]. For instance, continuous applications of widely used fungicides, such as strobilurins, fludioxonils, etc., to agricultural fields resulted in the development of fungal resistance to (and thus, escape from) the toxicities of fungicides. Moreover, applying fungicides at suboptimal concentrations or time-points of fungal growth can potentiate toxin production by mycotoxigenic fungi [9,10]. Fungicide-potentiation of mycotoxin production in fungi, especially those resistant to fungicides, has been reported in various aflatoxin-, trichothecene-, citrinin-, and patulin-producing fungal pathogens (See Table 1). Accordingly, there is an urgent demand to increase the efficacy of conventional antimycotic drugs/fungicides or develop new intervention strategies, which can secure the safe production of crops and food as well as public health.
Table
Table 1. Fungicide potentiation of mycotoxin production in fungal pathogens.
Table 1. Fungicide potentiation of mycotoxin production in fungal pathogens.
FungiFungicideKey Features (Potentiation of Mycotoxin Production)
Aspergillus parasiticusAnilinopyrimidineCorrelation between fitness parameters and aflatoxigenicity [11]
A. parasiticusFlusilazoleHighly aflatoxigenic, sterol demethylation inhibition-resistant isolates [12]
A. parasiticusPhenylpyrroleHighly aflatoxigenic, phenylpyrrole resistant isolates [13]
Fusarium graminearumCarbendazimIncreased trichothecene production with carbendazim resistance [14]
Fusarium sp.StrobilurinsIncreased deoxynivalenol production by sub-optimal application of strobilurin [9]
F. sporotrichioidesCarbendazimHigher mycotoxin production (T-2 toxin, 4,15-diacetoxyscirpenol, neosolaniol) with carbendazim resistance [15]
Penicillium expansumTebuconazole, Fludioxonil, etc.Adverse effect of fitness penalties on the mycotoxigenicity of resistant isolates [16]
P. expansumBenzimidazoleHighly mycotoxigenic field isolates resistant to the benzimidazoles [17]
P. verrucosumIprodioneStrong induction of mycotoxin biosynthesis by iprodione [18]
The cell wall integrity system of fungi is an effective target for control of fungal pathogens [19]. The cell wall integrity pathway is well elucidated and described in the model fungus Saccharomyces cerevisiae, where the operation of mitogen-activated protein kinase (MAPK) signaling pathway (viz., cell wall integrity pathway) is controlled by protein kinase C [20]. The BCK1 and SLT2 genes, which encode MAPK kinase kinase (MAPKKK) and MAPK, respectively (Table 2), in the pathway play crucial roles for maintaining cell wall integrity in fungi [20]. Studies have shown that genes in the cell wall integrity system in fungi, such as species in the genus Aspergillus and S. cerevisiae, are functionally well conserved [21,22]. Therefore, cell wall targeting drugs (e.g., echinocandins) could be administered as broad-spectrum antimycotic agents for control of filamentous or yeast fungal pathogens, including species of Candida ([23] and references therein).
Nevertheless, despite their usefulness as cell wall targeting drugs, echinocandins generally do not completely inhibit fungal growth ([24] and references therein), where the determination of precise endpoints for pathogen intervention is very rigorous [25]. Echinocandin treatment can also trigger a compensatory stimulation of chitin synthesis, which causes the development of fungal resistance to the drugs ([24] and references therein). Accordingly, development of new drugs or intervention strategies is continually required for effective control of fungal pathogens, especially the strains exhibiting drug or fungicide resistance.
Table
Table 2. Microbial strains used in this study.
Table 2. Microbial strains used in this study.
AspergillusCharacteristicsSource/References
A. flavus 3357Plant pathogen (aflatoxin), Human pathogen (aspergillosis), Reference aflatoxigenic strain used for genome sequencingNRRL a [26]
A. flavus 4212Plant pathogen (aflatoxin), Human pathogen (aspergillosis)NRRL
A. parasiticus 5862Plant pathogen (aflatoxin)NRRL
A. parasiticus 2999Plant pathogen (aflatoxin)NRRL
A. fumigatus AF293Human pathogen (aspergillosis), Parental strain, Reference clinical strain used for genome sequencing[26,27]
A. fumigatus sakAΔHuman pathogen (aspergillosis), Mitogen-Activated Protein Kinase (MAPK) gene deletion mutant derived from AF293[27]
A. fumigatus mpkCΔHuman pathogen (aspergillosis), Mitogen-Activated Protein Kinase (MAPK) gene deletion mutant derived from AF293[28]
P. expansum W1Plant pathogen (patulin), Parental strain[29]
P. expansum FR2Plant pathogen (patulin), Fludioxonil resistant mutant derived from P. expansum W1[29]
P. expansum W2Plant pathogen (patulin), Parental strain[29]
P. expansum FR3Plant pathogen (patulin), Fludioxonil resistant mutant derived from P. expansum W2[29]
SaccharomycesCharacteristicsSource/References
S. cerevisiae BY4741Model yeast, Parental strain (Mat a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0)[30]
S. cerevisiae slt2ΔMAPK mutant in cell wall integrity system derived from BY4741[30]
S. cerevisiae bck1ΔMAPK kinase kinase (MAPKKK) mutant derived from BY4741[30]
a NRRL, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL, USA.
Antifungal chemosensitization is an intervention scheme for effective control of pathogenic fungi, where co-application of a selected natural or synthetic compound (viz., a chemosensitizer or a chemosensitizing agent) with a conventional antifungal drug can intensify the drug efficacy [31]. Chemosensitization strategy makes the fungal pathogens highly susceptible to the drug co-administered, where the chemosensitizer significantly impaired fungal defense to the conventional drug. By definition, comparing to the traditional combination therapy (viz., combined application of two or more commercial drugs), a chemosensitizer itself does not have to exhibit a high extent of antifungal potency. However, chemosensitization not only magnifies the efficacy of the antimycotic drug co-applied, but also overcomes pathogen resistance to conventional drugs [31]. For example, co-application of piperazinyl quinolone with the azole drug fluconazole (FLC) resulted in overcoming FLC resistance of C. albicans, while the compound showed no antimycotic activity when applied alone [32]. Also, co-administration of cyclobutene-dione (squarile) derivatives with FLC elevated the drug activity during the treatment of C. albicans, where the chemosensitizer(s) modulated the major facilitator superfamily transporter (Mdr1p; responsible for FLC resistance) of the pathogen [33]. Chemosensitization mediated by a d-octapeptide derivative further overcame FLC resistance in S. cerevisiae and pathogenic fungi [34]. Collectively, fungal intervention via chemosensitization could be an alternative to (or complement) current antifungal practices, for example, combination therapy.
Natural products that present no significant medical or environmental side effects are potential sources of antimycotic or antimycotoxigenic agents, either in their nascent structure or as leads for more potent derivatives [35]. For instance, benzo derivatives (such as vanillic or caffeic acid) not only inhibited the growth of filamentous fungal pathogens, but also disrupted the production of mycotoxins [36]. The redox-active natural products, such as phenolic agents, can be potent redox cyclers that prevent fungal growth by interfering cellular redox homeostasis (thus, triggering fungal oxidative stress) or by disrupting the integrity of cellular components [37,38]. For defense, the fungal antioxidant system or cell wall/membrane integrity system play important roles for fungal tolerance to the phenolic agents administered [37,38].
Terpenoid phenols, such as carvacrol (5-isopropyl-2-methylphenol) and its structural isomer thymol (2-isopropyl-5-methylphenol) (Figure 1), have been demonstrated to be effective natural antimycotic agents by inhibiting the growth or activity of planktonic or biofilms of fungal pathogens ([39] and references therein). Carvacrol and thymol are generally regarded as safe (GRAS) reagents [40], and thus, are currently used as food additives. Genome-wide transcription profiling (microarray) study in the model fungus S. cerevisiae disclosed that genes in metabolic (energy, pyrimidine), biosynthetic, stress responses (oxidative, heat shock), etc., were highly up- or down-regulated with the treatment of carvacrol, where the ion homeostasis mutant (vmaΔ) was also hypersensitive to carvacrol treatment [39]. Similar microarray analysis in S. cerevisiae treated with thymol also revealed that genes involved in metabolism (sulfur, protein, thiamin, nucleic acid, etc.), mitochondrial function, organellar ribosome, cell proliferation, etc., were up- or down-regulated by thymol application [41]. However, effects of carvacrol or thymol on the function of cell wall integrity system were undetermined in these studies.
Ijms 16 25988 g001 1024
Figure 1. Structures of benzo derivatives, carvacrol, thymol, and aflatoxins used/detected in this study. (1) Benzaldehyde and derivatives: (1a) 2-Hydroxy-4-methoxybenzaldehyde, (1b) 2-Methyl-4-methoxybenzaldehyde, (1c) 3,5-Dimethoxybenzaldehyde, (1d) 2,3-Dimethoxybenzaldehyde, (1e) 2,5-Dimethoxybenzaldehyde, (1f) 2-Methoxybenzaldehyde, (1g) 2,4-Dimethoxybenzaldehyde, (1h) 3-Methoxybenzaldehyde, (1i) 4-Methoxybenzaldehyde, (1j) 2,4,5-Trimethoxybenzaldehyde, (1k) 3,4-Dimethoxybenzaldehyde, (1l) 4-Hydroxy-3-methoxybenzaldehyde, (1m) 4-Hydroxy-2-methoxybenzaldehyde, (1n) 3,5-Dimethoxy-4-hydroxybenzaldehyde; (2) Carvacrol (5-Isopropyl-2-methylphenol); (3) Thymol (2-Isopropyl-5-methylphenol); (4) Aflatoxin B1; (5) Aflatoxin B2; (6) Aflatoxin G1; (7) Aflatoxin G2.
Figure 1. Structures of benzo derivatives, carvacrol, thymol, and aflatoxins used/detected in this study. (1) Benzaldehyde and derivatives: (1a) 2-Hydroxy-4-methoxybenzaldehyde, (1b) 2-Methyl-4-methoxybenzaldehyde, (1c) 3,5-Dimethoxybenzaldehyde, (1d) 2,3-Dimethoxybenzaldehyde, (1e) 2,5-Dimethoxybenzaldehyde, (1f) 2-Methoxybenzaldehyde, (1g) 2,4-Dimethoxybenzaldehyde, (1h) 3-Methoxybenzaldehyde, (1i) 4-Methoxybenzaldehyde, (1j) 2,4,5-Trimethoxybenzaldehyde, (1k) 3,4-Dimethoxybenzaldehyde, (1l) 4-Hydroxy-3-methoxybenzaldehyde, (1m) 4-Hydroxy-2-methoxybenzaldehyde, (1n) 3,5-Dimethoxy-4-hydroxybenzaldehyde; (2) Carvacrol (5-Isopropyl-2-methylphenol); (3) Thymol (2-Isopropyl-5-methylphenol); (4) Aflatoxin B1; (5) Aflatoxin B2; (6) Aflatoxin G1; (7) Aflatoxin G2.
Ijms 16 25988 g001
The benzo derivative 2-hydroxy-4-methoxybenzaldehyde (2H4M) (Figure 1) is also a GRAS reagent [40], and hence, is currently used as a food additive. The 2H4M has been isolated from different plants as a natural compound, where it functioned as an insect repellent to protect food sources, etc. [42,43]. In this study, two S. cerevisiae cell wall integrity mutants (bck1Δ, slt2Δ), where genes in cell wall integrity MAPK pathway were deleted (Table 2), were examined to evaluate the efficacy of targeting cell wall integrity via natural product-based antifungal chemosensitization (namely, monoterpenoid phenols + 2H4M). The same intervention strategy was also investigated in filamentous fungal pathogens, such as Aspergillus and Penicillium, according to the Clinical Laboratory Standards Institute (CLSI) M38-A [44]. Results showed that: (1) 2H4M functioned as an effective antifungal chemosensitizer to augment the potency of monoterpenoid phenols; and (2) 2H4M overcame the tolerance of A. fumigatus MAPK mutants (sakAΔ, mpkCΔ) to fludioxonil, a phenylpyrrole fungicide.

2. Results and Discussion

2.1. Identification of 2-Hydroxy-4-methoxybenzaldehyde (2H4M) as the Most Potent Antifungal Benzaldehyde Analog via Yeast Screening: Structure-Activity Relationship

Antifungal efficacy of fourteen analogs of benzaldehyde (BA; basic structure) was investigated against the wild type (WT) and two cell wall integrity mutants (bck1Δ, slt2Δ) of S. cerevisiae, in in vitro agar plate (yeast dilution) bioassays. The bck1Δ and slt2Δ previously showed hypersensitivity to cell wall perturbing agents, such as caspofungin [45], and hence, can serve as screening tools for identifying new cell wall disrupting agents. Firstly, 2H4M was found to possess the highest antifungal activity against S. cervisiae strains (viz., no growth of bck1Δ, slt2Δ and WT at 5.0 mM cutoff) (Table 3). Test benzaldehydes were further classified into three groups based on the level of antifungal potency against bck1Δ and slt2Δ mutants as follows: Group 1, 2H4M, 2-methyl-4-methoxybenzaldehyde, 3,5-dimethoxybenzaldehyde (3,5-DMBA), 2,3-dimethoxybenzaldehyde (2,3-DMBA) (Complete growth inhibition of bck1Δ and slt2Δ mutants at 5.0 mM cutoff (viz., growth score = 0; See Experimental section)); Group 2, 2,5-dimethoxybenzaldehyde (2,5-DMBA), 2-methoxybenzaldehyde, 2,4-dimethoxybenzaldehyde (2,4-DMBA), 3-methoxybenzaldehyde, 4-methoxybenzaldehyde [Moderate growth inhibition of bck1Δ and slt2Δ mutants at 5.0 mM cutoff (viz., growth score = 1 to 4)]; and Group 3, 2,4,5-trimethoxybenzaldehyde, 3,4-dimethoxybenzaldehyde (3,4-DMBA), 4-hydroxy-3-methoxybenzaldehyde, 4-hydroxy-2-methoxybenzaldehyde (4H2M), 3,5-dimethoxy-4-hydroxybenzaldehyde, BA (No growth inhibition of bck1Δ and slt2Δ mutants at 5.0 mM cutoff (viz., growth score = 6)) (Table 3).
Table
Table 3. Growth scores of yeasts at 5 mM (cutoff) of benzaldehyde derivatives during yeast dilution bioassay (0, No growth; 6, Full growth; See Experimental section).
Table 3. Growth scores of yeasts at 5 mM (cutoff) of benzaldehyde derivatives during yeast dilution bioassay (0, No growth; 6, Full growth; See Experimental section).
Benzaldehyde DerivativesWTslt2Δbck1Δ
2-Hydroxy-4-methoxybenzaldehyde000
2-Methyl-4-methoxybenzaldehyde100
3,5-Dimethoxybenzaldehyde100
2,3-Dimethoxybenzaldehyde200
2,5-Dimethoxybenzaldehyde211
2-Methoxybenzaldehyde311
2,4-Dimethoxybenzaldehyde433
3-Methoxybenzaldehyde632
4-Methoxybenzaldehyde644
2,4,5-Trimethoxybenzaldehyde666
3,4-Dimethoxybenzaldehyde666
4-Hydroxy-3-methoxybenzaldehyde666
4-Hydroxy-2-methoxybenzaldehyde666
3,5-Dimethoxy-4-hydroxybenzaldehyde666
Benzaldehyde666
Structure-activity relationships were also found with the test compounds. Structural-activity relationship in this study is defined as the relationship between the structures of test compounds (benzaldehydes), i.e., kinds of chemical groups/side chains and/or their position on the benzene ring, and the level of antifungal activity. For example, the growth of S. cerevisiae bck1Δ and slt2Δ (and WT) was almost not affected by 3,4-DMBA (growth score = 6), while that of test strains treated with 3,5-DMBA (having a shift of a methoxyl residue from #4 to #5 position on the benzene ring) was greatly disrupted, where the level of growth inhibition was commensurate with compound concentration (1.0 to 5.0 mM) (viz., growth score = 0 to 5) (Figure 2; Table 3). Of note is that, while 2H4M was the most potent antifungal compound tested against WT, bck1Δ and slt2Δ (growth score = 0 at 5.0 mM cutoff; Table 3), the 4H2M, where the hydroxyl- and methoxyl-residues were reciprocally exchanged compared to 2H4M, exhibited almost no antimycotic activity in the same yeast strains (growth score = 6 at 5.0 mM cutoff; Table 3). Besides, the 2,3- and 2,5-DMBA exhibited higher antifungal potency (against the bck1Δ and slt2Δ) compared to 2,4-DMBA [order of activity (high to low): 2,3-DMBA (growth score = 0) > 2,5-DMBA (growth score = 1) > 2,4-DMBA (growth score = 6)]. Similar characteristics could be found in a prior study with quinone derivatives, where activities or functions of enzymes or cellular proteins were disrupted mainly by those analogs possessing an ortho- or para-quinonoid structure [46].
Ijms 16 25988 g002 1024
Figure 2. Differential susceptibility of S. cerevisiae strains to benzaldehyde derivatives. Exemplary yeast dilution bioassay showed that S. cerevisiae cell wall integrity mutants (slt2Δ, bck1Δ) were more susceptible to 3,5-DMBA compared to WT. Results also showed the structure-activity relationship, where 3,5-DMBA possessed potent antifungal activity (at 1 to 5 mM), while BA and 3,4-DMBA exhibited no antimycotic potency at the same concentrations. Numbers on right side of each row (0 to 6) indicate growth scores (See Experimental section).
Figure 2. Differential susceptibility of S. cerevisiae strains to benzaldehyde derivatives. Exemplary yeast dilution bioassay showed that S. cerevisiae cell wall integrity mutants (slt2Δ, bck1Δ) were more susceptible to 3,5-DMBA compared to WT. Results also showed the structure-activity relationship, where 3,5-DMBA possessed potent antifungal activity (at 1 to 5 mM), while BA and 3,4-DMBA exhibited no antimycotic potency at the same concentrations. Numbers on right side of each row (0 to 6) indicate growth scores (See Experimental section).
Ijms 16 25988 g002
Collectively, nine out of fifteen benzaldehydes (including BA as the basic structure) negatively affected the growth of bck1Δ and slt2Δ, the cell wall integrity mutants of S. cerevisiae, with structure-activity relationship. 2H4M possessed the highest antifungal activity among test compounds.

2.2. Growth Recovery of S. cerevisiae bck1Δ and slt2Δ Mutants by Sorbitol

In sorbitol remediation bioassay, sensitivity of slt2Δ and bck1Δ to carvacrol, thymol, 2H4M or 2,3-DMBA was alleviated by sorbitol (caffeine: positive control for cell wall perturbation) (See Experimental section and [47] for method). The level of growth of slt2Δ and bck1Δ on sorbitol-containing media was 10 to 100 times higher compared to controls without sorbitol (Figure 3). Therefore, the remediation by sorbitol indicates that disruption of cell wall integrity in fungi is one contributing mechanism of how the screened benzaldehydes (including thymol and carvacrol) exerted antimycotic activity (alone or in combination with monoterpenoid phenols; See below).
Ijms 16 25988 g003 1024
Figure 3. Yeast dilution bioassay showing sensitivity of S. cerevisiae slt2Δ and bck1Δ mutants to caffeine (5 mM; control) and test compounds (2H4M, 2,3-DMBA, thymol, carvacrol) was remediated by sorbitol. Results indicate the test compounds negatively affected cell wall integrity system of fungi.
Figure 3. Yeast dilution bioassay showing sensitivity of S. cerevisiae slt2Δ and bck1Δ mutants to caffeine (5 mM; control) and test compounds (2H4M, 2,3-DMBA, thymol, carvacrol) was remediated by sorbitol. Results indicate the test compounds negatively affected cell wall integrity system of fungi.
Ijms 16 25988 g003

2.3. Chemosensitization Test in S. cerevisiae: Co-Application of Thymol or Carvacrol with 2H4M Augmented the Antifungal Efficacy of Test Compounds in S. cerevisiae

Antifungal chemosensitization was examined based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth dilution protocol for yeasts [48], where both minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs), and thus, their respective Fractional Inhibitory Concentration Indices (FICI) and Fractional Fungicidal Concentration Indices (FFCI), of S. cerevisiae WT, slt2Δ and bck1Δ were calculated (See Experimental section for calculations and concentrations of test compounds).
For FFCIs of carvacrol, “synergistic” FFCIs (i.e., FFCI ≤ 0.5) were not identified between 2H4M and carvacrol for the test strains (Table 4). However, despite the absence of calculated synergism (as determined by “indifferent” interactions [49]) (Table 4), there was increased antifungal activity of 2H4M and carvacrol (viz., chemosensitizing effect; FFCIs = 0.6 to 0.8) in WT, slt2Δ and bck1Δ, which was reflected in lowered MFCs of test compounds when they were co-applied. For example, co-application of 2H4M (3.2, 1.6 or 1.6 mM for WT, slt2Δ and bck1Δ, respectively) with carvacrol (3.2 mM for all strains) completely prevented the yeast growth (on recovery agar plate), while individual treatment of each compound, alone, at the same dosages allowed the survival of yeast strains (See also Figure 4). Noteworthy is that slt2Δ (MAPK) and bck1Δ (MAPKKK) required much lower concentration of 2H4M (1.6 mM) compared to WT (3.2 mM) to achieve complete inhibition of yeast growth, thus demonstrating that the knockout strains are less capable of responding to the cell wall perturbation induced by 2H4M, and are therefore more susceptible to the molecule.
For FICIs of carvacrol, although no synergism was found for FICIs (Table 4), there was enhanced antifungal activity of 2H4M and carvacrol for WT (FICIs = 1.0), except slt2Δ and bck1Δ, for which FICI = 2.0 (neutral interaction). For example, co-application of 2H4M (0.8 mM) with carvacrol (0.4 mM) completely inhibited the growth of WT in liquid culture (microtiter plate), while individual treatment of each compound, alone, at the same concentrations allowed the growth of WT.
For FFCIs of thymol, there was enhanced antifungal activity of 2H4M and thymol for WT (FFCIs = 0.6), except slt2Δ and bck1Δ, for which FFCI = 2.0 (neutral interaction). For example, co-application of 2H4M (0.8 mM) with thymol (1.6 mM) resulted in complete inhibition of WT growth, while individual treatment of each compound, alone, at the same concentrations allowed the survival of the strain.
For FICIs of thymol, despite the absence of calculated synergism, there was increased antifungal activity of 2H4M and thymol (viz., chemosensitizing effect; FICIs = 0.8 to 1.0) in WT, slt2Δ and bck1Δ, which was reflected in lowered MICs of 2H4M and thymol when compounds were co-administered. For example, co-application of 2H4M (0.8 mM for all strains) with thymol (0.4, 0.4, 0.2 mM for WT, slt2Δ and bck1Δ, respectively) completely inhibited the growth of yeast strains, while individual treatment of each compound, alone, at the same concentrations allowed the survival of yeasts (Table 4).
Collectively, 2H4M possessed a chemosensitizing capability to carvacrol or thymol in yeast tests, where MICs and MFCs of test compounds were decreased (viz., antifungal potency of test compounds was increased) during chemosensitization. Thymol required much lower concentration (viz., 0.2 to 0.4 mM for FICIs; 1.6 mM for FFCI) during chemosensitization compared to carvacrol (viz., 0.4 mM for FICIs; 3.2 mM for FFCIs) for complete inhibition of yeast growth, indicating structure-activity relationship also existed in monoterpenoid phenols. The “neutral interaction” determined in slt2Δ and bck1Δ during chemosensitization (viz., FICICARVACROL or FFCITHYMOL = 2.0) indicated that the MICs of carvacrol for slt2Δ and bck1Δ (0.4 mM) or MFCs of thymol for the same mutants (1.6 mM) already reached the maximum antimycotic level without chemosensitization, while WT required chemosensitization for the improvement of potency of test compounds. Thus, results further reflect the higher susceptibility of slt2Δ and bck1Δ mutants to cell wall disrupting reagents (i.e., 2H4M, monoterpenoid phenols) compared to WT.
Table
Table 4. Antifungal chemosensitization of 2H4M (mM) to carvacrol or thymol (mM), tested against S. cerevisiae strains: summary of EUCAST-based microdilution bioassays. a
Table 4. Antifungal chemosensitization of 2H4M (mM) to carvacrol or thymol (mM), tested against S. cerevisiae strains: summary of EUCAST-based microdilution bioassays. a
Yeast Strains CarvacrolCompoundsMIC AloneMIC CombinedFICIMFC AloneMFC CombinedFFCI
S. cerevisiae WTCarvacrol0.80.41.06.4 b3.20.8
2H4M1.60.812.8 c3.2
S. cerevisiae slt2ΔCarvacrol0.40.42.06.43.20.6
2H4M1.61.612.81.6
S. cerevisiae bck1ΔCarvacrol0.40.42.06.43.20.6
2H4M0.80.812.81.6
MeanCarvacrol0.50.41.66.43.20.6
2H4M1.31.112.82.1
t-test dCarvacrol-p < 0.5--p < 0.005-
2H4M-p < 1.0--p < 0.005-
Yeast Strains ThymolCompoundsMIC AloneMIC CombinedFICIMFC AloneMFC CombinedFFCI
S. cerevisiae WTThymol1.60.40.83.21.60.6
2H4M1.60.812.80.8
S. cerevisiae slt2ΔThymol0.80.41.01.61.62.0
2H4M1.60.812.812.8
S. cerevisiae bck1ΔThymol0.80.20.81.61.62.0
2H4M1.60.812.812.8
MeanThymol1.10.30.82.11.61.5
2H4M1.60.812.88.8
t-test dThymol-p < 0.1--p < 0.5-
2H4M-p = 0.0--p < 0.5-
a MIC, Minimum inhibitory concentration; MFC, Minimum fungicidal concentration; FICI, Fractional Inhibitory Concentration Indices; FFCI, Fractional Fungicidal Concentration Indices; b Carvacrol was tested up to 3.2 mM. For calculation purpose, 6.4 mM (doubling of 3.2 mM) was used; c 2H4M was tested up to 6.4 mM. For calculation purpose, 12.8 mM (doubling of 6.4 mM) was used; d Student’s t-test for paired data (combined, i.e., chemosensitization) was vs. mean MIC or MFC of each compound (alone, i.e., no chemosensitization) determined in yeast strains.
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Figure 4. Chemosensitization test in S. cerevisiae (thymol or carvacrol + 2H4M). Exemplary plate bioassay showing co-application of thymol or carvacrol (1.6 to 3.2 mM) with 2H4M (0.8 to 3.2 mM) completely inhibited the growth of S. cerevisiae strains (WT, slt2Δ, bck1Δ).
Figure 4. Chemosensitization test in S. cerevisiae (thymol or carvacrol + 2H4M). Exemplary plate bioassay showing co-application of thymol or carvacrol (1.6 to 3.2 mM) with 2H4M (0.8 to 3.2 mM) completely inhibited the growth of S. cerevisiae strains (WT, slt2Δ, bck1Δ).
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2.4. Chemosensitization Test in Filamentous Fungi: Co-Application of Thymol or Carvacrol with 2H4M

Antifungal chemosensitization was further explored in filamentous fungal pathogens (Aspergillus, Penicillium) according to the CLSI microdilution bioassay protocol [44], where both MICs and MFCs, and thus, both FICIs and FFCIs, of fungal pathogens were determined (See Experimental section for concentrations of test compounds).
For FFCIs of carvacrol in Aspergillus, “synergistic” FFCI values (i.e., FFCI ≤ 0.5) were found between carvacrol and 2H4M for all Aspergillus strains examined (FFCIs = 0.3 to 0.4) (Table 5). Prior studies in yeasts showed that mutations in the antioxidant system, such as oxidative MAPK pathway, could result in the development of fungal resistance to cell wall perturbing agents [50,51,52,53]. Therefore, antioxidant mutants of A. fumigatus (sakAΔ, mpkCΔ) were also included in this study to determine whether antioxidant mutants of filamentous fungi develop resistance to 2H4M or monoterpenoid phenols.
Results showed that oxidative MAPK mutants (sakAΔ, mpkCΔ) were more sensitive to carvacrol compared to WT, where the carvacrol values of WT, sakAΔ or mpkCΔ were (12.8, 3.2, 3.2 mM) for MFCALONE or (1.6, 0.8, 0.8 mM) for MFCCOMBINED, respectively. It is surmised that, similar to other phenolics (See Introduction), carvacrol further exacerbated the vulnerability (namely, defects in countering oxidative stress or disruption of cellular redox homeostasis) of the antioxidant mutants (sakAΔ and mpkCΔ) [27,28] (See also Figure 5). Accordingly, besides the cell wall integrity MAPK system, the operation of the “intact” antioxidant MAPK pathway of A. fumigatus should also be important for pathogen’s defense against carvacrol. In Penicillium, “synergistic” FFCI values were also determined for all Penicillium strains tested (FFCIs = 0.3 to 0.4) (Table 5). Collectively, co-administration of 2H4M with carvacrol lowered the MFCs of either compound (2H4M, carvacrol), which resulted in the achievement of synergism in all Aspergillus and Penicillium strains tested (Mean FFCI of filamentous fungi = 0.3; Table 5).
Table
Table 5. Antifungal chemosensitization of 2H4M (mM) to carvacrol or thymol (mM), tested against Aspergillus and Penicillium strains: summary of CLSI-based microdilution bioassays. a
Table 5. Antifungal chemosensitization of 2H4M (mM) to carvacrol or thymol (mM), tested against Aspergillus and Penicillium strains: summary of CLSI-based microdilution bioassays. a
Fungal StrainsCompoundsMIC AloneMIC CombinedFICIMFC AloneMFC CombinedFFCI
A. fumigatus AF293Carvacrol1.60.40.812.8 b1.60.3
2H4M0.80.412.8 c1.6
A. fumigatus sakAΔCarvacrol1.60.40.83.20.80.4
2H4M0.80.412.81.6
A. fumigatus mpkCΔCarvacrol1.60.40.83.20.80.4
2H4M0.80.412.81.6
A. flavus 3357Carvacrol1.60.81.012.81.60.4
2H4M0.80.43.20.8
A. flavus 4212Carvacrol1.60.80.812.81.60.4
2H4M1.60.43.20.8
A. parasiticus 2999Carvacrol1.60.81.012.80.80.3
2H4M0.80.46.41.6
A. parasiticus 5862Carvacrol1.60.81.012.80.80.3
2H4M0.80.46.41.6
P. expansum W1Carvacrol0.80.20.812.81.60.4
2H4M0.40.212.83.2
P. expansum FR2Carvacrol0.80.41.012.81.60.3
2H4M0.40.212.81.6
P. expansum W2Carvacrol1.60.81.012.81.60.3
2H4M0.80.412.81.6
P. expansum FR3Carvacrol0.80.41.012.81.60.3
2H4M0.80.412.81.6
MeanCarvacrol1.40.60.911.11.30.3
2H4M0.80.49.91.6
t-test dCarvacrol–p < 0.005––p < 0.005–
2H4M–p < 0.005––p < 0.005–
Fungal StrainsCompoundsMIC AloneMIC CombinedFICIMFC AloneMFC CombinedFFCI
A. fumigatus AF293Thymol1.60.40.83.20.80.5
2H4M0.80.412.83.2
A. fumigatus sakAΔThymol1.60.40.81.60.40.5
2H4M0.80.412.83.2
A. fumigatus mpkCΔThymol1.60.40.81.60.40.5
2H4M0.80.412.83.2
A. flavus 3357Thymol1.60.40.83.21.60.6
2H4M1.60.83.20.4
A. flavus 4212Thymol1.60.40.83.21.60.6
2H4M1.60.83.20.4
A. parasiticus 2999Thymol1.60.81.012.8 e0.80.3
2H4M0.80.46.4 f1.6
A. parasiticus 5862Thymol1.60.81.012.80.80.3
2H4M0.80.46.4 f1.6
P. expansum W1Thymol0.80.41.012.81.60.3
2H4M0.40.212.81.6
P. expansum FR2Thymol0.80.41.012.81.60.3
2H4M0.40.212.81.6
P. expansum W2Thymol1.60.81.012.81.60.3
2H4M0.80.412.81.6
P. expansum FR3Thymol0.80.41.012.81.60.3
2H4M0.80.412.81.6
MeanThymol1.40.50.88.11.20.3
2H4M0.90.49.91.8
t-testThymol–p < 0.005--p < 0.005-
2H4M–p < 0.005--p < 0.005-
a MIC, Minimum inhibitory concentration; MFC, Minimum fungicidal concentration; FICI, Fractional Inhibitory Concentration Indices; FFCI, Fractional Fungicidal Concentration Indices. Synergistic FICIs and FFCI are in bold; b Carvacrol was tested up to 6.4 mM. For calculation purpose, 12.8 mM (doubling of 6.4 mM) was used; c 2H4M was tested up to 6.4 mM. For calculation purpose, 12.8 mM (doubling of 6.4 mM) was used; d Student’s t-test for paired data (combined, i.e., chemosensitization) was vs. mean MIC or MFC of each compound (alone, i.e., no chemosensitization) determined in strains; e Thymol was tested up to 6.4 mM. For calculation purpose, 12.8 mM (doubling of 6.4 mM) was used; f 99.8% killing.
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Figure 5. Chemosensitization test in aflatoxigenic Aspergillus or A. fumigatus (thymol or carvacrol + 2H4M). Exemplary bioassay showing co-application of thymol or carvacrol with 2H4M completely inhibited the growth of (a) aflatoxigenic A. flavus 4212 or (b) A. fumigatus. Results also showed that oxidative MAPK mutants (sakAΔ, mpkCΔ) were more sensitive to the chemosensitization compared to WT (Concentrations presented: 1.6 to 3.2 mM, carvacrol or thymol; 0.4 mM, 2H4M).
Figure 5. Chemosensitization test in aflatoxigenic Aspergillus or A. fumigatus (thymol or carvacrol + 2H4M). Exemplary bioassay showing co-application of thymol or carvacrol with 2H4M completely inhibited the growth of (a) aflatoxigenic A. flavus 4212 or (b) A. fumigatus. Results also showed that oxidative MAPK mutants (sakAΔ, mpkCΔ) were more sensitive to the chemosensitization compared to WT (Concentrations presented: 1.6 to 3.2 mM, carvacrol or thymol; 0.4 mM, 2H4M).
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Regarding FICIs of carvacrol in Aspergillus, enhanced activity of 2H4M or carvacrol was also identified during chemosensitization (FICIs = 0.8 to 1.0), although there was no calculated synergism. For example, co-application of 2H4M (0.4 mM) with carvacrol (0.8 mM) achieved complete inhibition of A. parasiticus growth, while individual treatment of each compound, alone, at the same concentrations resulted in the survival of A. parasiticus. However, unlike the MFC testing (See above), the sakAΔ and mpkCΔ mutants did not show higher sensitivity to carvacrol during MIC testing, when compared to WT (Table 5). In Penicillium, despite the no calculated synergism, enhanced activity of 2H4M and carvacrol was also found in all test strains, where FICIs were 0.8 to 1.0. Altogether, co-application of 2H4M and carvacrol resulted in enhancement of antifungal activity of either compound during FICI determination (Mean FICI of filamentous fungi = 0.9; See Table 5).
For FFCIs of thymol in Aspergillus, synergistic FFCIs were identified in A. fumigatus (FFCI = 0.5) and A. parasiticus (FFCI = 0.3). Although no synergism was identified, there was elevated antifungal activity of 2H4M and thymol in A. flavus strains (FFCI = 0.6) during chemosensitization (Table 5). As observed in carvacrol test (See above), A. fumigatus oxidative MAPK mutants (sakAΔ, mpkCΔ) were more susceptible to thymol compared to WT, where the thymol values of WT, sakAΔ or mpkCΔ were (3.2, 1.6, 1.6 mM) for MFCALONE or (0.8, 0.4, 0.4 mM) for MFCCOMBINED, respectively. Higher susceptibility to thymol (compared to carvacrol) was also observed in A. flavus (3357, 4212) (for example, MICCOMBINED or MFCALONE of thymol or carvacrol was (0.4 vs. 0.8 mM) for MICCOMBINED or (3.2 vs. 12.8 mM) for MFCALONE, respectively; Table 5). However, the level of antimycotic activity of carvacrol or thymol was vastly similar in A. parasiticus or P. expansum strains, except MICCOMBINED of P. expansum W1 (viz., MICCOMBINED for carvacrol or thymol was 0.2 or 0.4 mM, respectively). In Penicillium, “synergistic” FFCI values were also determined in all Penicillium strains tested (FFCIs = 0.3) (Table 5). Altogether, co-application of 2H4M and thymol resulted in enhancement of antifungal activity of either compound during FFCI determination (Mean FFCI of filamentous fungi = 0.3; See Table 5). The MFC values of thymol were, in general, lower than that of carvacrol, indicating that, as observed in S. cerevisiae (See above), thymol possessed higher antimycotic potency in selected fungi.
Regarding FICIs of thymol in Aspergillus, enhanced antimycotic activity of 2H4M or thymol was also identified during chemosensitization (FICIs = 0.8 to 1.0), despite the no calculated synergism. For example, co-application of 2H4M (0.4 mM) with thymol (0.8 mM) resulted in complete inhibition of A. parasiticus growth in liquid culture, while individual treatment of each compound, alone, at the same concentrations allowed the survival of A. parasiticus. However, unlike the MFC testing (See above), the sakAΔ and mpkCΔ mutants did not show higher sensitivity to thymol compared to WT in MIC testing (Table 5). In Penicillium, despite not achieving calculated synergism, enhanced activity of 2H4M and thymol was also identified in all test strains, where FICIs were 1.0. Collectively, co-application of 2H4M and thymol achieved the enhancement of antifungal activity of either compound during FICI determination (Mean FICI of filamentous fungi = 0.8; See Table 5).

2.5. Overcoming Fludioxonil Tolerance of A. fumigatus MAPK Mutants by 2H4M Co-Treatment

Fludioxonil, a conventional phenylpyrrole fungicide, elicits excessive stimulation of the intact MAPK signaling pathway, which is responsive to high osmotic/oxidative stress [54]. Thus, the immoderate activation of the osmotic/oxidative MAPK signaling system (HOG1 in S. cerevisiae) by fludioxonil results in an energy drain via metabolic shifts from normal growth to exhaustive stress response. Consequently, treatment of fungi with fludioxonil prevents the normal growth of fungal cells. However, fungal strains having mutations in genes of upstream signal transduction pathway, such as osmotic/oxidative MAPK signaling pathway, can evade toxicity exerted by fludioxonil [54]. We demonstrated that A. fumigatus MAPK mutants (sakAΔ, mpkCΔ) exhibited tolerance to 50 μM fludioxonil (thus, were able to develop radial growth on agar), while the growth of WT (AF293) was completely inhibited (Figure 6). However, co-application of sub-fungicidal concentration of 2H4M (at 0.8 mM, where the fungal growth rate was almost not inhibited) with fludioxonil achieved the prevention of fungal tolerance to the fungicide, which resulted in complete growth inhibition of MAPK mutants (sakAΔ, mpkCΔ) (Figure 6). Cell wall disrupting agents, such as echinocandin drugs, lyse the actively growing hyphal tips of fungi during filamentous fungal growth [23]. It is speculated that the cell wall disturbing capability of 2H4M can enhance the penetration of fludioxonil into the MAPK mutants through perturbed cell wall, which results in complete inhibition of the growth of A. fumigatus sakAΔ and mpkCΔ on fludioxonil-containing plates.
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Figure 6. Overcoming fludioxonil tolerance of A. fumigatus MAPK mutants (sakAΔ, mpkCΔ) by 2H4M (agar plate bioassay).
Figure 6. Overcoming fludioxonil tolerance of A. fumigatus MAPK mutants (sakAΔ, mpkCΔ) by 2H4M (agar plate bioassay).
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2.6. Antimycotoxigenic Property of 2H4M against A. parasiticus Strains

As mentioned previously (See Introduction), sub-optimal application of fungicide can potentiate the production of mycotoxins in various fungal pathogens. Therefore, the effect of the antimycotic 2H4M, thymol and carvacrol on the production of aflatoxins (AFs) was evaluated in the mycotoxigenic Aspergillus strains, i.e., A. flavus 3357, 4212 and A. parasiticus 2999, 5862. Test compounds were administered at sub-MICs or MFCs (0.125 to 1.000 mM), where the sclerotial development was also monitored.
In A. flavus, administration of thymol and carvacrol at 0.125 to 0.500 mM (0.125 to 0.250 mM for 2H4M) enhanced the production of aflatoxins (AFB1, AFB2; See Figure 1 for structures) compared to control (no treatment) (Table 6). Results indicated that, like other commercial fungicides (See Introduction), carvacrol, thymol or 2H4M could potentiate the biosynthesis of mycotoxins in fungi. However, mycotoxin production was lowered at >0.500 mM of monoterpenoid phenols (compared to no treatment control), while fungal growth (accordingly, mycotoxin production) was completely inhibited at ≥0.500 mM of 2H4M, reflecting higher antifungal potency of 2H4M compared to thymol or carvacrol (There was an observable growth defect across fungal species at 0.5 to 1.0 mM concentrations of each antifungal).
Table
Table 6. Effect of 2H4M and monoterpenoid phenols on the production of aflatoxins and sclerotia. Increased values compared to no treatment control are in bold characters. a
Table 6. Effect of 2H4M and monoterpenoid phenols on the production of aflatoxins and sclerotia. Increased values compared to no treatment control are in bold characters. a
Thymol
StrainsConcentrationAFB1AFB2AFG1AFG2Sclerotia
A. flavus 33570.0005.14 ± 0.870.07 ± 0.02– b–6 ± 6
0.1257.38 ± 0.310.14 ± 0.01––47 ± 5
0.2507.74 ± 1.160.14 ± 0.02––84 ± 7
0.5008.72 ± 0.360.19 ± 0.01––31 ± 9
1.0001.16 ± 0.480.01 ± 0.00––0 ± 0
A. flavus 42120.0004.55 ± 0.080.02 ± 0.00––0 ± 0
0.1255.86 ± 0.150.03 ± 0.01––0 ± 0
0.2506.77 ± 0.380.05 ± 0.01––3 ± 2
0.5007.15 ± 0.120.06 ± 0.01––0 ± 0
1.0001.56 ± 0.470.01 ± 0.00––0 ± 0
A. parasiticus 29990.00014.46 ± 0.310.25 ± 0.013.07 ± 0.180.12 ± 0.010 ± 0
0.12518.80 ± 0.060.50 ± 0.015.93 ± 0.060.30 ± 0.01171 ± 23
0.25023.16 ± 1.260.71 ± 0.067.65 ± 0.820.41 ± 0.0428 ± 13
0.50018.19 ± 0.500.49 ± 0.013.96 ± 0.350.17 ± 0.030 ± 0
1.0000.90 ± 0.440.01 ± 0.000.02 ± 0.010.00 ± 0.000 ± 0
A. parasiticus 58620.00015.01 ± 0.920.27 ± 0.023.39 ± 0.240.14 ± 0.010 ± 0
0.12518.64 ± 1.600.49 ± 0.065.98 ± 0.830.30 ± 0.03166 ± 21
0.25022.69 ± 1.380.69 ± 0.077.54 ± 0.820.40 ± 0.0617 ± 13
0.50016.34 ± 1.490.44 ± 0.053.32 ± 0.480.16 ± 0.040 ± 0
1.0000.69 ± 0.220.01 ± 0.000.01 ± 0.010.00 ± 0.000 ± 0
Carvacrol
StrainsConcentrationAFB1AFB2AFG1AFG2Sclerotia
A. flavus 33570.0004.80 ± 0.140.06 ± 0.00– b–2 ± 1
0.1255.78 ± 0.310.09 ± 0.01––64 ± 11
0.2505.73 ± 0.530.10 ± 0.01––59 ± 8
0.5005.96 ± 0.360.13 ± 0.01––0 ± 0
1.0001.23 ± 0.450.01 ± 0.01––0 ± 0
A. flavus 42120.0005.51 ± 0.560.03 ± 0.01––0 ± 0
0.1256.84 ± 0.660.05 ± 0.01––1 ± 2
0.2507.50 ± 0.660.07 ± 0.01––8 ± 4
0.5006.50 ± 0.290.07 ± 0.01––0 ± 0
1.0000.91 ± 0.320.01 ± 0.00––0 ± 0
A. parasiticus 29990.00014.02 ± 1.580.25 ± 0.062.75 ± 0.540.12 ± 0.031 ± 1
0.12517.30 ± 0.830.37 ± 0.033.22 ± 0.270.14 ± 0.0162 ± 4
0.25017.38 ± 0.260.43 ± 0.012.90 ± 0.120.13 ± 0.0146 ± 5
0.50019.54 ± 0.880.52 ± 0.042.20 ± 0.130.08 ± 0.000 ± 0
1.0000.09 ± 0.040.00 ± 0.000.00 ± 0.000.00 ± 0.000 ± 0
A. parasiticus 58620.00013.92 ± 0.360.23 ± 0.022.84 ± 0.100.10 ± 0.021 ± 1
0.12516.65 ± 0.880.35 ± 0.033.19 ± 0.280.12 ± 0.0144 ± 6
0.25016.51 ± 1.070.39 ± 0.042.68 ± 0.220.11 ± 0.0125 ± 3
0.50020.10 ± 0.490.52 ± 0.032.32 ± 0.100.07 ± 0.010 ± 0
1.0000.12 ± 0.120.00 ± 0.000.00 ± 0.000.00 ± 0.000 ± 0
2H4M
StrainsConcentrationAFB1AFB2AFG1AFG2Sclerotia
A. flavus 33570.0004.73 ± 0.230.06 ± 0.01– b–4 ± 3
0.1255.03 ± 0.350.08 ± 0.01––0 ± 0
0.2504.84 ± 0.060.10 ± 0.01––0 ± 0
0.500NG cNGNGNGNG
1.000NGNGNGNGNG
A. flavus 42120.0004.72 ± 0.240.02 ± 0.01––0 ± 0
0.1256.58 ± 1.020.05 ± 0.01––0 ± 0
0.2505.69 ± 0.550.04 ± 0.01––0 ± 0
0.500NGNGNGNGNG
1.000NGNGNGNGNG
A. parasiticus 29990.00015.92 ± 1.280.27 ± 0.043.20 ± 0.330.13 ± 0.030 ± 1
0.12512.80 ± 0.260.24 ± 0.012.62 ± 0.080.12 ± 0.000 ± 0
0.25010.16 ± 0.790.17 ± 0.022.23 ± 0.240.06 ± 0.020 ± 0
0.500NGNGNGNGNG
1.000NGNGNGNGNG
A. parasiticus 58620.00014.21 ± 1.020.23 ± 0.032.91 ± 0.350.11 ± 0.010 ± 1
0.12512.65 ± 0.530.23 ± 0.022.65 ± 0.070.10 ± 0.010 ± 0
0.25010.95 ± 1.190.19 ± 0.042.38 ± 0.430.06 ± 0.020 ± 0
0.500NGNGNGNGNG
1.000NGNGNGNGNG
a Thymol, Carvacrol, 2H4M: mM; aflatoxins: μg/cm2; Sclerotia: numbers per plate; b Below the detection limit; c NG, No growth (accordingly, no aflatoxin detected).
In A. parasiticus, treatment of fungi with thymol and carvacrol at 0.125 to 0.500 mM also enhanced the production of aflatoxins (AFB1, AFB2, AFG1, AFG2; See Figure 1 for structures) compared to control (no treatment) (Table 6). As observed in A. flavus, mycotoxin production was lowered at >0.500 mM of monoterpenoid phenols (compared to no treatment control), while fungal growth (accordingly, mycotoxin production) was also completely prevented at ≥0.500 mM of 2H4M. In general, A. parasiticus produced higher amounts of aflatoxins compared to A. flavus, when treated with thymol or carvacrol (See Table 6).
Notable observation is that, unlike the A. flavus strains, 2H4M actually inhibited the aflatoxin production in A. parasiticus at all test concentrations (Table 6), where the level of inhibition was commensurate with the 2H4M concentration. Therefore, results strongly indicated that “strain specificity” also existed for the “antimycotoxigenic activity” of 2H4M.
Of note, in a prior study, oxidative stress regulated the sclerotial differentiation, where application of antioxidant modulators of reactive oxygen species prohibited the development of sclerotia [55]. Furthermore, aflatoxin biosynthesis and sclerotial differentiation were co-modulated by oxidative stress [55]. Therefore, sclerotial development/differentiation is considered as the indicator of fungal stress response, especially to oxidative stress.
In this study, treatment of A. flavus or A. parasiticus strains with thymol or carvacrol also enhanced the sclerotial differentiation, depending on dosages and types of test compounds. A. flavus 3357 produced higher number of sclerotia compared to A. flavus 4212 (with both thymol and carvacrol), while A. parasiticus strains developed more sclerotia with thymol than with carvacrol (Table 6). Conversely, sclerotia development was almost not detected with the treatment of 2H4M, strongly indicating that: (1) the mechanism of action of 2H4M for the antifungal or antimycotoxigenic activity against Aspergillus strains is considered to be different from that of monoterpenoid phenols; and (2) antifungal or antimycotoxigenic activity is dependent upon types (or species) of Aspergillus strains examined. Noteworthy is the effect of cell wall stress in the production of the secondary metabolite pyomelanin in A. fumigatus. Previous study showed that pyomelanin production was enhanced when constitutive cell wall stress was applied to A. fumigatus [56], strongly indicating the link between secondary metabolite production and cell wall stress. Recent study further revealed that genes involved in the pyomelanin synthesis were all up-regulated when the cell wall integrity MAPK mutant ΔmpkA was cultured under cell wall stress [57].
The strain specificity for the differential antimycotoxigenic activity with 2H4M (i.e., A. flavus vs. A. parasiticus), as determined in this study, is a new observation, which may reflect the different level of cell wall stress triggered by 2H4M in pathogens, depending on types of fungal species. Precise determination of the mechanism of differential activity regarding the antifungal or antimycotoxigenic activity of 2H4M warrants future study.

3. Experimental Section

3.1. Fungal Strains and Culture Conditions

Fungal strains used in this study are summarized in Table 2. Aspergillus and Penicillium strains were grown at 35 °C and 28 °C (Forma Scientific, Marietta, OH, USA), respectively, on potato dextrose agar (PDA). Saccharomyces cerevisiae wild type (WT) BY4741 (Mat a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and selected single gene deletion mutants (bck1Δ, slt2Δ) were procured from Invitrogen (Carlsbad, CA, USA) and Open Biosystems (Huntsville, AL, USA; See also Saccharomyces Genome Database [30]). Yeast strains were cultured on Synthetic Glucose (SG; Yeast nitrogen base without amino acids 0.67%, glucose 2% with appropriate supplements: uracil 0.02 mg/mL, amino acids 0.03 mg/mL) or Yeast Peptone Dextrose (YPD; Bacto yeast extract 1%, Bacto peptone 2%, glucose 2%) medium at 30 °C. All chemicals for culturing fungi were procured from Sigma Co. (St. Louis, MO, USA).

3.2. Chemicals

Benzaldehyde (basal structure) and its structural analogs (i.e., fourteen benzaldehyde derivatives; See Figure 1), two monoterpenoid phenols (thymol, carvacrol; cell wall integrity disruptors), caffeine, sorbitol, and fludioxonil (fungicide) were procured from Sigma Co. (St. Louis, MO, USA). Each compound was dissolved in dimethylsulfoxide (DMSO from AMRESCO (Solon, OH, USA); absolute DMSO amount: <2% in media) before incorporation into culture media (except for those plates used in aflatoxin assays; see below). In all tests, control plates (i.e., No treatment) contained DMSO at levels equivalent to that of cohorts receiving antifungal agents, within the same set of experiments (See Tables and Figures).

3.3. Susceptibility Testing

3.3.1. Agar Plate Bioassay in S. cerevisiae

Petri plate-based yeast dilution bioassays were performed on the WT and mutants (slt2Δ, bck1Δ) to assess effects of screened compounds on the cell wall integrity system. Yeast strains were exposed to 1 to 5 mM of benzaldehyde analogs screened. 1 × 106 cells of the WT or cell wall integrity mutants (bck1Δ, slt2Δ) of S. cerevisiae, cultured on YPD plate, were serially diluted 10-fold in SG liquid medium supplemented with amino acids and uracil (See above) five times to yield cell dilution cohorts of 106, 105, 104, 103, 102 and 101 cells. Cells from each dilution of respective strains were spotted on SG agar incorporated with individual compounds, such as benzaldehyde analogs or other test reagents. Yeast cells were incubated at 30 °C. Results were monitored/evaluated based on a designated value of the highest dilution where colonies became visible after 5 to 7 days of incubation, as follows: Score “0”—no colonies were visible from any of the dilutions, Score “1”—only a colony from the spot with the undiluted cells (106 cells), Score “2” only colonies from the spots with the undiluted (106) and 105 cells were visible, etc., while Score “6”—colonies were visible from all dilution spots. Therefore, each unit (1 to 6) of numerical difference was equivalent to a 10-fold difference in the sensitivity of the yeast strain to the treatment.

3.3.2. Agar Plate Bioassay in Aspergillus: Overcoming Fludioxonil Resistance of A. fumigatus sakAΔ and mpkCΔ Mutants

Measurement of overcoming fungal (A. fumigatus sakAΔ, mpkCΔ) tolerance to fludioxonil was based on comparison of radial growth between treated and control fungal colonies (See Figure 6). For the above assays, fungal conidia (5 × 103) were diluted in phosphate buffered saline and applied as a drop onto the center of PDA plates containing: (1) No treatment (control); (2) 2H4M (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mM); (3) fludioxonil (50 μM); and (4) 2H4M + fludioxonil. Growth was observed for five to seven days at 35 °C.

3.3.3. Liquid Bioassay in Filamentous Fungi (CLSI) and S. cerevisiae (EUCAST)

To determine the precise level of compound interaction between 2H4M (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mM) and monoterpenoid phenols (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mM) in the strains of filamentous fungi (Aspergillus, Penicillium), checkerboard bioassays (triplicate) (0.4 × 104–5 × 104 CFU/mL) were performed in microtiter wells using a broth microdilution method (in RPMI 1640 medium; Sigma Co., St. Louis, MO, USA), according to protocols outlined by the Clinical and Laboratory Standards Institute (CLSI) M38-A [44]. RPMI 1640 medium was supplemented with 0.03% l-glutamine and buffered with 0.165 mM 3-(N-morpholino) propanesulfonic acid (Sigma Co., St. Louis, MO, USA). Minimum Inhibitory Concentrations (MICs), lowest concentration of agents showing no visible fungal growth in microtiter wells (200 μL per well), were assessed after 48 h. Minimum Fungicidal Concentrations (MFCs), lowest concentration of agents achieving ≥99.9% fungal death, were determined following completion of MIC assays by spreading entire volumes of microtiter wells (200 μL) onto individual PDA (recovery) plates, and culturing for additional 48 h (at 28 or 35 °C, depending on types of filamentous fungi). Compound interactions, i.e., Fractional Inhibitory Concentration Indices (FICIs) and Fractional Fungicidal Concentration Indices (FFCI), were calculated as follows: FICI or FFCI = (MIC or MFC of compound A in combination with compound B/MIC or MFC of compound A, alone) + (MIC or MFC of compound B in combination with compound A/MIC or MFC of compound B, alone). Levels and types of compound interactions between antifungal agents (2H4M and thymol or carvacrol) were defined as: synergistic (FICI or FFCI ≤ 0.5) or indifferent (FICI or FFCI > 0.5–4) [49].
Compound interaction between 2H4M (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mM) and thymol or carvacrol (0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mM) in S. cerevisiae was also determined by using checkerboard broth dilution bioassays in microtiter plates (with SG liquid medium; Sigma Co., St. Louis, MO, USA) according to methods outlined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [48].

3.4. Growth Recovery Bioassay for S. cerevisiae bck1Δ and slt2Δ Mutants

To examine the effect of antifungal agents (2-Hydroxy-4-methoxybenzaldehyde (2H4M), 2,3-Dimethoxybenzaldehyde (2,3-DMBA), thymol, carvacrol) on cell wall integrity system of fungi, sorbitol recovery bioassays were performed (See [47] for method). Tenfold serially diluted (See “Agar plate bioassays in S. cerevisiae” above) strains of S. cerevisiae BY4741, bck1Δ and slt2Δ were spotted on (1) SG only (No treatment control); (2) SG + caffeine (5.0 mM; Positive control), thymol or carvacrol (0.1 0.2, 0.3, 0.4, 0.5 mM), 2H4M (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.5 mM) or 2,3-DMBA (0.5, 1.0, 2.0, 3.0, 4.0, 5.0 mM) (viz., Testing sensitivity of bck1Δ and slt2Δ mutants to test compounds); and (3) SG + sorbitol (0.5 M) + caffeine, thymol, carvacrol, 2H4M or 2,3-DMBA (viz., Testing recovery of bck1Δ and slt2Δ mutants, by sorbitol, from sensitivity to antifungal reagents). Cell growth was monitored for 5 to 7 days. If the growth score of S. cerevisiae bck1Δ and slt2Δ on the sorbitol-containing medium was higher than that on the “no sorbitol” medium, the test compounds were considered to affect cell wall integrity system.

3.5. Aflatoxin Analysis of Fungal Cultures

Test compounds (thymol, carvacrol, 2H4M), dissolved in distilled water, were filter-sterilized (Millipore Millex GP, Billerica, MA, USA) before incorporation into PDA. Ten mL of PDA containing test compounds (See Table 6 and text for concentrations) were poured into Petri dish (60 mm) in triplicate for each concentration. Fungal spores of A. parasiticus and A. flavus strains, stored on PDA at 30 °C for 7 days, were collected on a sterile cotton swab and dispersed in 0.05% Tween 80 solution (Sigma Co., St. Louis, MO, USA). Fungal conidia (200 CFU/5 µL) from the prepared suspensions were applied as a drop onto the center of PDA plates, and were incubated at 30 °C for 5 days. The entire culture (agar medium plus fungal mat) was then extracted in 50 mL methanol (Fisher, Thermo Fisher Scientific, Waltham, MA, USA), and 1 mL aliquot was filtered through a 0.45 µm nylon syringe filter (Pall Acrodisc, Port Washington, NY, USA). An aliquot of 20 µL was analyzed for aflatoxins using an Agilent 1100 HPLC system (Santa Clara, CA, USA), which consists of a degasser, autosampler, quaternary pump, fluorescence detector, and a postcolumn photochemical reactor for enhanced detection (PHRED, Aura Industries, New York, NY, USA). Aflatoxins were separated on an Inertsil 4.6 mm × 250 mm ODS-3 column (GL Sciences, Torrance, CA, USA) using an isocratic mobile phase of water/acetonitrile/methanol (45/25/30) at a flow rate of 1 mL/min with fluorescence detection (365 nm excitation, 455 nm emission). Aflatoxin values were determined using standard curves prepared for each of the individual compounds.

3.6. Statistical Analysis

Statistical analysis (student’s t-test) was performed based on “Statistics to use” [58], where p < 0.05 was considered significant.

4. Conclusions

In this study, levels of compound interactions between monoterpenoid phenols (carvacrol, thymol) and a chemosensitizer (2H4M) were determined for the enhancement of antifungal efficacy. Key features identified for the antifungal or antimycotoxigenic potential of compounds are as follows: (1) Among fifteen benzaldehyde analogs examined, nine compounds inhibited the growth of bck1Δ and slt2Δ, the cell wall integrity mutants of S. cerevisiae, with structure-activity relationship. 2H4M exhibited the highest antifungal activity among test compounds; (2) 2H4M possessed a chemosensitizing capability to carvacrol or thymol in yeasts, where chemosensitization enhanced the antimycotic potency of test compounds. The 2H4M, a cell wall perturbing chemosensitizer, and monoterpenoid phenols could affect common cellular targets, i.e., cell wall integrity system of fungi, which results in synergistic inhibition of fungal growth; (3) In yeast chemosensitization, thymol required much lower concentration to achieve complete inhibition of yeast growth compared to carvacrol, thus reflecting structure-activity relationship; (4) In filamentous fungal tests, co-administration of 2H4M with carvacrol or thymol resulted in the achievement of synergism in Aspergillus and Penicillium strains; (5) Carvacrol or thymol further exacerbated the vulnerability (namely, defects in countering oxidative stress) of the oxidative MAPK mutants (sakAΔ, mpkCΔ) of A. fumigatus; (6) A. fumigatus or A. flavus were more susceptible to thymol compared to carvacrol, while the level of antimycotic activity of carvacrol or thymol was vastly similar in A. parasiticus or P. expansum (compound-strain relationship); (7) Co-application of 2H4M with fludioxonil overcame fungal tolerance to fludioxonil, where the cell wall interfering capability of 2H4M might enhance the susceptibility of the oxidative MAPK mutants (A. fumigatus sakAΔ, mpkCΔ) to fludioxonil, possibly by increased penetration of fludioxonil into the fungal cell through perturbed cell wall; (8) Thymol and carvacrol enhanced aflatoxin production in A. flavus and A. parasiticus. Although 2H4M also potentiated the aflatoxin production in A. flavus, this compound reduced the aflatoxin production in A. parasiticus at all concentrations examined (i.e., strain specificity for the antimycotoxigenic activity of 2H4M); (9) Thymol or carvacrol also enhanced the sclerotial differentiation in A. flavus or A. parasiticus, depending on dosages and types of test compounds. A. flavus 3357 produced higher number of sclerotia compared to A. flavus 4212 (w/carvacrol or thymol), while A. parasiticus developed more sclerotia with thymol than with carvacrol. However, sclerotia development was almost not detected with the treatment of 2H4M.
In previous studies in yeasts, another signaling pathway, namely the “intact” oxidative MAPK pathway, was also shown to be important for fungal susceptibility to cell wall perturbing agents [50,51,52,53]. Results showed that mutations in the antioxidant system could develop fungal resistance to cell wall disrupting agents. For example, the S. cerevisiae oxidative MAPK pathway mutants (e.g., MAPK or MAPK kinase mutants, the upstream transmembrane osmosensor or histidine kinase osmosensor mutants in the same signaling cascade, etc.) exhibited tolerance to the cell wall-perturbing agents, such as calcofluor white [51,52,53]. Therefore, existence of the “intact” oxidative MAPK pathway of fungi is necessary for effective control of pathogens. Nonetheless, the antioxidant mutants of A. fumigatus (sakAΔ, mpkCΔ) tested in this study did not develop tolerance to 2H4M or monoterpenoid phenols, while 2H4M further overcame fungal tolerance to the phenylpyrrole fungicide fludioxonil.
In conclusion, 2H4M, a natural phenolic compound, possesses a potential to serve as an antimycotic chemosensitizer in combination with monoterpenoid phenols. 2H4M-mediated chemosensitization, as described in this study, can modulate/debilitate the cell wall integrity system of fungal strains, which can lower effective doses of antimycotic agent co-administered. Future studies are needed for comprehensive determination of optimum chemosensitization in various fungal pathogens by including additional cell wall disrupting agents. It would also be interesting to know whether antifungal activity correlates with chemosensitizing activity, or whether a circumstance similar to that for piperazinyl quinolone with fluconazole [32], exists for benzaldehyde analogs with no antifungal activity.

Acknowledgments

This research was conducted under USDA-ARS CRIS Project 5325-42000-037-00D.

Author Contributions

Jong H. Kim designed and performed research including data analysis and interpretation, literature search, and wrote the manuscript. Kathleen L. Chan performed antifungal bioassays and prepared the figures. Noreen Mahoney analyzed aflatoxin production in Aspergillus strains.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Denning, D.W. Invasive aspergillosis. Clin. Infect. Dis. 1998, 26, 781–803. [Google Scholar] [CrossRef] [PubMed]
  2. Anderson, M.J.; Brookman, J.L.; Denning, D.W. Aspergillus. In Genomics of Plants and Fungi; Prade, R.A., Bohnert, H.J., Eds.; Marcel Dekker: New York, NY, USA, 2003; pp. 1–39. [Google Scholar]
  3. Marr, K.A.; Patterson, T.; Denning, D. Aspergillosis. Pathogenesis, clinical manifestations, and therapy. Infect. Dis. Clin. N. Am. 2002, 16, 875–894. [Google Scholar] [CrossRef]
  4. Roilides, E.; Holmes, A.; Blake, C.; Pizzo, P.A.; Walsh, T.J. Impairment of neutrophil antifungal activity against hyphae of Aspergillus fumigatus in children infected with human immunodeficiency virus. J. Infect. Dis. 1993, 167, 905–911. [Google Scholar] [CrossRef] [PubMed]
  5. Campbell, B.C.; Molyneux, R.J.; Schatzki, T.F. Current research on reducing pre- and post-harvest aflatoxin contamination of US almond, pistachio and walnut. J. Toxicol. Toxin Rev. 2003, 22, 225–266. [Google Scholar] [CrossRef]
  6. Frizzell, C.; Elliott, C.T.; Connolly, L. Effects of the mycotoxin patulin at the level of nuclear receptor transcriptional activity and steroidogenesis in vitro. Toxicol. Lett. 2014, 229, 366–373. [Google Scholar] [CrossRef] [PubMed]
  7. Cools, H.J.; Hammond-Kosack, K.E. Exploitation of genomics in fungicide research: Current status and future perspectives. Mol. Plant Pathol. 2013, 14, 197–210. [Google Scholar] [CrossRef] [PubMed]
  8. Possiede, Y.M.; Gabardo, J.; Kava-Cordeiro, V.; Galli-Terasawa, L.V.; Azevedo, J.L.; Glienke, C. Fungicide resistance and genetic variability in plant pathogenic strains of Guignardia citricarpa. Braz. J. Microbiol. 2009, 40, 308–313. [Google Scholar] [CrossRef] [PubMed]
  9. Ellner, F.M. Results of long-term field studies into the effect of strobilurin containing fungicides on the production of mycotoxins in several winter wheat varieties. Mycotoxin Res. 2005, 21, 112–115. [Google Scholar] [CrossRef] [PubMed]
  10. Bayer CropScience. Strobilurin Fungicides Increase DON Levels in Wheat, Reduce Quality. Available online: https://www.bayercropscience.us/~/media/BayerCropScience/Country-United-States-Internet/Documents/News/2014AgIssues/docs/FusariumHeadBlightFeatureStory.ashx (accessed on 25 August 2015).
  11. Markoglou, A.N.; Doukas, E.G.; Malandrakis, A.A. Effect of anilinopyrimidine resistance on aflatoxin production and fitness parameters in Aspergillus parasiticus Speare. Int. J. Food Microbiol. 2011, 146, 130–136. [Google Scholar] [CrossRef] [PubMed]
  12. Doukas, E.G.; Markoglou, A.N.; Vontas, J.G.; Ziogas, B.N. Effect of DMI-resistance mechanisms on cross-resistance patterns, fitness parameters and aflatoxin production in Aspergillus parasiticus Speare. Fungal Genet. Biol. 2012, 49, 792–801. [Google Scholar] [CrossRef] [PubMed]
  13. Markoglou, A.N.; Doukas, E.G.; Ziogas, B.N. Phenylpyrrole-resistance and aflatoxin production in Aspergillus parasiticus Speare. Int. J. Food Microbiol. 2008, 127, 268–275. [Google Scholar] [CrossRef] [PubMed]
  14. Zhang, Y.J.; Yu, J.J.; Zhang, Y.N.; Zhang, X.; Cheng, C.J.; Wang, J.X.; Hollomon, D.W.; Fan, P.S.; Zhou, M.G. Effect of carbendazim resistance on trichothecene production and aggressiveness of Fusarium graminearum. Mol. Plant Microbe Interact. 2009, 22, 1143–1150. [Google Scholar] [CrossRef] [PubMed]
  15. D’Mello, J.P.F.; Macdonald, A.M.C.; Briere, L. Mycotoxin production in a carbendazim-resistant strain of Fusarium sporotrichioides. Mycotoxin Res. 2000, 16, 101–111. [Google Scholar] [CrossRef] [PubMed]
  16. Karaoglanidis, G.S.; Markoglou, A.N.; Bardas, G.A.; Doukas, E.G.; Konstantinou, S.; Kalampokis, J.F. Sensitivity of Penicillium expansum field isolates to tebuconazole, iprodione, fludioxonil and cyprodinil and characterization of fitness parameters and patulin production. Int. J. Food Microbiol. 2011, 145, 195–204. [Google Scholar] [CrossRef] [PubMed]
  17. Malandrakis, A.A.; Markoglou, A.N.; Konstantinou, S.; Doukas, E.G.; Kalampokis, J.F.; Karaoglanidis, G.S. Molecular characterization, fitness and mycotoxin production of benzimidazole-resistant isolates of Penicillium expansum. Int. J. Food Microbiol. 2013, 162, 237–244. [Google Scholar] [CrossRef] [PubMed]
  18. Schmidt-Heydt, M.; Stoll, D.; Geisen, R. Fungicides effectively used for growth inhibition of several fungi could induce mycotoxin biosynthesis in toxigenic species. Int. J. Food Microbiol. 2013, 166, 407–412. [Google Scholar] [CrossRef] [PubMed]
  19. Beauvais, A.; Latgé, J.P. Membrane and cell wall targets in Aspergillus fumigatus. Drug Resist. Updat. 2001, 4, 38–49. [Google Scholar] [CrossRef] [PubMed]
  20. Levin, D.E.; Fields, F.O.; Kunisawa, R.; Bishop, J.M.; Thorner, J. A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle. Cell 1990, 62, 213–224. [Google Scholar] [CrossRef]
  21. Fuchs, B.B.; Mylonakis, E. Our paths might cross: The role of the fungal cell wall integrity pathway in stress response and cross talk with other stress response pathways. Eukaryot. Cell 2009, 8, 1616–1625. [Google Scholar] [CrossRef] [PubMed]
  22. Fujioka, T.; Mizutani, O.; Furukawa, K.; Sato, N.; Yoshimi, A.; Yamagata, Y.; Nakajima, T.; Abe, K. MpkA-Dependent and -independent cell wall integrity signaling in Aspergillus nidulans. Eukaryot. Cell 2007, 6, 1497–1510. [Google Scholar] [CrossRef] [PubMed]
  23. Perlin, D.S. Mechanisms of echinocandin antifungal drug resistance. Ann. N. Y. Acad. Sci. 2015. [Google Scholar] [CrossRef] [PubMed]
  24. Walker, L.A.; Lee, K.K.; Munro, C.A.; Gow, N.A. Caspofungin treatment of Aspergillus fumigatus results in ChsG-dependent upregulation of chitin synthesis and the formation of chitin-rich micro-colonies. Antimicrob. Agents Chemother. 2015. [Google Scholar] [CrossRef] [PubMed]
  25. Odds, F.C.; Brown, A.J.; Gow, N.A. Antifungal agents: Mechanisms of action. Trends Microbiol. 2003, 11, 272–279. [Google Scholar] [CrossRef]
  26. Aspergillus Comparative Database. Available online: http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html (accessed on 25 August 2015).
  27. Xue, T.; Nguyen, C.K.; Romans, A.; May, G.S. A mitogen-activated protein kinase that senses nitrogen regulates conidial germination and growth in Aspergillus fumigatus. Eukaryot. Cell 2004, 3, 557–560. [Google Scholar] [CrossRef] [PubMed]
  28. Reyes, G.; Romans, A.; Nguyen, C.K.; May, G.S. Novel mitogen-activated protein kinase MpkC of Aspergillus fumigatus is required for utilization of polyalcohol sugars. Eukaryot. Cell 2006, 5, 1934–1940. [Google Scholar] [CrossRef] [PubMed]
  29. Li, H.X.; Xiao, C.L. Characterization of fludioxonil-resistant and pyrimethanil-resistant phenotypes of Penicillium expansum from apple. Phytopathology 2008, 98, 427–435. [Google Scholar] [CrossRef] [PubMed]
  30. Saccharomyces Genome Database. Available online: http://www.yeastgenome.org (accessed on 22 August 2015).
  31. Campbell, B.C.; Chan, K.L.; Kim, J.H. Chemosensitization as a means to augment commercial antifungal agents. Front. Microbiol. 2012, 3, 79. [Google Scholar] [CrossRef] [PubMed]
  32. Youngsaye, W.; Hartland, C.L.; Morgan, B.J.; Ting, A.; Nag, P.P.; Vincent, B.; Mosher, C.A.; Bittker, J.A.; Dandapani, S.; Palmer, M.; et al. ML212: A small-molecule probe for investigating fluconazole resistance mechanisms in Candida albicans. Beilstein J. Org. Chem. 2013, 9, 1501–1507. [Google Scholar] [CrossRef] [PubMed]
  33. Keniya, M.V.; Fleischer, E.; Klinger, A.; Cannon, R.D.; Monk, B.C. Inhibitors of the Candida albicans major facilitator superfamily transporter Mdr1p responsible for fluconazole resistance. PLoS ONE 2015, 10, e0126350. [Google Scholar] [CrossRef] [PubMed]
  34. Niimi, K.; Harding, D.R.; Parshot, R.; King, A.; Lun, D.J.; Decottignies, A.; Niimi, M.; Lin, S.; Cannon, R.D.; Goffeau, A.; et al. Chemosensitization of fluconazole resistance in Saccharomyces cerevisiae and pathogenic fungi by a d-octapeptide derivative. Antimicrob. Agents Chemother. 2004, 48, 1256–1271. [Google Scholar] [CrossRef] [PubMed]
  35. Freiesleben, S.H.; Jäger, A.K. Correlation between plant secondary metabolites and their antifungal mechanisms—A review. Med. Aromat. Plants 2014, 3, 154. [Google Scholar]
  36. Beekrum, S.; Govinden, R.; Padayachee, T.; Odhav, B. Naturally occurring phenols: A detoxification strategy for fumonisin B1. Food Addit. Contam. 2003, 20, 490–493. [Google Scholar] [CrossRef] [PubMed]
  37. Guillen, F.; Evans, C.S. Anisaldehyde and veratraldehyde acting as redox cycling agents for H2O2 production by Pleurotus eryngii. Appl. Environ. Microbiol. 1994, 60, 2811–2817. [Google Scholar] [PubMed]
  38. Jacob, C. A scent of therapy: Pharmacological implications of natural products containing redox-active sulfur atoms. Nat. Prod. Rep. 2006, 23, 851–863. [Google Scholar] [CrossRef] [PubMed]
  39. Rao, A.; Zhang, Y.; Muend, S.; Rao, R. Mechanism of antifungal activity of terpenoid phenols resembles calcium stress and inhibition of the TOR pathway. Antimicrob. Agents Chemother. 2010, 54, 5062–5069. [Google Scholar] [CrossRef] [PubMed]
  40. U.S. Food and Drug Administration (FDA). Everything Added to Food in the United States. Available online: http://www.fda.gov/Food/IngredientsPackagingLabeling/FoodAdditivesIngredients/ucm115326.htm (accessed on 7 August 2015).
  41. Bi, X.; Guo, N.; Jin, J.; Liu, J.; Feng, H.; Shi, J.; Xiang, H.; Wu, X.; Dong, J.; Hu, H.; et al. The global gene expression profile of the model fungus Saccharomyces cerevisiae induced by thymol. J. Appl. Microbiol. 2010, 108, 712–722. [Google Scholar] [CrossRef] [PubMed]
  42. Jeong, E.Y.; Cho, K.S.; Lee, H.S. Food protective effects of Periploca sepium oil and its active component against stored food mites. J. Food Prot. 2012, 75, 118–122. [Google Scholar] [CrossRef] [PubMed]
  43. Shi, J.; Yamashita, T.; Todo, A.; Nitoda, T.; Izumi, M.; Baba, N.; Nakajima, S. Repellent from traditional Chinese medicine, Periploca sepium Bunge. Z. Naturforsch. C 2007, 62, 821–825. [Google Scholar] [CrossRef] [PubMed]
  44. Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi: Approved Standard–Second Edition. CLSI document M38-A2; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2008. [Google Scholar]
  45. Reinoso-Martín, C.; Schüller, C.; Schuetzer-Muehlbauer, M.; Kuchler, K. The yeast protein kinase C cell integrity pathway mediates tolerance to the antifungal drug caspofungin through activation of Slt2p mitogen-activated protein kinase signaling. Eukaryot. Cell 2003, 2, 1200–1210. [Google Scholar] [CrossRef] [PubMed]
  46. Senter, P.D.; Al-Abed, Y.; Metz, C.N.; Benigni, F.; Mitchell, R.A.; Chesney, J.; Han, J.; Gartner, C.G.; Nelson, S.D.; Todaro, G.J.; et al. Inhibition of macrophage migration inhibitory factor (MIF) tautomerase and biological activities by acetaminophen metabolites. Proc. Natl. Acad. Sci. USA 2002, 99, 144–149. [Google Scholar] [CrossRef] [PubMed]
  47. Ferreira, C.; Silva, S.; van Voorst, F.; Aguiar, C.; Kielland-Brandt, M.C.; Brandt, A.; Lucas, C. Absence of Gup1p in Saccharomyces cerevisiae results in defective cell wall composition, assembly, stability and morphology. FEMS Yeast Res. 2006, 6, 1027–1038. [Google Scholar] [CrossRef] [PubMed]
  48. Arendrup, M.C.; Cuenca-Estrella, M.; Lass-Flörl, C.; Hope, W. EUCAST technical note on the EUCAST definitive document EDef 7.2: Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST). Clin. Microbiol. Infect. 2012, 18, E246–E247. [Google Scholar] [CrossRef] [PubMed]
  49. Odds, F.C. Synergy, antagonism, and what the chequerboard puts between them. J. Antimicrob. Chemother. 2003, 52, 1. [Google Scholar] [CrossRef] [PubMed]
  50. Alonso-Monge, R.; Navarro-García, F.; Molero, G.; Diez-Orejas, R.; Gustin, M.; Pla, J.; Sánchez, M.; Nombela, C. Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans. J. Bacteriol. 1999, 181, 3058–3068. [Google Scholar] [PubMed]
  51. García-Rodriguez, L.J.; Durán, A.; Roncero, C. Calcofluor antifungal action depends on chitin and a functional high-osmolarity glycerol response (HOG) pathway: Evidence for a physiological role of the Saccharomyces cerevisiae HOG pathway under noninducing conditions. J. Bacteriol. 2000, 182, 2428–2437. [Google Scholar] [CrossRef] [PubMed]
  52. Jiang, B.; Ram, A.F.; Sheraton, J.; Klis, F.M.; Bussey, H. Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2 action in a pathway that includes modulation of EXG1 transcription. Mol. Gen. Genet. 1995, 248, 260–269. [Google Scholar] [CrossRef] [PubMed]
  53. Lai, M.H.; Silverman, S.J.; Gaughran, J.P.; Kirsch, D.R. Multiple copies of PBS2, MHP1 or LRE1 produce glucanase resistance and other cell wall effects in Saccharomyces cerevisiae. Yeast 1997, 13, 199–213. [Google Scholar] [CrossRef]
  54. Kojima, K.; Takano, Y.; Yoshimi, A.; Tanaka, C.; Kikuchi, T.; Okuno, T. Fungicide activity through activation of a fungal signalling pathway. Mol. Microbiol. 2004, 53, 1785–1796. [Google Scholar] [CrossRef] [PubMed]
  55. Grintzalis, K.; Vernardis, S.I.; Klapa, M.I.; Georgiou, C.D. Role of oxidative stress in sclerotial differentiation and aflatoxin B1 biosynthesis in Aspergillus flavus. Appl. Environ. Microbiol. 2014, 80, 5561–5571. [Google Scholar] [CrossRef] [PubMed]
  56. Valiante, V.; Jain, R.; Heinekamp, T.; Brakhage, A.A. The MpkA MAP kinase module regulates cell wall integrity signaling and pyomelanin formation in Aspergillus fumigatus. Fungal Genet. Biol. 2009, 46, 909–918. [Google Scholar] [CrossRef] [PubMed]
  57. Jain, R.; Valiante, V.; Remme, N.; Docimo, T.; Heinekamp, T.; Hertweck, C.; Gershenzon, J.; Haas, H.; Brakhage, A.A. The MAP kinase MpkA controls cell wall integrity, oxidative stress response, gliotoxin production and iron adaptation in Aspergillus fumigatus. Mol. Microbiol. 2011, 82, 39–53. [Google Scholar] [CrossRef] [PubMed]
  58. Kirkman, T.W. Statistics to Use. Available online: http://www.physics.csbsju.edu/stats/ (accessed on 12 August 2015).

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Kim, J.H.; Chan, K.L.; Mahoney, N. Augmenting the Activity of Monoterpenoid Phenols against Fungal Pathogens Using 2-Hydroxy-4-methoxybenzaldehyde that Target Cell Wall Integrity. Int. J. Mol. Sci. 2015, 16, 26850-26870. https://doi.org/10.3390/ijms161125988

AMA Style

Kim JH, Chan KL, Mahoney N. Augmenting the Activity of Monoterpenoid Phenols against Fungal Pathogens Using 2-Hydroxy-4-methoxybenzaldehyde that Target Cell Wall Integrity. International Journal of Molecular Sciences. 2015; 16(11):26850-26870. https://doi.org/10.3390/ijms161125988

Chicago/Turabian Style

Kim, Jong H., Kathleen L. Chan, and Noreen Mahoney. 2015. "Augmenting the Activity of Monoterpenoid Phenols against Fungal Pathogens Using 2-Hydroxy-4-methoxybenzaldehyde that Target Cell Wall Integrity" International Journal of Molecular Sciences 16, no. 11: 26850-26870. https://doi.org/10.3390/ijms161125988

APA Style

Kim, J. H., Chan, K. L., & Mahoney, N. (2015). Augmenting the Activity of Monoterpenoid Phenols against Fungal Pathogens Using 2-Hydroxy-4-methoxybenzaldehyde that Target Cell Wall Integrity. International Journal of Molecular Sciences, 16(11), 26850-26870. https://doi.org/10.3390/ijms161125988

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