Aspergillus fumigatus AF293, wild type, and A. fumigatus MAPK gene deletion mutants (sakAΔ and mpkCΔ) were grown at 35°C on potato dextrose agar (PDA) or Sabouraud dextrose agar (SDA; Sigma, St. Louis, MO, USA). A. terreus UAB673, UAB680, and UAB698 (clinical isolates) were procured from Centers for Disease Control and Prevention, Atlanta, GA, USA, and were grown at 35°C on PDA or SDA. A. flavus NRRL3357, procured from the National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL, USA, was grown at 35°C on PDA or SDA. Also, A. flavus CA14 (parental strain) and CA14msnAΔ (knockout mutant for msnA gene; Chang et al., 2011) strains were grown at 28°C on PDA.

C. albicans 90028 and C. krusei 6258 (reference strains) were procured from American Type Culture Collection (Manassas, VA, USA). C. albicans CAN276, C. krusei CAN75, C. tropicalis CAN286 and Cryptococcus neoformans CN24 (clinical isolates) were procured from Instituto de Higiene e Medicina Tropical/CREM, Universidade Nova de Lisboa, Portugal. S. cerevisiae wild type BY4741 (Mat a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and selected single gene deletion mutants, i.e., cytosolic superoxide dismutase (SOD; Cu, Zn-SOD; sod1Δ) mutant, mitochondrial SOD (Mn-SOD; sod2Δ) mutant, antioxidative transcription factor mutant (yap1Δ), glutathione reductase mutant (glr1Δ), vacuolar H⁺-ATPase (V-ATPase) assembly mutant (vph2Δ) and V-ATPase subunit A mutant (vma1Δ), were procured from Open Biosystems [Huntsville, AL, USA; see Saccharomyces Genome Database (www.yeastgenome.org; accessed February 2, 2012)]. 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⁻¹, amino acids 0.03 mg mL⁻¹) agar, yeast peptone dextrose (YPD; Bacto yeast extract 1%, Bacto peptone 2%, glucose 2%) agar or SDA at 30°C for S. cerevisiae or 35°C for yeast pathogens (Candida, Cryptococcus), respectively.

Antifungal chemosensitizing agents [2,3-dihydroxybenzaldehyde (2,3-DHBA), salicylaldehyde (SA), thymol (THY)], antifungal drugs [antimycin A (AntA), amphotericin B (AMB), itraconazole (ITZ)], strobilurins [pyraclostrobin (PCS), kresoxim methyl (Kre-Me)], and oxidizing agents [tert-butyl hydroperoxide (t-BuOOH), hydrogen peroxide (H₂O₂; Sigma product No. H1009, contained stabilizer)] were procured from Sigma Co. Hydrogen peroxide stock was prepared based on molar concentration provided by the manufacturer. Each compound was dissolved in dimethyl sulfoxide (DMSO; absolute DMSO amount: <1% in media), except oxidizing agents, which were dissolved in water, before incorporation into culture media. 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.

Determination of chemosensitizing activity of SA, a volatile benzaldehyde analog, to ITZ was performed in segmented Petri dishes (150 mm × 10 mm; VWR International Co., Radnor, PA, USA). These dishes are divided into four, isolated sections, and each of two sections was supplied with 6 mL of SDA for fungal growth (without or with 1–7 μg mL⁻¹ of ITZ in SDA). Then, conidial suspensions of A. fumigatus AF293 (5 × 10⁴ CFU mL⁻¹) were diluted in PBS and applied as a drop on the center of each section, providing two fungal inocula within each plate; see also Figure 3.).

SA was dissolved in DMSO, and was applied onto a round Whatman™ paper (2.5 cm in diameter; GE Healthcare Bio-science Co., Piscataway, NJ, USA; Final volume: Total 150 μL of SA plus DMSO on a Whatman™ paper per each plate based on SA concentration tested). Each SA preparation was then placed onto an empty section located between the fungal inocula. The fungal spores inside the Petri plate would be exposed to the volatilized SA. The SA-wet (or DMSO-wet control) Whatman™ paper was placed onto a piece of Parafilm^® (American National Can Co., Chicago, IL, USA) to avoid the direct contact of SA plus DMSO with the surface of a Petri dish. The plates containing SA (or DMSO control) and fungal inocula were then sealed with two layers of Parafilm^® to prevent escape of SA. The plates were incubated at 35°C (5–7 days). The antifungal treatments, therefore, consist of (1) control plates: A. fumigatus AF293 inocula without SA vapor (DMSO only) and (2) treated plates: A. fumigatus AF293 inocula with SA vapor (9.5–95 mM on a Whatman™ paper). Results were based on two replicated plates.

We initially investigated the phenotypic responses (i.e., level of sensitivity) of three Aspergillus mutant strains, i.e., deletion mutants for A. fumigatus MAPKs, sakA and mpkC, and A. flavus msnA genes, to t-BuOOH (an organic peroxide) and H₂O₂ (hydrogen peroxide). Sensitivities of fungi to the oxidizing agents were determined based on relative fungal radial growth on agar plates. The sakAΔ, mpkCΔ and msnAΔ mutants currently available were derived from two different species of infectious Aspergillus. However, we reasoned that results presented in this study would provide the basis for detecting signaling network(s) in Aspergillus, in general, under the stress/toxicity triggered by oxidizing agents and/or antifungal drugs. To our knowledge, the A. flavus msnA is the first MSN2 gene ortholog functionally characterized (with gene knockout) in Aspergillus.

In our tests, all three Aspergillus mutants, i.e., A. fumigatus sakAΔ and mpkCΔ, A. flavus CA14msnAΔ, showed marginally higher sensitivity to t-BuOOH and H₂O₂ compared to the wild type strains (Data not shown). Based on these results, we postulated that msnA (downstream regulator) and sakA/mpkC (upstream MAPKs) are located in the same stress response network in Aspergillus under the peroxide-mediated oxidative stress.

Next, we examined the level of sensitivity of Aspergillus mutants to ITZ or AMB in plate bioassays. We tried to determine if the function of the fungal antioxidant system is responsive to toxicities triggered by ITZ or AMB. As shown in Figure 1, the A. fumigatus sakAΔ mutant showed higher sensitivity to ITZ or AMB compared to AF293 or the mpkCΔ mutant. The A. fumigatus sakAΔ mutant did not grow at 0.5 μg mL⁻¹ of ITZ (i.e., sensitive), while AF293 and the mpkCΔ mutant did grow (Figure 1). Also, the A. fumigatus sakAΔ mutant was more sensitive to AMB compared to the AF293 or mpkCΔ strains. For example, when treated with AMB, the inhibition rate of fungal growth for the sakAΔ mutant was 32–54% (i.e., more sensitive; SD < 5%) at 1.0–2.0 μg mL⁻¹ of AMB, while inhibition of AF293 or the mpkCΔ mutant was around 8–34% (SD < 5%; Figure 1).

To evaluate the negative impact of the deletion of MAPK genes on cell survivability after AMB or ITZ treatment, we also monitored the minimum fungicidal concentrations (MFCs) of AMB or ITZ for A. fumigatus AF293, sakAΔ and mpkCΔ strains in a separate microdilution (microtiter plate) bioassay. Fungi were treated with 1–128 or 1–64 μg mL⁻¹ of AMB or ITZ, respectively. In this test, over 99.9% of fungicidality was achieved in the A. fumigatus sakAΔ mutant at dosage levels of ITZ or AMB lower than that for AF293 or the mpkCΔ mutant (Table 1). Altogether, we concluded that the antifungal activity of AMB or ITZ evoked a cellular oxidative stress response requiring participation of MAPK pathway genes in A. fumigatus. However, SakA must play a more significant role in response to AMB or ITZ than MpkC, as represented by the higher sensitivity of sakAΔ than mpkCΔ to either drug. A similar type of hypersensitivity of the sakAΔ mutant was previously observed when it was treated with redox-potent phenolic reagents, where the mpkCΔ mutant showed less sensitivity to the same treatment (Kim et al., 2010).

Sensitivity of the A. flavus CA14msnAΔ mutant to AMB was also examined using plate bioassays. As shown in Figure 1, the MIC of the A. flavus CA14msnAΔ mutant for AMB was between 4.0 and 8.0 μg mL⁻¹, while that of the parental strain, A. flavus CA14, was above 8.0 μg mL⁻¹ (the highest concentration of AMB tested). However, unlike its response to AMB, there was no difference in level of sensitivity of the A. flavus CA14msnAΔ mutant and the parental strain to ITZ (i.e., MICs for both strains were between 0.25 and 0.5 μg mL⁻¹; although, some discoloration of the parental strain occurred with ITZ treatment; Figure 1). One plausible explanation for this differential response to two different drugs would be that a regulator(s) other than MsnA is involved in fungal response/tolerance to ITZ.

Collectively, the experimental results from the A. fumigatus and A. flavus strains studied indicate that antifungal activity of ITZ or AMB involves the functioning/response of fungal antioxidant system. Among the molecular genetic components examined in these Aspergillus strains, it appears that SakA plays a more significant role for fungal tolerance/response to both ITZ- and AMB-induced toxicity than do either MpkC or MsnA.

In prior studies, the A. fumigatus sakAΔ mutant showed hypersensitivity to 2,3-DHBA or THY (Kim et al., 2008a,b). These compounds were effective as chemosensitizing agents to conventional antifungal drugs/fungicides (Kim et al., 2008a,b).

In the present study, the A. flavus CA14msnAΔ mutant was also more sensitive to 2,3-DHBA or THY compared to the parental strain in plate bioassays (Figure data not shown). For example, the MICs for 2,3-DHBA and THY were between 0.8 and 1.6 mM or 3.2 and 6.4 mM, respectively, in the parental strain, while those of the msnAΔ mutant were between 0.0 and 0.1 mM or 0.2 and 0.4 mM, respectively. Therefore, including results with the t-BuOOH and H₂O₂ experiments (see above), these findings with 2,3-DHBA and THY further provide evidence of antioxidative cellular components (i.e., MAPKs and MsnA) playing a role in fungal response to drugs (ITZ, AMB) or redox-potent compounds (2,3-DHBA, THY) [see Table 2 for the summary of responses of Aspergillus MAPKs (sakAΔ, mpkCΔ) and msnAΔ mutants to the test compounds].

We next investigated the potential role of redox-potent compounds (2,3-DHBA, THY) as chemosensitizing agents to oxidative stress drugs (AMB, ITZ) in different fungal pathogens. SA was also included in these tests. We reasoned there should be either an additive or synergistic antifungal interaction if the drug and compounds were co-applied, lowering dosages for control of fungi than sole treatment of each drug. At this stage of study, we tried to identify the most effective chemosensitization strategy with various combinations of redox-potent compounds and antifungal drugs.

Since positive correlation between the level of drug/compound sensitivity and antioxidation capacity was identified in the filamentous fungi (see above), we also investigated if such a relationship occurred in yeast pathogens using ITZ and t-BuOOH. As shown in Figure 4, C. albicans 90028, CAN276, C. krusei CAN75, and C. tropicalis CAN286 were relatively more tolerant (i.e., growth up to the 10⁵ dilution spot) to ITZ compared to C. krusei 6258 and C. neoformans CN24 (i.e., growth at no cellular dilution only). Noteworthy is that C. krusei 6258 and C. neoformans CN24, two ITZ-sensitive strains, were also sensitive to t-BuOOH (Figure 4). This finding indicated there might be a connection between ITZ sensitivity and antioxidation capacity in yeast pathogens [Our recent data with yeast pathogens also indicated the positive correlation between AMB sensitivity (of C. albicans CAN276) and thiol-oxidant sensitivity (Manuscript submitted)].

We examined the antifungal efficacy of three inhibitors of complex III of the MRC, i.e., AntA and strobilurins (Kre-Me, PCS), in four clinical yeast isolates to investigate whether yeast pathogens could also be chemosensitized by 2,3-DHBA to increase vulnerability to the complex III inhibitors.

As shown in Figure 5A (i.e., yeast dilution bioassay on SG agar containing 100 μg mL⁻¹ of MRC inhibitors), C. neoformans CN24 was relatively more innately sensitive to the MRC inhibitors compared to other pathogens. Results indicated disruption of complex III of MRC, alone, could be an effective strategy for control of C. neoformans CN24. The remaining three pathogens, relatively tolerant to the complex III inhibitors, were further examined for chemosensitization with 2,3-DHBA in plate bioassays.

Disruption of complex III of the MRC results in an abnormal release of electrons that additionally damage cellular components by oxidative stress (Takimoto et al., 1999). Therefore, antioxidant enzymes, such as Mn-SOD, play important roles in protecting cells from oxidative damage caused by MRC inhibitors. However, when fungal cells are treated with redox-potent chemosensitizers, cellular demand for Mn-SOD will continuously increase as more and more oxidative stress is applied. In this situation, the levels of antioxidant capacity in fungi, such as antioxidant enzymes, would not be sufficient for detoxifying the concerted activities of multiple oxidative stressors (e.g., MRC inhibitors/oxidative stress drug + redox-potent chemosensitizer), resulting in increased inhibition of fungal growth. Hence, we surmised redox-potent benzaldehydes could be useful chemosensitizing agents also in yeast pathogens when co-applied with the complex III inhibitors of the MRC. We used 2,3-DHBA as a representative molecule for this chemosensitization.

As shown in Figure 5B, co-application of 2,3-DHBA (0.1 mM) and PCS (100 μg mL⁻¹) enhanced growth inhibition of C. albicans CAN276 and C. tropicalis CAN286 compared to the control, while C. krusei CAN75 maintained robust growth under the same condition. Thus, C. krusei CAN75 seemed to be more tolerant to chemosensitization by 2,3-DHBA (A relatively lackluster response of C. krusei CAN75 to 2,3-DHBA chemosensitization was also observed above, with ITZ). However, slight enhancement [i.e., a 10-fold increase in number of yeast cells needed to survive (one cellular dilution less)] of growth inhibition of C. krusei CAN75 was achieved by increasing the concentration of 2,3-DHBA to 0.4 mM (see Figure 5B). In summary, these results indicated that: (1) The MRC (e.g., complex III) could be an alternative oxidative stress target for yeast pathogens, (2) Benzaldehyde analogs, such as 2,3-DHBA, could be developed as potent chemosensitizers in yeasts, especially with MRC inhibitors, and (3) Fungal sensitivity to chemosensitization (i.e., with 2,3-DHBA plus PCS), is strain specific, wherein C. krusei CAN75 was least sensitive.