Enhancement of Antimycotic Activity of Amphotericin B by Targeting the Oxidative Stress Response of Candida and Cryptococcus with Natural Dihydroxybenzaldehydes

In addition to the fungal cellular membrane, the cellular antioxidant system can also be a viable target in the antifungal action of amphotericin B (AMB). Co-application of certain redox-potent natural compounds with AMB actually increases efficacy of the drug through chemosensitization. Some redox-potent chemosensitizers and AMB perturb common cellular targets, resulting in synergistic inhibition of fungal growth. Chemosensitizing activities of four redox-potent benzaldehydes were tested against clinical and reference strains of Candida albicans, C. krusei, C. tropicalis, and Cryptococcus neoformans in combination with AMB, based on assays outlined by the European Committee on Antimicrobial Susceptibility Testing. Two dihydroxybenzaldehydes (DHBAs), i.e., 2,3-DHBA and 2,5-DHBA, significantly enhanced activity of AMB against most strains, as measured by lower minimum inhibitory concentrations and/or minimum fungicidal concentrations (MFCs). A non-hydroxylated benzaldehyde, trans-cinnamaldehyde, showed chemosensitizing activity through lower MFCs, only. Contrastingly, a methoxylated benzaldehyde (3,5-dimethoxybenzaldehyde) had no chemosensitizing activity, as all strains were hypertolerant to this compound. Bioassays using deletion mutants of the model yeast, Saccharomyces cerevisiae, indicated DHBAs exerted their chemosensitizing activity by targeting mitochondrial superoxide dismutase. This targeting, in turn, disrupted the ability of the yeast strains to respond to AMB-induced oxidative stress. These in vitro results indicate that certain DHBAs are potent chemosensitizing agents to AMB through co-disruption of the oxidative stress response capacity of yeasts. Such redox-potent compounds show promise for enhancing AMB-based antifungal therapy for candidiasis and cryptococcosis.


INTRODUCTION
There has been a persistent effort to improve efficacy of conventional antimycotic drugs, especially for treatment of human candidiasis and cryptococcosis. Currently, liposomal amphotericin B (LAMB), AMB lipid complex, etc., are preferred for clinical therapy of these mycoses, in that conventional AMB (e.g., AMB deoxycholate) is hepatotoxic/nephrotoxic (Patel et al., 2011). The lipid-based AMBs are generally recommended for patients who are intolerant to conventional AMB, which is still administered for treatment of mycoses, such as pediatric fungal infections (Allen, 2010 and references therein). However, high doses of LAMBs cause nephrosis and other tissue-damage in murine models of invasive pulmonary aspergillosis (Clemons et al., 2011). Thus, an antifungal therapeutic strategy to reduce side effects of AMB is warranted.
Amphotericin B binds to ergosterol in the fungal plasma membrane, undermining cell membrane integrity and causing ion leakage. However, formation of channels in the fungal membrane is not the sole mode of action of AMB (Palacios et al., 2007). There is ample literature showing AMB induces oxidative damage to both ascomycete and zygomycete fungal cells (Sokol-Anderson et al., 1986;Graybill et al., 1997, and references therein;Okamoto et al., 2004;An et al., 2009;González-Párraga et al., 2011). For example, Aspergillus terreus, a causative agent of human invasive aspergillosis, is intrinsically resistant to AMB, compared to other aspergilli. This resistance was thought to result from lower membrane ergosterol, thus offering fewer target sites for AMB (Walsh et al., 2003). However, this resistance was later found to result from higher catalase activity, an enzyme that protects against oxidative stress. This latter finding indicated there is an alternate or additional mode of action of AMB by causing oxidative damage (Blum et al., 2008). This was further confirmed by the finding that superoxide radical-mediated oxidative damage was caused by AMB activity (Okamoto et al., 2004).
Disrupting fungal redox homeostasis and/or the antioxidant system should augment antimycotic activity of AMB. Moreover, the antioxidant system plays an important role in pathogen virulence and defense against host cellular oxidative burst during infection (Washburn et al., 1987;Hamilton and Holdom, 1999;de Dios et al., 2010). Such disruption of the fungal redox homeostasis/antioxidant system could employ redox-potent natural products or their analogs (Jacob, 2006). The natural phenolic 2,3dihydroxybenzaldehyde (2,3-DHBA) augments antifungal activity of a number of fungicidal agents by interfering with the fungal oxidative stress response system (Kim et al., 2008(Kim et al., , 2011. In view that both 2,3-DHBA and AMB stress the fungal antioxidant system, their co-application should result in elevated antifungal activity. The aim of this study was to test the concept of using benzaldehydes, such as 2,3-DHBA and some of its structural derivatives, as chemosensitizing agents to AMB. As a proof-of-concept, we used clinical strains and species of Candida and Cryptococcus neoformans for this test. Specifically, we compared the chemosensitizing activity between two hydroxylated DHBAs (2,3-or 2,5-DHBA) and two non-hydroxylated benzaldehydes [non-DHBAs; transcinnamaldehyde or 3,5-dimethoxybenzaldehyde (3,5-DMBA)]. We reasoned that use of chemosensitizing agents from natural sources could enhance the activity of AMB, while lowering toxic side effects of this drug to human cells.

ANTIFUNGAL DRUGS AND COMPOUNDS
Amphotericin B, diamide, 2,3-or 2,5-DHBA, trans-cinnamaldehyde, and 3,5-DMBA were procured from Sigma Co. (St. Louis, MO, USA). Each compound was dissolved in dimethyl sulfoxide (DMSO; absolute DMSO amount: <2% in medium) before incorporation into the culture medium. 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.

SUSCEPTIBILITY TESTING: PLATE (AGAR) BIOASSAY
Petri plate-based yeast dilution bioassays were performed on the WT and antioxidant mutants of S. cerevisiae to assess the effects of AMB (0.0, 0.5, 1.0, 1.5, and 2.0 µg mL −1 ) on the fungal antioxidant system. These assays were performed in duplicate on SG agar following previously described protocols (Kim et al., 2008). Similar dilution bioassays were performed on Candida and Cryptococcus to assess their differential sensitivity to AMB (0.0, 0.5, 1.0 µg mL −1 ) or diamide (0.0, 0.2, 0.4, 0.6, 0.8 mM). Cell growth was observed for 3-5 days.
To measure changes in minimum fungicidal concentrations (MFCs), i.e., differences/changes of MFCs of each compound (AMB, benzaldehydes) alone compared to when they were combined, the entire volume of each microtiter well (200 µL), after determination of MICs, was spread onto individual YPD plates and cultured an additional 48 h (72 h for C. neoformans). The lowest concentration of agent showing ≥99.9% fungal death was defined as the MFC, except where noted (see tables). Student's t -test for paired data was used to determine significant differences between means of MICs or MFCs of each compound when combined (i.e., chemosensitization) vs alone (i.e., no chemosensitization) for six yeast pathogens (calculation was based on Kirkman, 1996). Compound interactions [for both fractional inhibitory concentration indices (FICI) and fractional fungicidal concentration indices (FFCI)] were calculated based on: 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). FICI or FFCI was defined as: "synergistic" (FICI or FFCI ≤0.5) or "indifferent" (FICI or FFCI >0.5-4;Odds, 2003).

RESULTS
We tested the hypothesis that benzaldehydes could act as chemosensitizing agents to AMB against clinical strains and species of Candida and C. neoformans. First, Petri plate-based yeast dilution bioassays were used to evaluate any relationship between AMB-sensitivity and lower antioxidant capacity. Duplicate assays were performed on SG agar containing AMB (0.0, 0.5, and 1.0 µg mL −1 ) according to described protocols (Kim et al., Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy 2008). In this test, C. albicans CAN276 was the most sensitive of all strains when exposed up to 1.0 µg mL −1 AMB (Figure 1). Next, we examined the effect of diamide (0.0, 0.2, 0.4, 0.6, and 0.8 mM) on these strains. Diamide causes stoichiometric oxidative stress by depleting cellular thiols, such as glutathione. CAN276 was also the most sensitive of Candida species or strains to diamide (up to 0.8 mM; Figure 1). C. krusei 6258, C. krusei CAN75, and C. tropicalis CAN286 grew similar to control (no diamide) cohorts (i.e., no antifungal activity against these strains at the given concentration). C. albicans 90028 and C. neoformans CN24 showed slight sensitivity to diamide, >100-fold less than CAN276 (Figure 1). The high sensitivity of CAN276 to both AMB and diamide indicated a diminished oxidative stress response system increases sensitivity to AMB.
The non-DHBAs tested were not potent chemosensitizing agents for AMB against the yeasts, as compared with the DHBAs. Interactions of cinnamaldehyde co-applied with AMB, in C. albicans 90028, CAN276, C. krusei 6258, and C. neoformans CN24, were "indifferent," although this co-application showed certain www.frontiersin.org  level of enhanced antifungal activity for MFCs (Table 3). Moreover, 3,5-DMBA did not show any antifungal activity in any of the yeast strains, even at the highest concentration tested (6.4 mM), nor any chemosensitization when co-applied with AMB (data not shown). Contrastingly, 3,5-DMBA had potent antifungal activity (average MIC: 1.17 mM) against filamentous fungal pathogens (i.e., species and strains of Aspergillus, Penicillium; Kim et al., 2011). Perhaps yeast pathogens possess an intrinsic capacity to detoxify 3,5-DMBA.

DISCUSSION
All compounds tested, except for 3,5-DMBA, are known natural volatiles or components of the essential oils of a number of plants, including almond and vanilla. Both 2,3-and 2,5-DHBAs and trans-cinnamaldehyde have been shown to have a moderate level (MICs 20-80 µg mL −1 ) of antibacterial activity (Wong et al., 2008). However, we found that the antifungal activity of these compounds, alone, is not particularly noteworthy.  However, as previously reported, certain phenolic antioxidants can prolong the activity of AMB against C. albicans by stabilizing the multiple double bonds of the polyene moiety. But, the mechanism by which the combination of such phenolics and AMB resulted in a synergistic interaction was unidentified (Beggs et al., 1978). Our results showed the DHBAs also augmented efficacy of AMB, in vitro, against yeast pathogens. Coapplication of DHBAs with AMB resulted in complete inhibition of fungal growth at lower doses than any of the individual components applied, alone. Based on gene deletion mutant bioassays, it now appears that this synergy between AMB and DHBAs is by targeting at least one common cellular component in the antioxidant system, Mn-SOD. SODs of C. albicans are involved in biofilm persistence against miconazole (Bink et al., 2011), further demonstrating the role of fungal SODs in drug resistance. The non-DHBAs tested were poor chemosensitizing agents of AMB against yeast pathogens, indicating hydroxyl (−OH) substituents on the aromatic ring contributed to improved www.frontiersin.org antifungal/chemosensitizing activity. Of note is benzaldehydes having ortho-and para-hydroxylation possessed higher antifungal activity than meta-or mono-hydroxyl analogs (Kim et al., 2008).
The results of this in vitro study demonstrate that chemically targeting the oxidative stress response system of fungi effectively augments antimycotic potency of AMB. DHBAs or their analogs could be developed as potent chemosensitizers to AMB in yeast pathogens. Chemosensitization by using natural compounds could enhance the efficacy of AMB to inhibit fungal growth, and lower the adverse side effects of AMB. Further in vivo studies are needed to determine if the activities of chemosensitizers shown in this in vitro study can translate to a clinically effective resolution of mycoses.