Targeting the Mitochondrial Respiratory Chain of Cryptococcus through Antifungal Chemosensitization: A Model for Control of Non-Fermentative Pathogens

Enhanced control of species of Cryptococcus, non-fermentative yeast pathogens, was achieved by chemosensitization through co-application of certain compounds with a conventional antimicrobial drug. The species of Cryptococcus tested showed higher sensitivity to mitochondrial respiratory chain (MRC) inhibition compared to species of Candida. This higher sensitivity results from the inability of Cryptococcus to generate cellular energy through fermentation. To heighten disruption of cellular MRC, octyl gallate (OG) or 2,3-dihydroxybenzaldehyde (2,3-DHBA), phenolic compounds inhibiting mitochondrial functions, were selected as chemosensitizers to pyraclostrobin (PCS; an inhibitor of complex III of MRC). The cryptococci were more susceptible to the chemosensitization (i.e., PCS + OG or 2,3-DHBA) than the Candida with all Cryptococcus strains tested being sensitive to this chemosensitization. Alternatively, only few of the Candida strains showed sensitivity. OG possessed higher chemosensitizing potency than 2,3-DHBA, where the concentration of OG required with the drug to achieve chemosensitizing synergism was much lower than that required of 2,3-DHBA. Bioassays with gene deletion mutants of the model yeast Saccharomyces cerevisiae showed that OG or 2,3-DHBA affect different cellular targets. These assays revealed mitochondrial superoxide dismutase or glutathione homeostasis plays a relatively greater role in fungal tolerance to 2,3-DHBA or OG, respectively. These findings show that application of chemosensitizing compounds that augment MRC debilitation is a promising strategy to antifungal control against yeast pathogens.

Various species of Cryptococcus and Candida are human and animal pathogens. For example, cryptococcal meningitis is reported to be the leading cause of death among those infected with HIV [25]. However, one of the key differences between the yeasts in these two genera is that species of Cryptococcus are non-fermentative, while the Candida species are fermentative [16]. Table 1. Mitochondrial targets and structures of compounds tested in this study.

Compounds
Target References

2,3-DHBA Mn-SOD (Saccharomyces cerevisiae
Mn-SOD gene deletion mutant is hypersensitive to 2,3-DHBA) [15] AcSA General mitochondrial function (Aspergillus fumigatus sakAΔ [oxidative stress-responsive mitogen-activated protein kinase gene deletion mutant] was also hypersensitive to AcSA [See [26]].) [16] PG AOX [13,27] OG AOX [28,29] Based on this difference, we reasoned the following: (1) When cellular MRC is disrupted by MRC-inhibitory drug(s), the Cryptococcus would show higher sensitivity than the Candida, (2) This higher sensitivity is due to the fact that the Candida can generate cellular energy also through fermentation (other than MRC), while the Cryptococcus, being non-fermentative, lack this ability, and (3) Thus, MRC could serve as an effective antifungal target especially for control of Cryptococcus pathogens.
In this study, we investigated if selected phenolic compounds/derivatives (See Table 1) could enhance the antifungal potency of pyraclostrobin (PCS), the most potent complex III inhibitor of MRC in our test, against Cryptococcus. Our hypothesis was that co-application of phenolic compounds/derivatives (as chemosensitizers) and PCS will negatively affect the common cellular target, i.e., functions of mitochondria, resulting in increased sensitivity of fungi. We also evaluated the potential of these chemosensitizing compounds to serve as active pharmaceutical "leads" against Cryptococcus yeasts, and compared the effectiveness of chemosensitization between Cryptococcus and Candida (See Table 2 for strains tested). Our results showed that the Cryptococcus were more susceptible to OG-or 2,3-DHBA-mediated chemosensitization to PCS than the Candida, where the chemosensitizing capacity of OG was found to be greater than that of 2,3-DHBA.

Results and Discussion
We initially tested the effect of chemosensitization by co-applying commercial antifungal/antimalarial drugs "ATQ + proguanil" on the growth of fermenting and non-fermenting yeast pathogens. For this test, we chose representative yeast pathogens, i.e., C. albicans 90028 as a fermentor, and C. neoformans 90112 and C. gatti 4560 as non-fermentors. In protozoan parasites, co-application of proguanil (a mitochondria-modulating chemosensitizer) increased anti-parasitic activity of ATQ [31]. Noteworthy is that proguanil-mediated chemosensitization was specific for ATQ.
In the model yeast Saccharomyces cerevisiae, nine cellular transporters required for sequestering toxic drugs/compounds out of the cell need to be knocked out to exhibit ATQ sensitivity [33]. This indicates active drug-detoxification systems do operate in S. cerevisiae. We surmised that pathogenic yeasts, i.e., Cryptococcus or Candida, might also operate similar type(s) of detoxification system(s), enabling these pathogens to escape from ATQ/proguanil-triggered toxicity. Therefore, we performed chemosensitization tests using other types of MRC inhibitors with co-application of phenolic compounds (i.e., OG, PG, 2,3-DHBA, AcSA; See Table 1) as chemosensitizers.

PCS Is the Most Potent MRC Inhibitor in Cryptococcus
First, we identified the potency of MRC inhibitors tested against yeast pathogens. Antifungal efficacy was compared among 12 different MRC inhibitors disrupting one of the five different components of MRC, i.e., complexes I to IV or AOX (See Table 3, Figure 1). The level of differential sensitivity between fermenting (Candida) and non-fermenting (Cryptococcus) yeasts to the MRC inhibitors was determined by an agar plate-based yeast dilution bioassay (See Experimental section). We initially examined (1) three fermentors: C. albicans 90028, C. glabrata 90030, C. krusei 6258 and (2) two non-fermentors: C. neoformans 90112, C. gatti MYA-4560.
MRC inhibitors targeting complex I, III or IV reduced the growth of the Cryptococcus, with differing levels of fungal sensitivity (Table 3, Figure 2a). For example, when C. neoformans 90112 was treated with complex III inhibitors, growth was inhibited by 100 to 1000 times more compared to controls (i.e., log 10 dilution score of "no treatment" controls was "6" [i.e., yeast cells appeared at the highest dilution level of 10 6 ] vs. log 10 score of "treatments" was "3-4" [i.e., cells did not appear at dilution levels greater than10 3 -10 4 ]), depending on types of complex III inhibitors applied. Also, rotenone (a complex I inhibitor) and Na-azide (a complex IV inhibitor) inhibited the growth of C. neoformans 90112 at cell dilution levels above 10 3 times.
Growth of C. gatti 4560 was also decreased by four different MRC inhibitors (Table 3). However, unlike the results of C. neoformans 90112, AntA and AZS did not inhibit the growth of C. gatti 4560 (Table 3). Moreover, besides carboxin, which inhibited C. neoformans 90112, both complex II and AOX inhibitors did not discernably inhibit the growth of C. neoformans 90112 or C. gatti 4560 (See also Figure 2a).
Based on these initial bioassays, rotenone, Kre-Me, PCS and Na-azide (exhibiting antifungal activity against both C. neoformans 90112 and C. gatti 4560) were selected for further evaluation for antifungal potency against additional test strains (i.e., seven additional Cryptococcus and six additional Candida strains) (See Figure 2b). A similar trend of growth inhibition was found in these additional Cryptococcus strains with PCS and rotenone, while Kre-Me and Na-azide showed almost no effect. The growth of the additional Candida strains was not noticeably affected by any of these same treatments. In summary: (1) PCS possessed the highest antifungal activity (Average log 10 dilution score = 3.4 ± 0.9, this lowest average log 10 score was based upon the results using nine Cryptococcus strains shown in Figure 2a,b), followed by rotenone (average log 10 score = 4.6 ± 1.2); (2) as expected, the growth of the Candida (fermenting yeasts) was not affected by any of the MRC inhibitors tested (Table 3; Figure 2a,b), and (3) thus, we chose PCS as the most potent MRC-inhibitory drug against Cryptococcus in our chemosensitization study. TTFA, Thenoyltrifluoroacetone; 3-NPA, 3-Nitropropionic acid; BHAM, Benzhydroxamic acid; SHAM, Salicylhydroxamic acid; AntA, Antimycin A; Kre-Me, Kresoxim methyl; PCS, Pyraclostrobin; AZS, Azoxystrobin; KCN, Potassium cyanide; Na-azide, Sodium azide. Numbers represented highest dilution level (log 10 ) where cell growth was visible. Numbers in bold show cell growth was inhibited (viz., < 6).

Figure 2. Differential antifungal activity of MRC inhibitors, targeting complexes I -IV or AOX, in yeast pathogens. (a) Representative yeast dilution bioassay showing
Cryptococcus strains, non-fermentors, are relatively more sensitive to MRC inhibitors (rotenone, carboxin, PCS, Na-azide) than Candida strains, fermentors. 10 0 to 10 −5 , yeast-cell dilution level; Numbers on the right side of each row (0 to 6), log 10 score of cell numbers visible (survived) (See Experimental section and Table 3). (b) Yeast dilution bioassay showing PCS is the most potent MRC inhibitor tested, followed by rotenone (Cell dilution level showing visible growth equates to antifungal potency [higher dilution score with visible growth = lower potency]; PCS, average log 10 score = 3.4 ± 0.9 vs. rotenone, average log 10 score = 4.6 ± 1.2. Average log 10 score was determined in nine Cryptococcus strains). As observed in panel (a), Candida strains did not exhibit growth inhibition to any of the MRC inhibitors tested.

Selection of OG and 2,3-DHBA as the Most Potent Chemosensitizers
Next, we identified the most potent antifungal chemosensitizer(s), among four phenolic compounds listed in Table 1, which inhibit functions of fungal mitochondria. Based on agar plate-based yeast dilution bioassay (See Experimental section), we determined minimum inhibitory concentrations (MICs) of OG, PG, 2,3-DHBA and AcSA. As shown in Table 4, OG and 2,3-DHBA possessed the highest antifungal activity (i.e., low MIC values) compared to PG or AcSA. Of note, Candida strains were generally more tolerant to the phenolic compounds tested compared to Cryptococcus strains. Based on these results, OG and 2,3-DHBA were selected as the chemosensitizers to test with PCS.

Chemosensitization in Cryptococcus: OG + PCS
Next, chemosensitization efficacy of "OG + PCS" in Cryptococcus was evaluated. For MICs, synergistic FICIs were achieved for most of the Cryptococcus strains (Table 7). Similar to the results for "2,3-DHBA + PCS", the only exception for achieving synergism was C. neoformans 4567, which was determined to be an "indifferent" interaction. However, increased antifungal activity of OG and PCS (i.e., chemosensitization; FICI = 0.6) could be achieved in C. neoformans 4567, resulting in lowered MICs of OG or PCS when co-applied.
For MFCs, synergistic FFCIs (at the level of ≥ 99.9% fungal death) between OG and PCS occurred in all Cryptococcus strains (Table 7), reflecting the most potent antifungal activity of OG, as determined in Table 4. Most notable is that the concentration of OG needed to achieve synergism with PCS was much lower than that for 2,3-DHBA, i.e., chemosensitizing potency (higher to lower, as indicated by lower concentrations required) = OG (0.01-0.02 mM) > 2,3-DHBA (0.2-1.6 mM; See also Table 5).

Chemosensitization in Candida: OG + PCS
The chemosensitization effect of "OG + PCS" was further examined in the Candida strains. For MICs, "synergistic" FICIs were found in four strains, i.e., all three C. krusei strains and C. glabrata CAN252 (Table 8). This synergism was not detected with "2,3-DHBA + PCS", further reflecting the higher antifungal activity of OG than 2,3-DHBA (See also Table 6). Despite the "indifferent" interaction, increased antifungal activity of OG and PCS (i.e., chemosensitization; FICI = 0.6) occurred in C. albicans CAN242, as determined in lowered MICs of OG or PCS when combined (Table 8).
Of note, the trends of compound interactions of OG + PCS for MICs in Table 8 were congruent with "2,3-DHBA + PCS" chemosensitization ( Table 6). For both "2,3-DHBA + PCS" and "OG + PCS", incremental increase of growth inhibition occurred in five common strains, i.e., all three C. krusei strains, C. albicans CAN242 and C. glabrata CAN252 (Tables 6 and 8). This indicated that strain specificity to chemosensitization also exists. This is reflected in the level of differential vulnerability of each strain to chemosensitization (See also FICIs of C. neoformans 4567 in Tables 5 and 7, showing lower sensitivity of this strain to both OG-and 2,3-DHBA-mediated chemosensitization compared to other Cryptococcus strains).
Of the five deletion mutants, yap1Δ was hypersensitive to OG (log 10 = 4), while sod2Δ was hypersensitive to 2,3-DHBA (log 10 = 5) ( Figure 4) (see also [15]). These results indicate OG or 2,3-DHBA affect different cellular components in fungi, where Mn-SOD plays a relatively greater role in fungal tolerance to 2,3-DHBA, while glutathione homeostasis, etc., protects cells from OG-induced toxicity, compared to the other genes represented. Further studies, such as microarray-based chemogenomic analysis, inclusion of more gene deletion mutants, etc., are warranted to determine the precise mechanism of action of OG or 2,3-DHBA during chemosensitization.

Chemicals
The

Antifungal Bioassay: Agar Plate (Yeast Dilution) Bioassay
Petri plate-based yeast dilution bioassays were performed on the wild type and antioxidant mutants (trr1Δ, trr2Δ, tsa1Δ, sod2Δ, yap1Δ)  In conclusion, OG and/or 2,3-DHBA show potential to serve as antifungal chemosensitizers that in combination with PCS greatly enhance antifungal activity. This capacity was shown to be most effective against Cryptococcus, etiologic agents for the leading cause of death among those suffering from immunocompromised disorders. Chemosensitizers, especially those proven to be safe compounds, such as natural phenolic agents or their structural derivatives, could serve as potential "leads" against yeast pathogens for more effective treatment of mycoses using MRC inhibitory drugs. Determination of precise mechanisms of action of chemosensitization as well as identification of effective MRC-inhibitory drugs which selectively interfere with fungal mitochondrial function, and not human (mammalian), must be ensured through future study.