Respiratory Deficiency Enhances the Sensitivity of the Pathogenic Fungus Candida to Photodynamic Treatment

Written By: Sam Savage

Published Date: October 5, 2008 Last Edited: October 5, 2008

By Chabrier-Rosello, Yeissa Foster, Thomas H; Mitra, Soumya; Haidaris, Constantine G

ABSTRACT Mucosal infections caused by the pathogenic fungus Candida are a significant infectious disease problem and are often difficult to eradicate because of the high frequency of resistance to conventional antifungal agents. Photodynamic treatment (PDT) offers an attractive therapeutic alternative. Previous studies demonstrated that filamentous forms and biofilms of Candida albicans were sensitive to PDT using Photofrin as a photosensitizer. However, early stationary phase yeast forms of C. albicans and Candida glabrata were not adversely affected by treatment. We report that the cationic porphyrin photosensitizer meso-tetra (N-methyl- 4pyridyl) porphine tetra tosylate (TMP-1363) is effective in PDT against yeast forms of C. albicans and C. glabrata. Respiratory- deficient (RD) strains of C. albicans and C. glabrata display a pleiotropic resistance pattern, including resistance to members of the azole family of antifungals, the salivary antimicrobial peptides histatins and other types of toxic stresses. In contrast to this pattern, RD mutants of both C. albicans and C. glabrata were significantly more sensitive to PDT compared to parental strains. These data suggest that intact mitochondrial function may provide a basal level of anti-oxidant defense against PDT-induced phototoxicity in Candida, and reveals pathways of resistance to oxidative stress that can potentially be targeted to increase the efficacy of PDT against this pathogenic fungus.

INTRODUCTION

Species of the fungus Candida commonly colonize the epithelial surfaces of the body, with the alimentary canal considered as the primary site of colonization (1). However, few healthy carriers develop clinical signs of candidiasis (2). Oropharyngeal candidiasis (OPC) results from fungal overgrowth and penetration of oral tissues when the body’s physical and immunological defenses are compromised (2). Patients with diseases such as cancer, HIV/AIDS or diabetes, as well as premature infants and patients requiring intensive care, are at risk of developing infection from Candida, including OPC (1,3). Impairment of salivary gland function by disease or medical treatment is correlated with a high incidence of OPC (2). Spread of Candida albicans infection from the oropharynx to the esophagus makes swallowing painful and difficult.

While C. albicans is the predominant species in OPC, Candida glabrata (4) and Candida krusei (5) are also seen. Infections with non-albirans species often emerge after treatment for an initial C. albicans infection (6), or during prophylaxis for C. albicans infection, by virtue of their inherent resistance to commonly used antifungals. An example of this trend is that fluconazole-resistant Candida species colonize approximately 81% of AIDS patients receiving therapy for oral candidiasis (7). The conversion from harmless commensal to the development of OPC is a key initial step in the progression to life-threatening disseminated candidiasis (1). As the microbiology and resistance patterns of clinical isolates evolve in response to selective pressures of current antifungal therapy, the importance of developing novel strategies for treatment of OPC becomes paramount.

Photodynamic treatment (PDT) is a process in which cells are treated with an agent that makes them susceptible to killing by exposure to light. Photosensitizing agents are generally macrocyclic compounds that exhibit no or minimal inherent toxicity, but result in the generation of cytotoxic reactive oxygen species (ROS) when optical excitation occurs with light of the appropriate wavelength (8). PDT has been applied most extensively in the treatment of neoplasia (8,9) and shows promise as a novel therapy for some nonneoplastic disorders (10-12). The application of PDT to the treatment of microbial infection is also gaining widespread interest as an alternative or adjunct to conventional antimicrobial therapy (reviewed in Jori et al [13]), including PDT of fungal infections.

The photosensitizing agent Green 2W demonstrated an in vitro fungicidal effect against Aspergillus fumigatus that was both light- dose and inoculum-dependent (14), suggesting that PDT may be an effective treatment option for localized cavitary infections with this organism. Porphyrin derivatives inactivated the fungal dermatophyte Trichophyton rubrum (15), and different Candida species in our studies (16,17) and those of others (18). The cationic phenothiazine photosensitizers toluidine blue, methylene blue (19,20) and the cationic porphyrin TriP(4) (21) have also been used in PDT of Candida in vitro. The effectiveness of PDT for fungal infections in vivo is largely untested. One study investigated the effect of topical methylene blue followed by laser light in a murine model of oral candidiasis (22). In this study, SCID mice were infected orally with C. albicans and treated topically with increasing concentrations of methylene blue followed by illumination with laser light at 664 nm. Eradication of the infection in a dose- dependent manner supports the feasibility of this approach for mucosal infections, including OPC.

Successful PDT of C. glabrata has not yet been reported. The sensitivity of C. glabrala to PDT has relevance to OPC in that this species of Candida has inherent resistance to both the histatins (23), cationic antifungal proteins found in saliva, and to the azole class of antifungal agents (24). Our initial studies (16) showed that Photofrin was ineffective in PDT against either C. glabrala or C. albicans early stationary phase yeast. Here we report the efficacy of the cationic porphyrin photosensitizer TMP-1363 against the yeast form of these two pathogenic Candida species.

In addition to wild-type “grande” (rho ^sup +^ ) yeast strains, Baker’s yeast Saccharomyces cerevisiae (25), C. albicans (26), and C. glahrata (27) can exist as respiratory-deficient (RD), “petite” strains as a consequence of nuclear mutations, deletions of segments of mitochondrial DNA (referred to as rho^sup 0^), or total absence of mitochondrial DNA (rho^sup 0^). RD strains generated by exposure to ethidium bromide (28) are characterized functionally by smaller colony size and an inability to grow on a nonfermentable substrate such as glycerol. More recently, it has been noted that RD strains of these fungi display a pleiotropic resistance pattern, including resistance to members of the azole family of antifungals, histatins, and other types of toxic stresses (25,29,30). During the course of our investigations on the susceptibility of Candida to PDT using TMP- 1363, we made the surprising observation that RD mutants of C. albicans and C. glabrata are hypersensitive to PDT compared to their respective wildtype parental strains, in contrast to the pleiotropic resistance seen using other stressors.

MATERIALS AND METHODS

Organisms. C. albicans laboratory strains 3153A (16,17,31,32) and SC5314 (17,33), C. glabrata clinical isolate MRO-084-R (L6) were used for the bulk of the studies. C. albicans clinical isolates TW 07229 and TW 072243 (34,35) were generously provided by Theodore C. White, Seattle. WA.

Culture conditions. To obtain early stationary phase cells, organisms were grown overnight at 37[degrees]C in yeast extract- peptone-dextrose (YPD) broth (Difco, Detroit, Ml). Organisms were washed twice with dH2O and diluted to 10^sup 7^ cells mL^sup -1^ in dH2O prior to PDT. Germ tube formation was initiated from early stationary phase yeast using a previously described method (16,17). Briefly, 1 mL of overnight YPD culture was washed twice with dH2O and diluted to 3 x 10^sup 5^ cells mL^sup -1^ in RPMI 1640 supplemented with 1% glucose (RPMI/G; BioWhittaker, Walkersville, MD). To induce filament formation, 3 mL of the diluted cell suspension was grown statically in six-well tissue culture dishes (VWR) at 37[degrees]C for 3 h. Prior to incubation with photosensitizer, germ tubes were washed with dH2O. C. albicans strain 3153A biofilms were grown as described previously (17). Briefly. 2 mL of fetal bovine serum was added to each well of a six- well tissue culture plate and incubated at 37[degrees]C overnight with gentle rocking to provide a substrate for organism adhesion (36). C. albicans 3153A early stationary phase yeast were washed twice with dH2O and diluted in RPM1/G as described for germ tube formation. To each well, 3 mL of the diluted cell suspension was added and incubated for 90 min with gentle rocking to promote initial attachment. Non-adherent cells were removed by washing with RPMI/G, and 3 mL of fresh RPMI/G was added to each well. Cells were incubated at 37[degrees]C with gentle rocking for 48 h for biofilm formation.

Photodynamic treatment conditions. For PDT, organisms in sixwell dishes were incubated with a range of concentrations of either Photofrin (Axcan Pharma, Birmingham, AL) or meso-tetra (N-methyl-4- pyridyl) porphine tetra tosylate (TMP-1363; Frontier Scientific, Logan. UT) for 10 min at 37[degrees]C (17). For Photofrin incubation, wash and irradiation steps were performed in phosphate buffered saline. pH 7.0; for TMP-1363 all treatment steps were performed in dH2O. Organisms were washed twice after incubation to remove excess photosensitizer, and 2 mL of wash solution was added for the irradiation step. Organisms were irradiated at room temperature with visible light from a 48 cm x 48 cm light box equipped with a bank of fluorescent lamps (Sylvania GRO-LUX, 15 W, part no. F15T8/GRO). The irradiance at the surface of the light box was 4.0 mW cm^sup -2^, and the spectrum of the light was such that approximately 67% of the power was emitted within the range of 575- 700 nm, where the absorption spectra of Photofrin and TMP-1363 are very similar. For each experiment, an identical plate that was not irradiated (shielded) was used as a control. XTT phototoxicity assay. Following irradiation, organisms were incubated in fresh RPMI/ G for 30 min to permit recovery of surviving organisms. Phototoxicity was determined using a metabolic assay (37) in which (2,3)-bis (2-methoxy-4-nitro-5-sulfenyl)-(2H)-tetrazolium- 5carboxanilide (XTT; Sigma-Aldrich, St. Louis, MO) is converted by mitochondrial dehydrogenases to a soluble, orange-colored formazan product that diffuses into the medium. Plates were incubated at 37[degrees]C for 1 h to allow the assay to develop. A 100 [mu]L aliquot from the reaction supernatant was removed and serially diluted in PBS in a 96well microtiter plate. The intensity of the colorimetric reaction was determined by measuring the absorbance at 450 nm (Abs^sub 450^) using an automated microplate reader (Bio-Rad Laboratories, Hercules, CA). A reduction in the ABs^sub 450^ of irradiated cultures compared to nonirradiated cultures was used as a measure of phototoxic damage (16,17,37).

Colony forming unit (CFU) phototoxicity assay. Candida early stationary phase yeasts were subjected to PDT using TMP-1363 as described above. Following irradiation, organism suspensions were diluted in dH2O. For screening of a range of fluences. dilutions of the different experimental groups were spotted (2 [mu]L/spot) on YPD plates and grown overnight to assay phototoxicity. To assay organism phototoxicity following PDT quantitatively, dilutions of treated cells were plated on YPD agar and incubated 24-48 h at 37[degrees]C to allow colony formation. Data were expressed as CFU mL^sup -1^.

Generation of respiratory-deficient mutants. C. albicans SC5314 and C. glabrata MRO-084-R respiratory-deficient (RD) mutants were generated and characterized by selection on YPD agar supplemented with ethidium bromide (40 [mu]g mL^sup -1^) as described in (29). The plates were incubaled at 30[degrees]C for 72 h. Respiratory deficiency was corroborated by plating on YP-Glycerol and Eosin Y/ Trypan blue plates (29).

Antifungal susceptibility testing. Broth microdilution for fluconazole sensitivity was performed by the NCCLS reference method (38) using a final inoculum of 0.5-2.5 x 10^sup 5^ cells per mL in RPMI 1640 medium supplemented with 2% glucose (BioWhittaker) and 0.165 M MOPS (3-(N-morpholino) propanesulfonic acid) buffer and adjusted to pH 7.0. Organism growth was determined spectrophotometrically at Abs^sup 450^ after 48 h to determine the 50% minimum inhibitory concentration (MlC^sub 50^).

Statisticul analysis. Each experimental group was assayed in duplicate (biofilms) or triplicate and all experiments were performed lhree times. These data represent lhe mean from combined replicate experiments +- SD. In all cases, P-values of

RESULTS

C. albicans germ tubes and biofilms arc sensitive to PDT using the hydrophobic photosensitizer Photofrin and the cationic photosensitizer TMP-1363

Previous studies demonstrated that C. albicans germ tubes and biofilms were sensitive to PDT using the clinically approved hydrophobic photosensitizer Photofrin (16,17). However, early stationary phase yeast of C. albicans and C. glahrata were not susceptible to PDT using Photofrin (16). Consequently, we sought to identify photosensitizers with broader efficacy against Candida. Since cationic photosensitizers have been applied successfully to antimicrobial PDT (39) including C. albicans (21), we tested the caiionic porphyrin photosensitizer TMP-1363 (40) (Frontier Scientific) against C. albicans and C. glabrata grown under different conditions. Initially, we compared Photofrin and TMP-1363 in PDT of germ tubes and biofilms of the strain used in our earlier studies, C. albicans 3153A (16,17). Candida albicans 3153A germ tubes were incubated with increasing concentrations of each photosensitizer ranging from 0.1- 3.0 [mu]g mL^sup -1^. To compensate for an increase in organism biomass, biofilms were incubated with a higher range of concentrations (1.0-20 [mu]g mL^sup -1^) compared to germ tubes. In each case, excess photosensitizer was removed by washing prior to irradiation. Organisms treated identically but shielded from irradiation served as a negative control. Since filamentous forms of C. albicans are multicellular structures, determination of CPU as a measure of viability is not quantitative. Therefore, we utilized a metabolic activity assay (37) based on the conversion of XTT (Sigma-Aldrich) as a measure of phototoxicity (17).

Irradiated biofilms were highly sensitive to both the hydrophobic photosensitizer Photofrin and the cationic photosensitizer TMP- 1363, as shown by a concentration-dependent reduction in their metabolic activity compared to shielded controls (Fig. 1). TMP-1363- treated, irradiated biofilms demonstrated a significant reduction (P 0.06) in metabolic activity between samples treated with I and 3 [mu]g mL^sup -1^ and irradiated compared to shielded samples, the overall trend was similar to TMP-1363. A significant reduction (P

TMP-1363 is effective in PDT of C. albicans and C. glabrata early stationary phase yeast

We next examined whether TMP-1363 was effective against Candida growth forms that were not susceptible to Photofrin phototoxicity (16). Using the more virulent C. albicans strain SC5314 (33) and a clinical isolate of C. glabrata MRO-084-R (16), we performed PDT of early stationary phase yeast with the photosensitizer TMP-1363, and assessed its efficacy using the XTT assay. Both C. albicans and C. glabrata displayed a photosensitizer concentration-dependent reduction in metabolic activity (data not shown). Since the viability of the yeast form of Candida is accurately quantitated by a CFU assay, and the CFU assay has a greater dynamic range than the spectrophoto metric XTT assay, we utilized the colony forming ability of Candida yeast to obtain a more accurate measure of phototoxicity. Spotting dilutions of organisms on agar plates following PDT was used as a screen to evaluate phototoxicity of TMP- 1363 (IO [mu]g mL^sup -1^) over a range of fluences (Fig. 2A). Candida albicans SC5314 and C. glabrala MRO-084-R demonstrated a similar pattern of sensitivity with increased fluence. To more accurately quantify TMP-1363-induced phototoxicity against Candida, a set fluence of 2.4 J cm^sup -2^ and a TMP-1363 concentration of 10 [mu]mL^sup -1^ were used in PDT and evaluated by the CFU assay (Fig. 2B). Candida albicans SC5314 (open bars) and C, glabrata MRO-084-R (closed bars) exhibited a significant reduction (P 0.5) in CFU between the Candida species in the irradiated group, and no significant (P > 0.5) dark toxicity was exerted by TMP-1363. These data confirm that both C albicans SC5314 and C. glabrata MRO-084-R early stationary phase yeast are sensitive to PDT using TMP-1363, and represents the first example of the successful application of PDT against C. glabrata.

Figure 1. Photodynamic treatment (PDT) of C. albicans biofilms and germ tubes. C. albicans germ tubes were grown in M199 for 3 h at 37[degrees]C. C. albicans biofilms were grown in RPMI 1640 supplemented with 1% glucose for 24 h at 37[degrees]C with gentle rocking. Germ tubes and biofilms were treated with either increasing doses of Photofrin (left panels) or TMP-1363 (right panels). After incubation with photosensitizer, samples were irradiated with broadband light at a fluence of 2.4 J cm^sup -2^ for germ lubes and 4.8 J cm^sup -2^ for biofilms (open bars); photosensitizer-treated cells shielded from light served as a negative control (closed bars). Following PDT. organism metabolic activity was determined by XTT assay and used as an indicator of cell damage. Conversion of soluble, colorless XTT to orange-colored formazan product was measured spectrophotometrically at Abs^sub 450^, (Y-axis). These data represent the mean of two separate experiments using triplicate samples +- SD.

Figure 2. Plate-based killing assay for C. albicans and C. glabrata stationary phase yeasts treated with TMP 1363. Panel (A) corresponds to C. albicans and C. glabrata early stationary phase yeast treated with 10 [mu]g mL^sup -1^ of TMP-1363 and irradiated at increasing fluences (0.242.4 J cm^sup -2^). Organisms were serially diluted 10-fold (undiluted to 10^sup -4^), 2 [mu]L were spotted on YPD plates and incubated at 37[degrees]C for 24 h. Panel (B) corresponds to C. albicans and C. glabrata early stationary phase yeast treated with 10 [mu]g mL^sup -1^ of TMP-1363 and irradiated at 2.4 J cm^sup -2^. Phototoxic damage induced by TMP-1363 at 2.4 J cm^sup -2^ was assessed by viable plate counts (CFU mL^sup -1^) on YPD. Data represents the mean of three separate experiments using duplicate samples +- SD. Open bars, C. albicans SC5314. Closed bars, C. glabrata MRO-084-R. Figure 3. Phenotypic characterization of C. albicans and C. glabrata respiratory-deficient (RD) “petite” mutants. C. albicans SC5314 and C. glabrata MRO-084-R RD mutants were selected on YPD plates containing ethidium bromide (40 [mu]g mL^sup -1^) after 72 h at 30[degrees]C (7). “Petite” colony types were identified for both Candida species. To confirm the RD phenotype, mutants of C. albicans 6p (panel A) and C. glabrata 1p (panel B) were streaked on the right side of YPDextrose and YPGlycerol agar plates. The corresponding wild-type respiratorycompetent (RC) strains were streaked on the left side of the same plates. As predicted, both wild-type RC and the putative RD mutants grew on YPDexlrose plates (left column), but RD mutants were unable to grow on plates containing the nonfermenlable carbon source glycerol (right column).

Table 1. Susceptibility of Candida albicans and Candida glabrata strains to fluconazole.

Respiratory-deficient mutants of Candida are hypersensitive to PDT compared to wild-type

RD strains of C. albicans and C. glabrata display a pleiotropic resistance pattern, including resistance to members of the azole family of antifungals, the cationic salivary antimicrobial peptides termed histatins, and other types of toxic stresses (25,29,30). We sought to determine whether RD mutants of C. albicans SC5314 and C. glabrata MRO-084-R acquired a similar resistance phenotype to PDT using TMP-1363. Confirmation of the RD phenotype was demonstrated by the inability of the mutants to grow on nutrient agar using the nonfermentable substrate glycerol as the carbon source, but they could ferment glucose to support growth. Representative mutants are shown in Fig. 3. Furthermore, both C. albicans and C. glabrata RD mutants displayed enhanced uptake of the dyes eosin Y and trypan blue, as reported by Gyurko et al. (29) for C. albricans (data not shown). Similar to previous reports (25,26,41), RD mutants displayed an increase in azole resistance compared to parental strains (Table 1). The increase in the MIC^sub 50^ to fluconazole was particularly striking in the C. albicans RD mutant 6p (> 128 [mu]g mL^sup -1^) compared to wildtype SC5314 (0.25 [mu]g mL-1). Although wild-type C. glabrata MRO-084-R was inherently more resistant to fluconazole compared to C. albicans SC5314 (42), the MIC^sub 50^ of C. glabrata RD mutant 1p (128 [mu]g mL^sup -1^) also increased compared to wild- type (>64 mg mL^sup -1^). The RD mutants from C. albicans and C. glabrata were then compared to their respective parental strains for susceptibility to PDT using TMP-1363.

Surprisingly, rather than displaying the resistance phenotype observed in response to other stressors, RD mutants of both C. albicans and C. glabrata were significantly more sensitive to PDT compared to their respective wild-type parental strains. For both C. albicans (Fig. 4, panel A) and C. glabrata (Fig. 4, panel B), wild- type parental and RD strains treated with 10 [mu]g mL^sup -1^ TMP- 1363, but shielded from light, showed a level of viability comparable to untreated organisms. The wild-type strain of C. albicans showed approximately a 1-log^sub 10^ reduction in CFU when treated with 0.5 [mu]g mL^sup -1^ TMP-1363 and irradiated compared to controls (P = 0.07; not significant). For C. glabrata RC strain MRO-084-R, slightly less killing was observed under the same PDT conditions (P = 0.08; not significant). In contrast, RD mutants of each Candida species treated with 0.5 [mu]g mL^sup -1^ TMP-1363 and irradiated showed over a 4-log^sub 10^ reduction in CFU compared to controls. For parental strains, PDT using 10 [mu]g mL^sup -1^ TMP- 1363 was needed to achieve a 4-log^sub 10^ reduction in CFU compared to controls (P

Figure 4. Increased sensitivity of Candida RD mutants to the photosensitizer TMP 1363 compared to wild-type parental strains. Early stationary phase yeast of C. albicans SC5314 (panel A) and C. glabrata MRO-084-R (panel B) with their corresponding respiratory- deficient mutants of early stationary phase yeast were incubated with either 0.5 or 10 [mu]g mL^sup -1^ TMP-1363 for 10 min and irradiated at 2.4 J cm^sup -2^ with broadband visible light. Untreated organisms and organisms treated with TMP-13263 but shielded from light were used as controls. Organism killing was determined by the colony forming unit (CFU) assay and represented as a log^sub 10^ reduction compared to the untreated control.

Acquired azole resistance contributes to sensitivity to PDT in respiratory competent C. albicans

It is unknown what metabolic alterations in RD mutants of Candida resulted in their increased sensitivity to PDT using TMP-1363. To assess the contribution of acquired fluconazole resistance to PDT sensitivity, we tested a matched pair of respiratory-competent C. albicans isolates (34,35) derived from the oral cavity of the same AIDS patient over a 2-year period. C. albicans TW 07229 was isolated early in the course of fluconazole treatment and is fluconazole- sensitive; strain TW 072243 was isolated at the end of the 2-year period and is fluconazole-resistant. We corroborated these phenotypes (Table 1), with C. albicans TW 07229 having an MIC^sub 50^ of 1 [mu]g mL^sup -1^ and C. albicans TW 072243 having an MIC^sub 50^ of >64 [mu]g mL-1. Early stationary phase yeast were sensitized with 0.5 and 10 [mu]g mL^sup -1^ of TMP-1363 and irradiated at a fluence of 2.4 J cm^sup -2^. As shown in Fig. 5, the two strains exhibited no significant difference in log^sub 10^ reduction of CFU after PDT with 0.5 [mu]g mL^sup -1^ of TMP-1363. At 10 [mu]g mL^sup -1^ TMP-1363, there was a significant difference (P

Figure 5. Fluconazole resistance contributes to the sensitivity of C. albicans to PDT using TMP-363. Early stationary phase yeast from a matched pair of fluconazole-sensitive (TW 07229) and fluconazole-resistant (TW 072243) C. albicans strains were treated with either 0.5 or 10 [mu]g mL^sup -1^ TMP-1363 and irradiated at a fluence of 2.4 J cm^sup -2^ with broadband visible light. Untreated organisms and organisms treated with TMP-13263 but shielded from light were used as controls. Organism killing was determined by the colony forming unit (CFU) assay and represented as a log^sub 10^ reduction compared to the untreated control.

DISCUSSION

The importance of oropharyngeal and esophageal candidiasis as a medical problem (2) and a therapeutic challenge (35) make PDT an attractive alternative for treatment. Experimental successes against oral candidiasis (22) increase confidence that application of PDT to treatment will be translated to the clinic. In this study, we describe the efficacy of a cationic porphyrin photosensitizer TMP- 1363 against morphological forms of Candida refractile to PDT using Photofrin. The sensitivity of C. glabrata to TMP-1363 phototoxicity is significant since, compared to C. albicans, this species of Candida is inherently more resistant to the widely used azole class of antifungals that target ergosterol synthesis (42). C. glabrata is also comparatively more resistant to the cationic salivary antimicrobial peptides of the histatin family (23), which comprise an innate oral defense mechanism.

The primary biological finding in our studies was the demonstration of significantly increased sensitivity of RD mutants of C. albicans and C glabrata to PDT with TMP1363. Unlike mammalian cells, certain fungi, including S. cerevisiae (28), C. albicans (29) and C. glabrata (27) can survive without functional mitochondria, using fermentation to generate ATP. Adaptation to stress induced by drug treatment modulates mitochondrial function in Candida. C. glabrata may switch reversibly between states of mitochondrial competence and incompetence in response to fluconazole exposure (43). The clinical relevance of these observations is that uncoupling of oxidative phosphorylation enables C. albicans to resist killing by phagocytes and persist in tissue (30). Further, azoleresistant, RD mutants of C. glabrata can be selected in vivo (44).

Thus, PDT may be effective under conditions that allow Candida to escape both host defenses and conventional therapeutic intervention. Importantly, the increased sensitivity of RD Candida mutants to PDT with TMP-1363 reveals pathways of resistance to oxidative stress that can be targeted to increase the efficacy of PDT. The potential advantage of inhibiting these pathways to increase the sensitivity of the fungus to PDT would be diminished phototoxicity to surrounding host tissue as a result of the application of milder treatment parameters, such as reduced photosensitizer concentration or reduced fluence.

While the mechanisms of fungal resistance to toxic stress are not fully understood, in some cases, resistance has been associated with an increased expression of drug efflux pumps (25,41,45,46). In C. glabrata. the zinc cluster transcriptional activator Pdr1p is a key regulator of a pleiotropic drug resistance network that mediates azole resistance in clinical isolates and RD mutants, at least in part, via increased expression of drug efflux pumps (45,47). Elevated drug pump activity is also a contributing mechanism to azole resistance in C albicans (35,48). The increased sensitivity to PDT with TMP-1363 of C. albicans and C. glabrata RD mutants, as well as azole-resistant C. albicans. would suggest that this photosensitizer is not a substrate for the drug pumps contributing to azole resistance. Azole-resistant, respiratory-competent mutants of C. albicans display changes in membrane lipid fluidity and asymmetry (49). These changes in membrane composition may have contributed to the observation (50) that azole-resistant strains of C. glabrata with an ERG11 deletion in the ergostcrol synthesis pathway demonstrated enhanced susceptibility to oxidative killing. Furthermore, treatment of C. albicans with miconazole or fluconazole significantly increased endogenous ROS (51). In our studies, comparison of the sensitivity of matched fluconazole-sensitive and fiuconazote-resistant strains of C. albicans (34,35) to PDT showed a measurable increase in the sensitivity of the fluconazole-resistant strain. However, the differential in sensitivity was not as significant as the difference between RD mutants of C. albicans and C. glabrata and their respective wild-type parental strains.

There are several potential explanations for the marked increase in the sensitivity of the RD mutants to PDT compared to wild-type and fluconazole resistant, respiratory-competent strains of Candida. One possible contributing factor for the increased sensitivity of the RD strains to PDT is that alterations in cell wall structure and/ or permeability results in increased levels of cell-associated photosensitizer compared to wild-type. Recent studies indicate that access of the photosensitizer to the plasma membrane is a prerequisite for phototoxicity against Candida (21) and other microbes (13). Hence, increased penetration of the cell wall by photosensitizers would be expected to increase the sensitivity of the fungus to PDT by allowing interaction with the plasma membrane. The ability of photosensitizers to damage or traverse the plasma membrane could lead to phototoxicity of cytoplasmic constituents or intracellular organelles. The colonies of RD mutants grown on eosin Y-Trypan blue plates demonstrated increased dye association compared to parental strains (data not shown; [29,30]), suggesting increased cell binding/penetration of these polar, heterocyclic dyes in RD mutants, reflective of an altered cell wall. Furthermore, RD mutants of both S. cerevisiae (52,53) and C. glabrata (54) display wall alterations that increased concanavalin A binding to the cell surface. In S. cerevisiae, RD mutants also displayed increased sensitivity to calcoflour white, suggesting a weakened cell wall in these strains (55).

Mitochondrial electron transport contributes to maintenance of appropriate plasma membrane permeability in S. cerevisiae (56). RD strains frequently acquire resistance to ftuconazole and other azoles (25,29,30,57). suggesting a relationship between mitochondria and ergosterol metabolism. In fluconazole-resistant RD mutants of C. glabrata. increased free ergosterol content was proposed to account for increased susceptibility to polyene antifungals (41). Interestingly, in S. cerevisiae, a deficiency in the synthesis of the mitochondrial anionic phospholipid cardiolipin results in a growth defect at elevated temperature that can be suppressed by a loss-of-function mutation in KRE5, a gene involved in cell wall biogenesis (58). Taken together, the observations underscore the relationship between mitochondrial function, membrane composition and cell wall integrity in fungi.

Phototoxicity following membrane photosensitization can also lead to the production of secondary ROS, probably as a result of lipid peroxidation (59). Furthermore, because of the large amounts of ROS produced during oxidative phosphorylation occurring in its inner membrane, the mitochondrion has mechanisms to detoxify ROS, primarily superoxide anion and hydrogen peroxide. In S. cerevisiae (60) and Candida (61), manganese-superoxide dismutase (SOD) Mn- Sod2p specifically localizes in the mitochondrial matrix and contributes to protection against oxidative stress. C. albicans encodes five additional Cu-Zn SODs located cytoplasmically (61). In addition, there are secondary defenses of enzymes that repair oxidatively damaged components (62). Our previous studies have indicated that catalase induction docs not participate significantly in protection against PDT-induced phototoxicity in C. albicans (17). However, the role of SODs and secondary oxidative defenses in protection against antimicrobial PDT has not been explored extensively. The importance of identifying specific mechanisms of protection against ROS induced by PDT, primarily singlet oxygen, is underscored by the recent work of Dawes and colleagues in S. cerevisiae demonstrating that cells have constitutive defense systems that are largely unique to each oxidant (63,64).

Our studies with the Candida RD mutants suggest intact mitochondrial function may provide a basal level of antioxidant defense against PDT-induced phototoxicity. We suggest that increased endogenous oxidative stress as a consequence of mitochondrial dysfunction combined with the added oxidative stress induced by PDT resulted in the increased sensitivity of the respective RD mutants compared to wild-type C. albicans and C. glabrata. Future efforts will be directed at identifying the genetic alterations that contribute to the increased sensitivity of the Candida RD mutants to PDT.

Acknowledgements-This work was supported by grant DE016537 from the National Institutes of Health. The authors thank David Kessel for generously providing the broadband light source used in these studies.

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Yeissa Chabrier-Rosello1, Thomas H. Foster2, Soumya Mitra2 and Constantine G. Haidaris*1,3

1 Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY

2 Department of Imaging Sciences, University of Rochester Medical Center, Rochester, NY

3 Center for Oral Biology, University of Rochester Medical Center, Rochester, NY

Received 23 October 2007, accepted 6 December 2007, DOI: 10.1111/ j.1751-1097.2007.00280.x

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