Respiratory Deficiency Enhances the Sensitivity of the Pathogenic Fungus Candida

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Respiratory Deficiency Enhances the Sensitivity of the Pathogenic

Fungus Candida to Photodynamic Treatment

Posted on: Sunday, 5 October 2008

RedOrbit - Dallas,TX*

By Chabrier-Rosello, Yeissa , H; Mitra, Soumya;

Haidaris, Constantine G

http://www.redorbit.com/news/health/1578167/respiratory_deficiency_en

hances_the_sensitivity_of_the_pathogenic_fungus_candida/

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, sville,

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 <0.05 were

considered significant.

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

< 0.001) in metabolic activity at all photosensitizer concentrations

tested, demonstrating that TMP-1363 is as efficient at inducing

phototoxicity against C. albicans biofilms as Photofrin. Although

Photofrintreated biofilms exhibited no significant difference (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 <

0.001) in metabolic activity was observed between the irradiated

samples treated with 5 to 20 [mu]g mL^sup -1^ of Photofrin compared

to shielded samples. Candida albicans 3153A germ tubes exhibited a

similar, concentration-dependent pattern of phototoxicity with

Photofrin and TMP-1363. For either photosensitizer, a minimal degree

of dark toxicity was observed.

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 < 0.04) in

viability of three to four logs compared to the shielded control.

There was no significant difference (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 ( 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 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 , 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 < 0.001). In the RD mutants of both C. albicans and

C. glabrata, PDT using 10 [mu]g mL^sup -1^ TMP-1363 resulted in an

additional 2- to 3-log^sub 10^ reduction in CFU compared to the

respective parental strains; over a 6-log^sub 10^ reduction compared

to controls.

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 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

< 0.05) in CFU log^sub 10^ reduction between the fluconazole-

sensitive and fluconazole-resistant C. albicans clinical isolates.

The fluconazole-sensitive C. albicans TW 07229 strain exhibited

approximately a 4.5 log^sub 10^ reduction in CFU and the fluconazole-

resistant C. albicans TW 07224? strain exhibited a 5.5 log^sub 10^

reduction in CFU. There was no significant dark toxicity observed in

those samples treated with TMP-1363, but shielded from the

irradiation. Despite a statistically significant increase in

sensitivity to PDT in the fluconazole-resistant strain compared to

the fluconazole-sensitive strain at higher TMP1363 concentrations,

the differential was not as great as observed between wild-type C.

albicans and the RD mutant (Fig. 4).

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 Kessel for

generously providing the broadband light source used in these

studies.

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Yeissa Chabrier-Rosello1, H. 2, 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

*Corresponding author email; haid@mail, rochester.edu (Constantine

G. Haidaris) © 2008 The Authors. Journal Compilation. The American

Society of Photobiology 0031-8655/08

Copyright American Society for Photobiology Sep/Oct 2008

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