Aim: To evaluate the capacity of hematoporphyrin monomethyl ether (HMME) in the presence of light to cause photodynamic inactivation (PDI) of Candida albicans. Materials & methods: HMME photoactivity was evaluated against azole-susceptible and -resistant C. albicans. The mechanisms by which PDI of C. albicans occurred were also investigated. Results: HMME-mediated PACT caused a dose-dependent inactivation of azole-susceptible and -resistant C. albicans. Incubation with 10 μM HMME and irradiation with 72 J cm-2 light decreased the viability of C. albicans by 7 log10, induced damage of genomic DNA, led to loss of cellular proteins and damaged the cell wall, membrane and intracellular targets. Conclusion: Candida albicans can be effectively inactivated by HMME in the presence of light, and HMME-mediated PACT shows its potential as an antifungal treatment.

First draft submitted: 27 July 2015; Accepted for publication: 10 November 2015; Published online: 2 March 2016

Candida albicans, which is present in the normal microbiota of healthy individuals, is an opportunistic commensal pathogen that lives in the oral cavity, GI tract and vagina [1]. It causes a wide range of human diseases ranging from superficial mucosal infections to life-threatening invasive candidiasis in immunocompromised patients [2–4]. Most treatments available for systemic and invasive candidiasis are based on antifungal drugs including azoles, polyenes, pyrimidine and echinocandins. However, these drugs can be toxic to the host [5] and can damage and interrupt cellular functions [6]. Moreover, the extensive and repetitive use of antifungal drugs has resulted in the development of drug-resistant C. albicans strains, and the occurrence of infections refractory to standard antifungal therapy has increased [7,8]. Thus, development of alternative methods to manage drug resistance is imperative. One promising therapeutic approach is photodynamic antimicrobial chemotherapy (PACT), which uses a photosensitizer (PS) that is excited from a ground state to a triplet state upon illumination with light of an appropriate wavelength. The triplet state PS reacts with oxygen in and around the cells, thereby forming singlet oxygen (1 O2 ) or other reactive oxygen species (ROS) [9] that rapidly react with nonspecific microbial targets and irreversibly destroy microbial cells through chemical and phototoxic reactions [10]. PACT has several advantages over traditional therapies, including high target specificity through direct application of PS and light irradiation to the target sites [11], low risk of chemical and thermal side effects [12], biocompatibility with human cells [13] and low potential for development of drug resistance due to the nonspecific action of liberated 1 O2 or other ROS [5].

Hematoporphyrin monomethyl ether (HMME) is a second-generation, porphyrin-related PS developed in China [14]. It consists of a mixture of the two positional isomers 3-(1-methyloxyethyl)- 8-(1-hydroxyethyl) deuteroporphyrin IX and 8-(1-methyloxyethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX (Figure 1) [15]. Compared with first-generation PS, for example, photofrin and hematoporphyrin derivative (HpD), HMME has a known structure, higher photoactivity, stronger photodynamic efficiency, lower toxicity and a faster clearance rate. Moreover, HMME is less costly than other photoactive drugs [16–20]. In previous studies, it has been shown that in the presence of light HMME is effective at killing several types of cancer cells [19–22] and some Gram-positive and Gram-negative bacteria [23,24]. However, there are no reported studies on the capacity of this porphyrin to cause photodynamic inactivation (PDI) of C. albicans. Therefore, we conducted this study to assess the potential of HMME to mediate PDI of drug-resistant and drug-sensitive strains of C. albicans and evaluated the effects of treatment on cell macromolecular structure, DNA and protein.

Material & methods

● Candida albicans strains & culture conditions

A standard C. albicans strain (ATCC 10231) and an azole-resistant clinical isolate of C. albicans were obtained from the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China. The strains were grown aerobically on Sabouraud dextrose agar (SDA, Nisuvi Sehuu Biotech, China) at 37°C for 48 h. The colonies were transferred into 15 ml Sabouraud dextrose broth (SDB, Nisuvi Sehuu Biotech, China) and incubated overnight at 37°C. Cell pellets were collected by centrifugation (4000 rpm for 10 min, Thermo Fisher D–37520, Germany) and washed three-times with sterile phosphatebuffered saline (PBS, pH 7.0). The pellets were resuspended to a cell density of 1 × 107 colony forming units (CFU)/ml before the experiments.

● PS & light source.

Drug-grade HMME was purchased from Shanghai Xianhui Pharmaceutical Co., China. A 1 mM stock solution was freshly prepared by dissolving the PS in PBS and stored in the dark. The stock solution was filtered through a 0.22 μm filter disk and diluted to the desired concentration with PBS before use. All illuminations were performed with white light from a 300 W xenon lamp (Ceaulight CEL-HXF300, China) with a wavelength range 400–780 nm selected by an optical filter (Ceaulight CELUVIRCUT PD–145, China). To avoid sample heating, the light was passed through a 1 cm water filter. The fluence rate at the level of the samples was 40 mW cm-2, as measured by a power meter (Ceaulight CEL-NP2000, China)

● PDI on C. albicans

Samples of the yeast suspension (2 ml, 1 × 107 CFU/ml) were centrifuged at 4000 rpm for 10 min. The pellets were resuspended in 2 ml of PBS containing HMME at various concentrations (0.01–10 μM) and incubated at 37°C

in the dark for 30 min in a shaking incubator (100 rpm). The samples were transferred to sterile 35-mm polystyrene culture dishes and irradiated for 30 min (total energy dose of 72 J cm-2). After irradiation, yeast suspensions were centrifuged at 4000 rpm for 10 min. The pellets were resuspended, serially diluted 10-fold with PBS and 20 μl of each dilution was spread in triplicate on SDA. Colonies were counted after 24-h incubation at 37°C. The fraction of surviving yeast was calculated as the CFU/ml after exposure to light divided by the CFU/ml before light exposure. All experiments were performed three times.

● Genomic DNA purification & electrophoresis.

To determine if PDI of C. albicans occurred through DNA damage, genomic DNA was extracted and analyzed by agarose gel electrophoresis. After PDI treatment (10 μM HMME and 72 J cm-2 white light), genomic DNA was immediately extracted using a Genomic DNA Purification Kit (Promega, USA). DNA samples were mixed with 6× loading buffer (0.25% w/v bromophenol blue, 40% w/v sucrose, 1.15% acetic acid, 40 mM Tris, 1 mM EDTA) and analyzed by electrophoresis in a 1% agarose gel in Tris/Borate/EDTA buffer (TBE; 90 mM TrisHCl, 90 mM boric acid and 2 mM EDTA, pH 8) at 2.9 V cm-1 for 1.5 h. Ethidium bromide (1 μg/ml) was incorporated into the agarose gel, and a Lambda DNA/HindIII digest marker with 125–23,130-bp DNA fragments (Promega, USA) was used as a molecular weight marker.

● Protein extraction & SDS-PAGE

Whole-cell protein extraction was performed according to a previously described method [25]. After PDI treatment, yeast suspensions were centrifuged at 4000 rpm for 10 min. The pellets were washed twice with PBS and resuspended in 200 μl sample buffer containing 0.06 M Tris-HCl, pH 6.8, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 10% (v/v) glycerol, 1 mM phenylmethylsulfonylfluoride and 0.5% (w/v) bromophenol blue. The samples were boiled for 20 min, and 10 μl of each sample was loaded onto a 10% (w/v) polyacrylamide gel and subjected to electrophoresis at 80 V for 10 h. The reservoir buffer consisted of 0.25 M Tris-HCl, 1.92 M glycine and 1% (w/v) SDS. A Biostep Prestained Protein Marker (Tanon, China) with a range of proteins 10–170 kDa was used as molecular weight marker. The gel was stained with 0.05% (w/v) Coomassie Brilliant Blue R 250 for 4 h and destained in 10% (v/v) acetic acid and 20% (v/v) methanol.

● Fluorescence labeling For intracellular localization of HMME, the yeast cells were incubated with HMME and a DNAspecific fluorescent dye, Hoechst 33342 (Sigma– Aldrich, China). After PDI treatment, yeast suspensions were centrifuged at 4000 rpm for 10 min. The pellets were resuspended in 2 ml Hoechst 33342 (1 μg/ml) in PBS and incubated in the dark at room temperature for 10 min in a shaking incubator (100 rpm). Labeled cells were washed three-times with PBS, spotted on glass slides and immobilized by the coverslips. Cell imaging was conducted on a confocal microscope (Olympus FluoView FV1000, Japan). Images were captured with CFI VC 60× oil immersed optics. Confocal images of HMME and Hoechst 33342 fluorescence were collected using solid-state diode lasers, with 543 and 351 nm excitation wavelengths, respectively, and with appropriate emission filters.

● Transmission electron microscopy

Transmission electron microscopy (TEM) samples were prepared according to a previously described method [26]. After PDI treatment, yeast cells were centrifuged at 4000 rpm for 10 min and fixed in 2.5% glutaraldehyde at 4°C for 2 h. The pellets were washed with PBS three-times and fixed in 1% osmium tetroxide at 4°C for 2 h. Thereafter, the pellets were dehydrated with ethanol gradients and embedded in Epon 812 epoxy resin (SPI-Chem, USA) at 60°C for 24 h. Thin-section samples of 50–70 nm were prepared using an LKB-V ultratome (LKB, Sweden). The samples were stained with uranyl acetate and lead citrate for 15 min, respectively. Finally, the samples were viewed and digitally photographed using a TEM (Hitachi H-7650, Japan).

● Scanning electron microscopy

After PDI treatment, yeast suspensions were transferred into the wells of a sterile 24-well polystyrene microplate (Corning, USA) that contained sterile glass coverslips and incubated at 37°C for 1 h. The coverslips were gently washed with PBS three times and fixed in 2.5% glutaraldehyde at 4°C for 2 h. Then, the coverslips were washed with PBS three times and fixed in 1% osmium tetroxide at 4°C for 2 h. After dehydration with ethanol gradients, the samples were freeze dried, sputter coated with gold and observed using an SEM (Hitachi TM-1000, Japan).

Results

PDI

Before and after irradiation, sample temperatures were 35.1 and 32.3°C, respectively, as measured by a thermocouple (IKA EST-D5, Germany) at room temperature, indicating that the light had no heating effect on these samples. HMME did not have any dark toxicity toward the two C. albicans strains at the concentrations and times tested (Figure 2). Furthermore, direct exposure of these strains to light in the absence of HMME produced no cytotoxic effect (data not shown). By contrast, treatment with 0.01 μM HMME and irradiation with 72 J cm-2 white light achieved 0.90 and 0.78 log10 reductions in the number of viable C. albicans ATCC 10231 and the azole-resistant clinical isolate of C. albicans, respectively. The number of viable yeast was further reduced with increasing concentrations of HMME; treatment with 1 μM HMME in the presence of 72 J cm-2 white light yielded 4.26 and 3.88 log10 reductions in the number of viable C. albicans ATCC 10231 and the azole-resistant clinical isolate of C. albicans, respectively. Furthermore, no viable cells were detected after irradiation in the presence of 10 μM HMME, representing a 7 log10 reduction (Figure 2).