Our previous work (3
) demonstrated the susceptibility of the fungus Candida
to the photodynamic effects of Photofrin. In the present studies, we defined conditions of photodynamic treatment that resulted in significant phototoxicity to C. albicans
while reducing the time of exposure of the organism to Photofrin, minimizing the concentration of Photofrin needed, and minimizing the fluence of irradiation applied to the organism. Similar to efforts directed against the treatment of tumors, the success of PDT against candidiasis in vivo will require maximizing the differential between the phototoxicity against the organism and that against normal host tissue. In tumor systems, this differential is based, in part, on the selective retention of photosensitizing agent by tumor cells compared to that by normal cells.
Exposure times of as short as 1 to 5 min were adequate for the effective treatment against C. albicans
germ tubes, and the short incubation time did not require a substantially higher drug concentration to exert a significant phototoxic effect. The brief exposure time to Photofrin and the low concentration required to mediate a significant degree of damage to C. albicans
germ tubes indicate that it may be possible to establish effective PDT treatment regimens for candidiasis that will result in minimal damage to surrounding host tissue and to establish that effective phototoxicity against C. albicans
can be achieved under conditions comparable to a brief topical application of Photofrin and irradiation in vivo. Although these data strongly suggest that PDT will be useful clinically against C. albicans
, the complexity of the in vivo environment makes it difficult to extrapolate directly from in vitro studies the drug and light doses that would be optimal in vivo; it is possible that higher doses may be required for effective PDT. Nonetheless, the in vitro studies described here will provide guidelines for testing in animal models of mucocutaneous (28
) and cutaneous (13
Similar to what is seen for other microbes, two mechanisms contributing to drug resistance in Candida
and other fungi are the enzymatic alteration of the active agent and the induction of drug efflux pumps (35
). Therefore, we sought to determine whether organisms preloaded with Photofrin lost photosensitivity over time, which would suggest either the degradation or the efflux of the photosensitizer. Incubation of preloaded organisms for up to 2 h in a hydrophilic environment of either buffer or tissue culture medium supplemented with glucose prior to irradiation did not diminish the phototoxic effect of 10 μg/ml of Photofrin (Fig. ). The retention of photosensitivity in the absence of serum and the gradual loss of photosensitivity in the presence of serum suggest that C. albicans
germ tubes do not extensively modify the photosensitizing properties of Photofrin, nor do they rapidly pump the photosensitizer from the cell. Rather, the presence of serum in the incubation medium may result in the gradual leaching of Photofrin to the external milieu, similar to what is seen in tumor cells (1
In many cell types, including C. albicans
, exposure to sublethal concentrations of hydrogen peroxide or superoxide anion results in the acquisition of resistance to more toxic concentrations of these oxidative species. This is largely due to the induction of enzymes capable of neutralizing these oxidative species: catalase and superoxide dismutase, respectively (15
). The primary oxidative species generated by the interaction of Photofrin, oxygen, and light is singlet oxygen (40
). A number of biomolecules, including thioredoxin (7
), histidine (32
), vitamin B6
), vasoactive intestinal peptide (30
), and melatonin (22
), are capable of scavenging or quenching singlet oxygen. However, a search of the literature database uncovered no reports of an inducible enzymatic scavenger of singlet oxygen. Furthermore, resistance to singlet oxygen is rare in biological systems, with only the Cercospora
fungi, a group of plant pathogens, reported as being tolerant to photosensitizers that generate singlet oxygen. Resistance to singlet oxygen is under the control of the SOR1
gene in Cercospora
, and orthologs are found in a wide range of species, including yeast (Saccharomyces cerevisiae
and Schizosaccharomyces pombe
). Two Cercospora
genes involved in singlet oxygen detoxification include a multidrug ABC transporter protein and a member of the pyridine nucleotide-disulfide oxidoreductase family (39
). Therefore, we reasoned that an as-yet-undiscovered inducible mechanism of resistance to singlet oxygen could be expressed in C. albicans
Since C. albicans produces catalase, we confirmed previous observations that resistance to subsequent H2O2 challenge could be induced in germ tubes after initial exposure to subtoxic levels of H2O2. In contrast to this observation, primary treatment of C. albicans germ tubes with a subtoxic concentration of Photofrin did not result in a comparable acquisition of resistance to increasing concentrations of Photofrin in a secondary treatment. Hence, no adaptation to a primary singlet oxygen stress similar to that seen with hydrogen peroxide was observed in C. albicans germ tubes. Furthermore, no cross talk was observed in the responses of C. albicans to the two different treatments used in sequence, regardless of the order of administration. These data suggest that reactive oxygen intermediates produced by the photodynamic activation of Photofrin internalized by C. albicans does not include an amount of hydrogen peroxide sufficient to induce significant catalase production; the data also demonstrate that the response to oxidative stress induced by H2O2 does not elicit protection against subsequent challenge with Photofrin.
Both in the environment and during the course of infection, organisms frequently exist in adherent, organized communities termed biofilms rather than as the independent entities usually seen during planktonic growth (37
). Of particular importance clinically, biofilm populations are more resistant to antibiotic concentrations that are effective against the same population if the biofilm is dispersed (14
). We sought to determine whether biofilm development rendered C. albicans
more resistant to Photofrin-mediated phototoxicity.
Under the experimental conditions used, the sensitivity of C. albicans to Photofrin-mediated phototoxicity did not diminish during the course of mature (48-h) biofilm formation (Fig. ). Attempts to examine photodynamic efficacy against mature C. albicans biofilms over longer time periods of development were thwarted by the fragility of the older biofilms, which did not hold up well to the necessary wash steps. Intermediate-stage biofilms proved to be fairly resilient to manipulation and were used in subsequent experiments. The intermediate-stage (24-h) C. albicans biofilms were as sensitive as germ tubes to Photofrin-mediated phototoxicity in terms of the fluence of irradiation applied.
Intermediate biofilms treated with a high concentration of amphotericin B (10 μg/ml) over the time period required to complete the photosensitization and irradiation protocol against parallel cultures did not show a significant reduction in XTT metabolism compared to Photofrin-treated, nonirradiated cultures. Treatment of planktonic C. albicans
blastoconidia with 1 to 8 μg/ml of amphotericin B for a comparable time period significantly reduced the plating efficiency of the organism (26
), demonstrating that, under the proper conditions, amphotericin B can exert a toxic effect in a relatively brief time frame. In contrast to the sensitivity of planktonic cells to both fluconazole and amphotericin B, Kuhn et al. (25
) demonstrated increased resistance of C. albicans
biofilms to both the azole and the free amphotericin B, as measured by the XTT assay. In the same study, only liposome-encapsulated amphotericin B and the cell wall synthesis inhibitor echinocandin (25
) displayed efficacies against C. albicans
biofilms. Thus, Photofrin-mediated phototoxicity against C. albicans
biofilms in vitro appeared at least comparable to the most effective antifungal formulations in current use.
We have begun to define conditions for effective photodynamic treatment against C. albicans
in vitro that will serve as guidelines for investigating the efficacy of Photofrin in PDT for candidiasis in experimental models of superficial infection. It is difficult to mimic the complex milieu of a cutaneous or mucocutaneous infection in vitro. However, knowing the lower limits of photosensitizer exposure time, photosensitizer concentration, and total fluence required to mediate significant damage against the organism in vitro will allow for the rational design of animal studies similar to those described by Teichert et al. (38
), who used methylene blue in PDT of experimental oral candidiasis. Using these in vitro parameters of effective photosensitization as a baseline, we have also shown that several of the mechanisms that microorganisms use to subvert either antimicrobial oxidative defenses or antimicrobial therapy are apparently not operative during Photofrin-mediated phototoxicity of C. albicans
. These observations provide support and rationale for the continued investigation of PDT as an adjunctive, or possibly alternative, mode of therapy against cutaneous and mucocutaneous candidiasis.