Chemical features of the photosensitizers new methylene blue N and S137 influence their subcellular localization and photoinactivation efficiency in Candida albicans

https://doi.org/10.1016/j.jphotobiol.2020.111942Get rights and content

Highlights

  • Mechanism of C. albicans photoinactivation with photosensitizers NMBN and S137

  • Photosensitizer lipophilicity influences subcellular localization and photokilling.

  • S137 is an extremely lipophilic photosensitizer that targets the cell membrane.

  • NMBN is moderately lipophilic and targets mitochondria.

  • S137 is more effective at lower fluences when compared to NMBN.

Abstract

Antimicrobial photodynamic treatment (APDT) has emerged as an effective therapy against pathogenic fungi with both acquired and intrinsic resistance to commonly used antifungal agents. Success of APDT depends on the availability of effective photosensitizers capable of acting on different fungal structures and species. Among the phenothiazinium dyes tested as photoantifungals, new methylene blue N (NMBN) and the novel pentacyclic compound S137 are the most efficient. In the present study we compared the effects of APDT with NMBN and S137 on the survival of Candida albicans and employed a set of fluorescent probes (propidium iodide, FUN-1, JC-1, DHR-123 and DHE) together with confocal microscopy and flow cytometry to evaluate the effects of these two chemically diverse photosensitizers on cell membrane permeability, metabolism and redox status, and mitochondrial activity. Taken together, our results indicate that, due to chemical features resulting in different lipophilicity, NMBN and S137 localize to distinct subcellular structures and hence inactivate C. albicans cells via different mechanisms. S137 localizes mostly to the cell membrane and, upon light exposure, photo-oxidizes membrane lipids. NMBN readily localizes to mitochondria and exerts its photodynamic effects there, which was observed to be a less effective way to achieve cell death at lower light fluences.

Introduction

Several procedures in modern medicine, such as solid organ and hematopoietic stem cell transplantations, surgeries, autoimmune disease therapies, and uncontrolled HIV infection make millions of patients vulnerable to lethal fungal diseases [1,2]. Candida albicans, usually a harmless commensal fungus, is also an opportunistic pathogen for immunocompromised people and the major human fungal pathogen in the USA and several other countries [3]. Today, fungal infections are among the most difficult diseases to treat in humans [1]. One of the factors that makes treatment so difficult is the rapid acquisition of resistance to all of the only four major classes of antifungal agents clinically available: azoles, polyenes, echinocandins, and a nucleotide analog [[4], [5], [6]]. Additionally, many species of Candida, such as Candida auris and Candida glabrata are intrinsically resistant to some antifungal classes [3,4,7]. Multidrug resistance can eliminate treatment options completely, which has a serious effect on patient survival [5].

The emergence of resistance to currently used antifungals has promoted the development of novel antifungal approaches, such as antimicrobial photodynamic treatment (APDT). The basic principle behind antimicrobial photodynamic inactivation is the combination of three factors: (1) visible or near-infrared light, (2) molecular oxygen, and (3) a photosensitizer (PS). Light exposure excites the photosensitizer to a singlet state. Then, intersystem crossing results in a photosensitizer in an excited triplet state which can interact with molecular oxygen either via electron or via energy transfer. Electron transfer, also called Type I reaction, usually results in the formation of radicals such as the superoxide anion radical. Conversely, energy transfer or Type II reaction results in the formation of singlet oxygen. In either case, reactive oxygen species (ROS) such as singlet oxygen, superoxide, and hydroxyl radicals have a broad spectrum of activity and can damage several microbial targets such as proteins, lipids, and nucleic acids encountered, therefore making selection of resistant strains unlikely [8,9]. Among photoantimicrobials evaluated as antifungals, the phenothiazinium dyes methylene blue and toluidine blue are the most commonly used, mainly due to their low toxicity and their long-established use for other clinical applications [9,10]. Phenothiazinium derivatives with improved photoantimicrobial activity against yeasts and filamentous fungi such as new methylene blue N (NMBN) and the novel pentacyclic compound S137, have been identified [10,11]. APDT with NMBN and S137 has been shown to be highly effective against fungi of the genera Aspergillus [12], Candida [10,11], Colletotrichum [12], Neoscytalidium [13], and Trichophyton [14].

The most important factor determining the outcome of APDT is how a photosensitizer interacts with cells of the target microorganism, with its subcellular localization being of particular interest [12,15,16]. This is because ROS have a short half-life and therefore exert their action in the vicinity of their production site [17]. Cellular uptake and intracellular localization is determined by chemical and structural features of the PS (e.g. molecular mass, lipophilicity, charge distribution, number of H-bond donors and acceptors, etc.), the concentration of the PS, the incubation time, and the phenotypic characteristics of the target cells [17]. PS characteristics such as charge type and distribution as well as lipophilicity may be controlled by informed synthesis [18].

The use of confocal laser scanning fluorescence microscopy has made the determination of intracellular localization of PS much easier. Colocalization of subcellular organelle-specific fluorescent probes with differing fluorescence emission peak to that of the PS can be used to more closely identify the site of localization and these probes can also be used to identify sites of damage after illumination [17].

The photosensitizers NMBN and S137 are chemically and structurally distinct, and consequently present different outcomes when used in APDT. For instance, use of S137 usually results in cell damage even in the dark (dark toxicity) and its microbial photoinactivation tends to be higher at lower light fluences when compared to NMBN. As previously mentioned, PS subcellular localization can greatly influence the results of APDT. Therefore, here we compared NMBN and S137 by employing a set of fluorescent probes (propidium iodide, FUN-1, JC-1, DHR-123, and DHE) together with confocal microscopy and flow cytometry in order to evaluate PS subcellular localization as well as the mechanism behind APDT with these PS.

Section snippets

C. albicans Strain and Growth Conditions

C. albicans strain ATCC 64548 was obtained from the American Type Culture Collection (ATCC) (Manassas, USA). Cells were grown on Sabouraud Dextrose Agar (SDA) medium (BD Difco, USA) in the dark, at 35 °C, for 48 h. Cells from isolated colonies were transferred to 150-mL Erlenmeyer flasks containing 50 mL of YPD medium [1% Yeast Extract (BD Difco, Sparks, USA), 2% Peptone (BD Difco) and 2% Dextrose (Vetec, Duque de Caxias, Brazil)]. Cultures were incubated in the dark at 35 °C for 6 h under

C. albicans Survival After APDT

The PS NMBN and S137 were compared in terms of cell mortality after APDT with fluences of 3, 9, and 14 J cm−2. Importantly, treatment with PS alone or light exposure alone did not result in cell mortality (Fig. 3). At 3 J cm−2, S137 was a much more effective PS, reducing cell viability by 99.98% (3.70 log10) whereas NMBN achieved only 85.2% (0.83 log10) under the same conditions (Fig. 3). Increasing fluence to 9 and to 14 J cm−2 allowed NMBN and S137 to achieve similar cell mortality, which was

Discussion

Understanding the mechanism behind microbial photoinactivation with different PS is a key step in improving the efficiency of APDT and in selecting the most appropriate PS based on target microorganism and condition. APDT of C. albicans with the PS NMBN and S137 revealed that the latter achieves increased cell mortality at lower fluences when compared to the former (Fig. 3). Under the experimental conditions used here, NMBN is expected to produce more singlet oxygen compared to S137 as its peak

Conclusion

Taken together, our results indicate that S137 and NMBN localize to different subcellular structures and hence inactivate C. albicans cells via different mechanisms. S137 localizes mostly to cell membrane and, upon light exposure, photo oxidizes membrane lipids, which in turn could change membrane permeability to S137 itself and allow the PS to reach other intracellular sites [26]. On the other hand, NMBN readily localizes to mitochondria and exerts its photodynamic effects there, which was

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank Eduardo Tozatto and Fabiana Rossetto de Moraes, both from Faculdade de Ciências Farmacêuticas de Ribeirão Preto, for confocal microscopy and flow cytometry analyses, respectively. This work was supported by the State of São Paulo Research Foundation (FAPESP) grants 2012/15204-8 and 2016/11386 as well as by the National Council for Scientific and Technological Development (CNPq) grants 425998/2018-5 and 307738/2018-3 to G.UL.B. We sincerely thank FAPESP for a post-doc fellowship to

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These authors contributed equally to this work.

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