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A recyclable and light-triggered nanofibrous membrane against the emerging fungal pathogen Candida auris [1]

['Xinyao Liu', 'Department Of Dermatovenereology', 'West China Hospital', 'Sichuan University', 'Chengdu', 'Laboratory Of Dermatology', 'Clinical Institute Of Inflammation', 'Immunology', 'Ciii', 'Frontiers Science Center For Disease-Related Molecular Network']

Date: 2022-07 

The emerging "super fungus" Candida auris has become an important threat to human health due to its pandrug resistance and high lethality. Therefore, the development of novel antimicrobial strategy is essential. Antimicrobial photodynamic therapy (aPDT) has excellent performance in clinical applications. However, the relevant study on antifungal activity and the mechanism involved against C. auris remains scarce. Herein, a recyclable and biodegradable polylactic acid-hypocrellin A (PLA-HA) nanofibrous membrane is newly developed. In vitro PLA-HA-aPDT could significantly reduce the survival rate of C. auris plankton and its biofilms, and the fungicidal effect of the membrane is still significant after four repeated uses. Simultaneously, PLA-HA exhibits good biocompatibility and low hemolysis. In vivo experiments show that PLA-HA-aPDT can promote C. auris-infected wound healing, reduce inflammatory response, and without obvious toxic side-effects. Further results reveal that PLA-HA-aPDT could increase endogenous reactive oxygen species (ROS) levels, leading to mitochondrial dysfunction, release of cytochrome C, activation of metacaspase, and nuclear fragmentation, thereby triggering apoptosis of C. auris. Compared with HA, PLA-HA shows stronger controllability and reusability, which can greatly improve the utilization efficiency of HA alone. Taken together, the efficacy, safety and antifungal activity make PLA-HA-aPDT a highly promising antifungal candidate for skin or mucous membrane C. auris infection.

It is urgent to develop new antifungal strategies to address the problem of Candida auris infection and drug resistance. Previous studies have revealed that antimicrobial photodynamic therapy (aPDT) based on natural products, such as hypocrellin A (HA), is a promising method in clinical applications. However, equivalent studies of aPDT on antifungal activity and its mechanism against C. auris remain scarce. Herein, we successfully prepared a recyclable, biodegradable, and light-driven antifungal PLA-HA nanofibrous membrane through the electrospinning technique. C. auris infection has been treated by aPDT in vitro and in vivo for the first time, especially HA-mediated aPDT. In vitro and in vivo experiments have provided sufficient lines of evidence that PLA-HA is a promising antifungal material for superficial C. auris infections due to its antifungal effect and excellent biocompatibility. Notably, there still remains a very high antifungal activity after utilizing PLA-HA four times. In addition, this study clarifies that the anti-C. auris mechanism of PLA-HA, namely, PLA-HA-mediated aPDT, is attributed to the formation of intracellular ROS, resulting in mitochondrial dysfunction and a decline in the mitochondrial transmembrane potential, releasing cytochrome C from mitochondria to the cytoplasm, promoting the activation of metacaspase, and inducing nuclear condensation and fragmentation of C. auris, thus triggering yeast cell apoptosis. This study lays a foundation for developing new antimicrobial nanofibrous dressings mediated by aPDT and provides an alternative strategy for the treatment of local fungal infectious diseases.

Funding: This work was financially sponsored by the National Natural Science Foundation of China (Nos. 81773343 (Y.P.R.), 51803128 (L.T.), 52073186 (L.T.), 81803150 (K.W.Z.). ( https://www.nsfc.gov.cn/ ) The authors also thank the support by the PostDoctor Research Project, West China Hospital, Sichuan University (2020HXBH152 (X.Y.L.)), 1.3.5 project for disciplines of excellence of West China Hospital, Sichuan University (ZYJC18033 (Y.P.R.)), and HX-Academician project of West China Hospital, Sichuan University (HXYS19003 (Y.P.R.)). ( http://www.wchscu.cn/index.html , the funder website of West China Hospital, Sichuan University). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Fig 1. A schematic illustration of the synthetic route of the PLA-HA membrane and its antifungal applications in C. auris planktonic cells and biofilms, as well as the corresponding infected wound healing.

In this study, we have prepared a novel type of polylactic acid-hypocrellin A (PLA-HA) nanofibrous membrane and tested the PLA-HA based aPDT effects on C. auris treatment. First, fungal culture, biofilm construction, morphological observation, and animal models of skin wound infection were used to clarify the role of PLA-HA nanofibrous membrane-aPDT on C. auris. Second, the ROS levels and apoptosis hallmarks phosphatidylserine ectropion, mitochondrial transmembrane potential, DNA fragmentation, nuclear condensation, metacaspase and cytochrome C activity in C. auris were examined to investigate the cellular mechanism of PLA-HA-aPDT killing ( Fig 1 ). This is the first application of nanofibrous membrane with non-toxic photosensitizer for photodynamic therapy against C. auris.

Acute and chronic skin wounds are important infective routes for C. auris to invade human localized tissue or cause systemic infection [ 5 ]. Therefore, anti-infective wound dressings are beneficial to heal wounds and promote tissue repair [ 25 , 26 ]. In antifungal-drug delivery materials, electrospun nano-membranes offer greater air permeability, higher porosity and surface area and higher loading capabilities of PS, making them an excellent choice for withstanding the invasion of exogenous microorganisms [ 27 , 28 ]. Severyukhina et al. used chitosan and a second-generation PS to prepare an electrospun nanofiber membrane, and achieved local PDT sterilization with visible light irradiation [ 29 ]. In addition, Si et al. reported a green bio-based nanofibrous membrane with antimicrobial activity that could be repeatedly sterilized by sunlight excitation [ 30 ]. Due to its readily spinnability, drawing-induced crystallization, excellent biocompatibility and biodegradability, the synthetic polymer polylactic acid (PLA) has the largest application in fiber and film manufacturing and biomedical engineering [ 31 , 32 ]. Compared to natural biodegradable polymers, PLA has more controllable mechanical and processible properties [ 33 ]. Therefore, PLA has been extensively applied as a substrate in diversified biomedical applications. Electrospinning technology can reconstruct the structure of PLA, transforming it from solid particles into a three-dimensional reticular nanofibrous membrane, which can mimic the architecture and morphology of the extracellular matrix around cells [ 34 ]. Moreover, the incorporation of bioactive materials can overcome the insufficiencies and enrich the functions of PLA for particular applications [ 31 ].

Hypocrellin A (HA), which is generally regarded as a new type of PS, belongs to the class of perylenequinonoid compounds [ 17 ]. This lipid-soluble pigment is one of the main secondary metabolites (SMs) produced by the Chinese medicinal fungus Shiraia bambusicola P. Henn. and Hypocrella bambusae (Berk. & Broome) Sacc., the fruit bodies of which can unblock meridians, dissolve phlegm and arrest cough [ 17 , 18 ]. It is well known that HA plays an important role in PDT for anticancer [ 19 ], antiviral [ 20 ] and antimicrobial [ 21 – 23 ] treatments, due to its production of singlet oxygen and semi-quinone free radicals under light irradiation. However, studies on the antifungal photodynamic activity of HA are limited, only focused on Candida albicans [ 22 , 24 ]. As a newly discovered “Super fungus”, aPDT or HA treatment on C. auris infection has never been reported previously.

Antimicrobial photodynamic therapy (aPDT) involves non-toxic photosensitizers (PSs) and suitable light sources to induce the production of reactive oxygen species (ROS), which can kill the microbial pathogens [ 7 , 8 ]. Whether in vitro or in vivo, numerous studies have demonstrated that ROS can inactivate many microorganisms regardless of the drug-resistance [ 9 – 11 ]. To date, many studies report that aPDT can overcome the resistance in fungi and bacteria, even if microbial pathogens can protect against the damaging effects of ROS and overcome the toxicity of photooxidative stress generated by aPDT to some degree [ 12 – 14 ]. In addition, the type of PS is the decisive factor of aPDT efficiency [ 15 , 16 ].

With the widespread application of broad-spectrum antimicrobial agents and immunosuppression, fungal infection has become a major global health threat [ 1 ]. The related challenge of the emergence of fungicide resistance is also an increasingly serious problem worldwide [ 2 ]. Compared with various antibiotics for bacterial treatment, azoles, polyenes (cell membrane-targeting [ 2 ]) and echinocandins (cell wall-targeting [ 3 ]) are the only three kinds of antifungal drugs clinically available. The long-term use of antifungal drugs can easily cause the emergence of new and multidrug-resistant strains. Candida auris, first isolated from the ear discharge of a female Japanese patient in 2009, is a newly emerged member of the Candida family [ 4 ]. As a new opportunistic pathogen, C. auris can cause both superficial and life-threatening infections in immunocompromised hosts, especially nosocomial infections. In the past decade, infections caused by C. auris have become a global threat due to their rapid emergence worldwide and multidrug resistance properties [ 5 ]. Based on the above reasons, C. auris is also called “Super fungus”, and it has been reported that the proportion of fluconazole-resistant strains even exceeds 90% [ 6 ]. Therefore, it is imperative to identify alternative antifungal agents or strategies that are effective against C. auris infections.

Results and discussion

In vitro antifungal activity To observe the antifungal activity of PLA-HA, C. auris (BJCA001, the first isolated strain in China) was selected [41]. The BJCA001 strain was treated with PLA and PLA-HA with or without light irradiation. Agar plate images and quantitative histograms of the colonies are shown in Fig 3A and 3C. Our results show that the PLA and PLA-HA treatments without illumination did not cause any significant toxicity in C. auris. However, when illumination was employed for 30 min, the survival rate of the PLA-HA treated strain decreased dramatically, with a survival rate lower than 0.1%. Notably, the antifungal activity of PLA-HA is still very effective after reuse 4 times. Compared with the recently reported antimicrobial materials on HA and its derivatives which fixed on powdery nanocarriers [19,42]. Our material exhibits excellent repeatable antifungal activity. PPT PowerPoint slide

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TIFF original image Download: Fig 3. In vitro antifungal activities and biosafety of PLA-HA and PLA. (A) Spread plate results of C. auris growth under different conditions. The images from left to right represent PLA and PLA-HA were repeatedly used for four times. (B) SEM and TEM images of C. auris under different conditions. CM indicates the cytoplasmic membrane, CW represents the cell wall, N indicates the nucleus and L refers to lipid inclusion. (C) Corresponding survival rate results of C. auris with PLA and PLA-HA treatment (n = 3). (D) Hemolysis assay. The inset shows the image directly observed after adding PLA and PLA-HA for 2 h. Distilled water was used as a positive control, and PBS was used as a negative control. (E) Cell toxicity evaluation of PLA and PLA-HA on mouse fibroblast L929 cells for 24 h, 48 h and 72 h. Data are presented as the mean ± s.d. https://doi.org/10.1371/journal.ppat.1010534.g003 Moreover, the Live/Dead double staining fluorescent dye Syto9/PI was used to further evaluate the antifungal activity of PLA-HA (S2 Fig). C. auris was stained with Syto9 and PI. The Syto9 with green fluorescence could stain all fungal cells, whereas PI only stained cells with damaged walls. When Syto9 and PI dyes stain a cell at the same time, the fluorescence intensity of Syto9 becomes weaker [43]. As shown in S2 Fig, PLA and PLA-HA treated C. auris without illumination, and PLA treated C. auris with illumination were stained with bright green fluorescence, indicating that the fungi had an integrated wall and membrane structure. However, the PLA-HA treated strain exhibited red fluorescence, which suggested that the cell wall had been damaged. SEM and TEM were used to observe how PLA-HA kills C. auris (Fig 3B). SEM of C. auris shows that the surfaces of the healthy blastospores are smooth with elliptic in shape, and the boundaries between blastospores are clearly visible. However, PLA-HA with illumination could destroy the cell wall and membrane of the fungus, resulting in a shrinkage of cells, and conglutination occurred in the adjacent spores. Additionally, fragments of blastospores are clearly visible in SEM images. From the TEM photographs, the healthy blastospores of C. auris in the three control groups showed regular shapes, with a clear outlined cell walls and cytoplasmic membranes. In the cytoplasm, organelles such as nucleus and lipid inclusions were regularly distributed. Meanwhile, the shape of cells of PLA-HA treated C. auris is irregular and severely damaged as seen by TEM, the permeability of cell was significantly increased, contents of destroyed fungal flowed out of the cell. Furthermore, some organelles disappeared, some cytoplasms were disintegrated into empty bubbles, the nucleus was fragmented, and some cell walls and membranes were broken and discontinuous. In addition, the cytocompatibility of the wound dressings plays a pivotal role on wound healing. We used mouse fibroblast (L929) cells to examine the toxicity of PLA and PLA-HA with and without illumination. The results of the CCK-8 assay indicated that neither PLA nor PLA-HA caused obvious cytotoxicity toward L929 cells after 24, 48 and 72 h of incubation without illumination (Fig 3E). However, compared with the control group, the cell viability of L929 cells in the PLA-HA group, declined 59.32%, 22.38% and 27.51% after 24, 48 and 72 h under 470 nm laser irradiation, which means PLA-HA has a slight phototoxicity to L929 cells (Fig 3E). Hemolysis ratio (HR) is an important factor for the hemo-compatibility of biomaterials. There is no obvious hemolysis of wound dressing when the dressing directly contacted with blood in vivo [44]. Therefore, a hemolysis experiment was used to investigate the effect of PLA and PLA-HA on the rupture and lysis of red blood cells (RBCs). As shown in Fig 3D, HR of PLA is 1.51% and the other is 0.89%, indicating that neither PLA nor PLA-HA caused obvious hemolysis and had an excellent blood biocompatibility.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010534

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