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Background: Sporotrichosis is a zoonotic disease caused by the dimorphic fungus Sporothrix spp., leading to skin lesions that can, in some cases, progress and result in the death of infected individuals. Candida albicans is another fungus involved in several skin, oral, and vaginal mucosal infections. Fungal diseases are concerning due to increasing incidence and the limited variety of antifungal classes available for treatment. Furthermore, antifungal medications can cause various side effects, exacerbated by their prolonged use during infection treatment. There is a need to explore alternatives to conventional drugs that are effective, fast, and safe in combating sporotrichosis. This study aimed to achieve in vitro elimination of the fungi Sporothrix brasiliensis and Sporothrix schenckii through Photodynamic Inactivation (PDI), using curcumin as a photosensitizer and in combination with antifungal agents used in the treatment of sporotrichosis., Methodology: Yeasts of Candida albicans, Sporothrix brasiliensis, and Sporothrix schenckii were subjected to Photodynamic Inactivation (PDI) using light at a wavelength of 450 ± 10 nm, irradiance of 35 mW/cm2, delivering a fluence of 31.5 J/cm2, with curcumin as the photosensitizer at doses ranging from 0.75 to 150 μg/mL. After determining the Minimum Inhibitory Concentration (MIC) values of the antifungal drugs itraconazole, ketoconazole, and potassium iodide, sub-MIC doses of these antifungals were combined with sub-MIC doses of curcumin in a new PDI session., Conclusion: Photodynamic inactivation is a promising technique in the treatment of sporotrichosis, as well as its combination with antifungals. The combination of curcumin in concentrations ranging from 0.75 g/mL a 7.5 g/mL with sub-MIC concentrations of itraconazole, ketoconazole, and potassium iodide was able to completely inactivate the fungi C. albicans, S. brasiliensis and S. schenckii, indicating that PDI may increase the effectiveness of antifungals. However, further studies are needed to establish protocols for future clinical applications., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rocha 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.)

Objective: The goal of this study is to investigate the efficacy, safety, and mechanism of ABL for inactivating Candida albicans (C. albicans), and to determine the best wavelength for treating candida infected disease, by experimental measurements and dynamic modeling., Methods: The changes in reactive oxygen species (ROS) in C. albicans and human host cells under the irradiation of 385, 405, and 415 nm wavelengths light with irradiance of 50 mW/cm 2 were measured. Moreover, a kernel-based nonlinear dynamic model, i.e., nonlinear autoregressive with exogenous inputs (NARX), was developed and applied to predict the concentration of light-induced ROS, whose kernels were selected by a newly developed algorithm based on particle swarm optimization (PSO)., Results: The ROS concentration was increased respectively about 10-12 times in C. albicans and about 3-6 times in human epithelial cells by the ABL treatment with the same fluence of 90 J/cm 2 . The NARX models were respectively fitted to the data from the experiments on both types of cells. Besides, four different kernel functions, including Gaussian, Laplace, linear and polynomial kernels, were compared in their fitting accuracies. The errors with the Laplace kernel turned out to be only 0.2704 and 0.0593, as respectively fitted to the experimental data of the C. albicans and human host cells., Conclusion: The results demonstrated the effectiveness of the NARX modeling approach, and revealed that the 415 nm light was more effective as an anti-fungal treatment with less damage to the host cells than the 405 or 385 nm light., Significance: The kernel-based NARX model identification algorithm offers opportunities for determining the effective and safe light dosages in treating various fungal infection diseases.

In the pathogenic yeast Candida albicans , the DNA damage response contributes to pathogenicity by regulating cell morphology transitions and maintaining survival in response to DNA damage induced by reactive oxygen species (ROS) in host cells. However, the function of nucleotide excision repair (NER) in C. albicans has not been extensively investigated. To better understand the DNA damage response and its role in virulence, we studied the function of the Rad23 nucleotide excision repair protein in detail. The RAD23 deletion strain and overexpression strain both exhibit UV sensitivity, confirming the critical role of RAD23 in the nucleotide excision repair pathway. Genetic interaction assays revealed that the role of RAD23 in the UV response relies on RAD4 but is independent of RAD53 , MMS22 , and RAD18 RAD4 and RAD23 have similar roles in regulating cell morphogenesis and biofilm formation; however, only RAD23 , but not RAD4 , plays a negative role in virulence regulation in a mouse model. We found that the RAD23 deletion strain showed decreased survival in a Candida -macrophage interaction assay. Transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) data further revealed that RAD23 , but not RAD4 , regulates the transcription of a virulence factor, SUN41 , suggesting a unique role of RAD23 in virulence regulation. Taking these observations together, our work reveals that the RAD23 -related nucleotide excision pathway plays a critical role in the UV response but may not play a direct role in virulence. The virulence-related role of RAD23 may rely on the regulation of several virulence factors, which may give us further understanding about the linkage between DNA damage repair and virulence regulation in C. albicans IMPORTANCE Candida albicans remains a significant threat to the lives of immunocompromised people. An understanding of the virulence and infection ability of C. albicans cells in the mammalian host may help with clinical treatment and drug discovery. The DNA damage response pathway is closely related to morphology regulation and virulence, as well as the ability to survive in host cells. In this study, we checked the role of the nucleotide excision repair (NER) pathway, the key repair system that functions to remove a large variety of DNA lesions such as those caused by UV light, but whose function has not been well studied in C. albicans We found that Rad23, but not Rad4, plays a role in virulence that appears independent of the function of the NER pathway. Our research revealed that the NER pathway represented by Rad4/Rad23 may not play a direct role in virulence but that Rad23 may play a unique role in regulating the transcription of virulence genes that may contribute to the virulence of C. albicans ., (Copyright © 2020 Feng et al.)

During symmetry breaking, the highly conserved Rho GTPase Cdc42 becomes stabilized at a defined site via an amplification process. However, little is known about how a new polarity site is established in an already asymmetric cell-a critical process in a changing environment. The human fungal pathogen Candida albicans switches from budding to filamentous growth in response to external cues, a transition controlled by Cdc42. Here, we have used optogenetic manipulation of cell polarity to reset growth in asymmetric filamentous C. albicans cells. We show that increasing the level of active Cdc42 on the plasma membrane results in disruption of the exocyst subunit Sec3 localization and a striking de novo clustering of secretory vesicles. This new cluster of secretory vesicles is highly dynamic, moving by hops and jumps, until a new growth site is established. Our results reveal that secretory vesicle clustering can occur in the absence of directional growth., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Candida albicans is responsible for the majority of nosocomial infections affecting immunocompromised patients. Systemic antifungals may promote microbial resistance, which has led to the search for alternative treatments, such as photothermal therapy (PTT). PTT assumes that the interaction of electromagnetic radiation with a photothermal agent generates heat that can lead to the destruction of tumor cells and the death of microorganisms. Carbon nanotubes (CNTs) have the potential for applications in biomedical systems, including acting as controlled deliverers of drugs, biosensors and scaffolds for tissue engineering and regenerative medicine. Furthermore, the absorption of radiation by CNTs in the infrared region induces an increase in temperature, which makes CNTs candidates for photothermal agents. In this work, the photothermal inactivation of C. albicans was evaluated by multiple wall CNTs associated with laser radiation in the near-infrared region. The mechanisms that are involved in inactivation were evaluated through cell susceptibility studies and an analysis of microscopic images that are associated with mathematical models and fractal concepts. The results indicate that direct contact between the cells and CNTs without irradiation does not lead to cell death, whereas the laser-mediated process is effective in inactivation. The application of the laws of scale and fractal concepts indicate that in the control groups, there are two distinct regimes that are delimited by the mean diameter of the microorganisms, as described by the Eden model and by the quasi-Euclidean surface. For the irradiated groups, the surfaces present only one regime described by Kardar-Parisi-Zhang, KPZ. The analysis of the fractality of the system by mathematical models can help in the identification of new strategies for the inactivation of microorganisms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Candida albicans is a predominant cause of fungal infections in mucosal tissues as well as life-threatening bloodstream infections in immunocompromised patients. Within the human body, C. albicans is mostly embedded in biofilms, which provides increased resistance to antifungal drugs. The glyoxalase Glx3 is an abundant proteomic component of the biofilm extracellular matrix. Here, we document phenotypic studies of a glx3Δ null mutant concerning its role in biofilm formation, filamentation, antifungal drug resistance, cell wall integrity and virulence. First, consistent with its function as glyoxalase, the glx3 null mutant showed impaired growth on media containing glycerol as the carbon source and in the presence of low concentrations of hydrogen peroxide. Importantly, the glx3Δ mutant showed decreased fitness at 37°C and formed less biofilm as compared to wild type and a reintegrant strain. At the permissive temperature of 28°C, the glx3Δ mutant showed impaired filamentation as well as increased sensitivity to Calcofluor white, Congo red, sodium dodecyl sulfate and zymolyase, indicating subtle alterations in wall architecture even though gross quantitative compositional changes were not detected. Interestingly, and consistent with its impaired filamentation, biofilm formation and growth at 37°C, the glx3Δ mutant is avirulent. Our results underline the role of Glx3 in fungal pathogenesis and the involvement of the fungal wall in this process.

We have analysed the role of homologous recombination (HR) genes on the repair of double-strand breaks induced by γ-ionising radiation in Candida albicans. Depletion of either CaRad51 or CaRad52 caused a dramatic drop in the number of survivors compared with wild type, whereas depletion of CaRad59 caused a moderate decrease. Besides, compared with Saccharomyces cerevisiae, C. albicans relies more on HR proteins for repair of ionising radiation lesions. Pulse-field electrokaryotypes of survivors identified genetic alterations mainly in the form of aneuploidy in HR mutants and chromosome length polymorphism and ectopic translocation in wild type. Increasing irradiation (4 to 80 krad) of both cycling and nocodazole-treated (G2/M-arrested) cells revealed a gradual loss of chromosomes, larger chromosomes being more affected than smaller ones. For cycling wild-type cells, shattered chromosomes were progressively restored following incubation in yeast extract, peptone, dextrose medium, but not in phosphate-buffered saline, and this accompanied by a moderate increase in colony-forming units, suggesting that repair was followed by replication of survivors. Irradiated G2/M arrested cells from wild type but not from HR mutants partially restored the chromosome ladder following incubation (4-8 hr) in yeast peptone dextrose-nocodazole. However, HR mutants showed a chromosome shattering pattern similar to wild type, an indication that lesions other than double-strand breaks, likely single-strand break, are responsible for their drastically reduced survivability., (© 2018 John Wiley & Sons Ltd.)

Antimicrobial photodynamic therapy (aPDT) has been proposed as an alternative method for oral candidiasis (OC), while nanocarriers have been used to improve the water solubility of curcumin (CUR). The aim of this study is to encapsulate CUR in polymeric nanoparticles (NPs) and to evaluate its photodynamic effects on a murine model of OC. Anionic and cationic CUR-NP is synthesized using poly-lactic acid and dextran sulfate and then characterized. Female mice are immunosuppressed and inoculated with Candida albicans (Ca) to induce OC. aPDT is performed by applying CUR-NP or free CUR on the dorsum of the tongue, followed by blue light irradiation for five consecutive days. Nystatin is used as positive control. Afterward, Ca are recovered and cultivated. Animals are euthanized for histological, immunohistochemical, and DNA damage evaluation. Encapsulation in NP improves the water solubility of CUR. Nystatin shows the highest reduction of Ca, followed by aPDT mediated by free CUR, which results in immunolabelling of cytokeratins closer to those observed for healthy animals. Anionic CUR-NP does not show antifungal effect, and cationic CUR-NP reduces Ca even in the absence of light. DNA damage is associated with Ca infection. Consecutive aPDT application is a safe treatment for OC.

Photodynamic inactivation (PDI) has been shown to be a potential treatment modality against Candida infection. However, limited light penetration might leave some cells alive and undergoing regrowth. In this study, we explored the possibility of combining PDI and antifungal agents to enhance the therapeutic efficacy of Candida albicans and drug-resistant clinical isolates. We found that planktonic cells that had survived toluidine blue O (TBO)-mediated PDI were significantly susceptible to fluconazole within the first 2 h post PDI. Following PDI, the killing efficacy of antifungal agents relates to the PDI dose in wild-type and drug-resistant clinical isolates. However, only a 3-log reduction was found in the biofilm cells, suggesting limited therapeutic efficacy under the combined treatment of PDI and azole antifungal drugs. Using confocal microscopic analysis, we showed that TBO-mediated PDI could partially remove the extracellular polymeric substance (EPS) of biofilm. Finally, we showed that a combination of PDI with caspofungin could result in the complete killing of biofilms compared to those treated with caspofungin or PDI alone. These results clearly indicate that the combination of PDI and antifungal agents could be a promising treatment against C. albicans infections., Competing Interests: The authors declare no conflict of interest.

Candida albicans is the most commonly encountered human fungal pathogen, and it is traditionally treated with antimicrobial chemical agents. The antimicrobial effect of these agents is largely weakened by drug resistance and biofilm-associated virulence. Enhancement of the antimicrobial activity of existing agents is needed for effective candidiasis treatment. Our aim was to develop a therapy that combined biofilm disruption with existing antimicrobial agents. Photodynamic therapy (PDT) utilizing curcumin and blue light was tested as an independent therapy and in combination with fluconazole treatment. Viability assays and morphology analysis were used to assess the effectiveness of C. albicans treatment. Results showed that fluconazole treatment decreased the viability of planktonic C. albicans , but the decrease was not as pronounced in adherent C. albicans because its biofilm form was markedly more resistant to the antimicrobiotic. PDT effectively eradicated C. albicans biofilms, and when combined with fluconazole, PDT significantly inhibited C. albicans to a greater extent. This study suggests that the addition of PDT to fluconazole to treat C. albicans infection enhances its effectiveness and can potentially be used clinically., Competing Interests: The authors declare no conflict of interest.

Background: Ultraviolet irradiation is effectively used as a disinfection method for inactivating microorganisms. Methods: We investigated the bactericidal effects by irradiation with a deep-ultraviolet light-emitting diode (DUV-LED) on the causative microorganisms of catheter related blood stream infection contaminating the solution for intravenous infusion. For irradiation, prototype modules for water disinfection with a DUV-LED were used. Experiments were conducted on five kinds of microorganisms. We examined the dependence of bactericidal action on eleven solutions. Administration sets were carried out three types. Results: When the administration set JY-PB343L containing the infusion tube made of polybutadiene was used, the bactericidal action of the DUV-LED against all tested microorganisms in the physiological saline solutions was considered to be effective. We confirmed that the number of viable bacteria decreased in 5% glucose solution and electrolyte infusions with DUV-LED irradiation. Conclusions: These results indicate that the DUV-LED irradiation has bactericidal effects in glucose infusion and electrolyte infusions by irradiating via a plasticizer-free polybutadiene administration set. We consider DUV-LED irradiation to be clinically applicable., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The impact of substituents on the photochemical and biological properties of tetraphenylporphyrin-based photosensitizers for photodynamic therapy of cancer (PDT) as well as photodynamic inactivation of microorganisms (PDI) was examined. Spectroscopic and physicochemical properties were related with therapeutic efficacy in PDT of cancer and PDI of microbial cells in vitro. Less polar halogenated, sulfonamide porphyrins were most readily taken up by cells compared to hydrophilic and anionic porphyrins. The uptake and PDT of a hydrophilic porphyrin was significantly enhanced with incorporation in polymeric micelles (Pluronic L121). Photodynamic inactivation studies were performed against Gram-positive (S. aureus, E. faecalis), Gram-negative bacteria (E. coli, P. aeruginosa, S. marcescens) and fungal yeast (C. albicans). We observed a 6 logs reduction of S. aureus after irradiation (10 J/cm2) in the presence of 20 μM of hydrophilic porphyrin, but this was not improved with incorporation in Pluronic L121. A 2-3 logs reduction was obtained for E. coli using similar doses, and a decrease of 3-4 logs was achieved for C. albicans. Rational substitution of tetraphenylporphyrins improves their photodynamic properties and informs on strategies to obtain photosensitizers for efficient PDT and PDI. However, the design of the photosensitizers must be accompanied by the development of tailored drug formulations.

Genomic instability in Candida albicans is believed to play a crucial role in fungal pathogenesis. DNA polymerases contribute significantly to stability of any genome. Although Candida Genome database predicts presence of S. cerevisiae DNA polymerase orthologs; functional and structural characterizations of Candida DNA polymerases are still unexplored. DNA polymerase eta (Polη) is unique as it promotes efficient bypass of cyclobutane pyrimidine dimers. Interestingly, C. albicans is heterozygous in carrying two Polη genes and the nucleotide substitutions were found only in the ORFs. As allelic differences often result in functional differences of the encoded proteins, comparative analyses of structural models and molecular dynamic simulations were performed to characterize these orthologs of DNA Polη. Overall structures of both the ORFs remain conserved except subtle differences in the palm and PAD domains. The complementation analysis showed that both the ORFs equally suppressed UV sensitivity of yeast rad30 deletion strain. Our study has predicted two novel molecular interactions, a highly conserved molecular tetrad of salt bridges and a series of π-π interactions spanning from thumb to PAD. This study suggests these ORFs as the homologues of yeast Polη, and due to its heterogeneity in C. albicans they may play a significant role in pathogenicity.

The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state., Importance: Epigenetic transitions are an important mechanism by which microbes adapt to external stimuli. For Candida albicans, such transitions are crucial for adaptation to complex, fluctuating environments, and therefore contribute to its success as a human pathogen. The white-opaque switch modulates multiple C. albicans attributes, from sexual competency to niche specificity. Here, we demonstrate that metabolic circuits are extensively rewired between white and opaque states, so that the two cell types exhibit optimal fitness under different nutritional conditions and at different temperatures. We thereby establish that epigenetic events can profoundly alter the metabolism of fungal cells. We also demonstrate that epigenetic switching regulates filamentation and biofilm formation, two phenotypes closely associated with pathogenesis. These experiments reveal that white cells, considered the most clinically relevant form of C. albicans, are a "general-purpose" state suited to many environments, whereas opaque cells appear to represent a more metabolically specialized form of the species., (Copyright © 2016 Ene et al.)

Fungal infections are a common cause of morbidity, mortality and cost in critical care populations. The increasing emergence of antimicrobial resistance necessitates the development of new therapeutic approaches for fungal infections. In the present study, we investigated the effectiveness of an innovative approach, antimicrobial blue light (aBL), for inactivation of Candida albicans in vitro and in infected mouse burns. A bioluminescent strain of C. albicans was used. The susceptibilities to aBL (415 nm) were compared between C. albicans and human keratinocytes. The potential development of aBL resistance by C. albicans was investigated via 10 serial passages of C. albicans on aBL exposure. For the animal study, a mouse model of thermal burn infected with the bioluminescent C. albicans strain was used. aBL was delivered to mouse burns approximately 12 h after fungal inoculation. Bioluminescence imaging was performed to monitor in real time the extent of infection in mice. The results obtained from the studies demonstrated that C. albicans was approximately 42-fold more susceptible to aBL than human keratinocytes. Serial passaging of C. albicans on aBL exposure implied a tendency of reduced aBL susceptibility of C. albicans with increasing numbers of passages; however, no statistically significant difference was observed in the post-aBL survival rate of C. albicans between the first and the last passage (P>0.05). A single exposure of 432 J/cm(2) aBL reduced the fungal burden in infected mouse burns by 1.75-log10 (P=0.015). Taken together, our findings suggest aBL is a potential therapeutic for C. albicans infections.

Photoactivated disinfection has a strong local antimicrobial effect. In the field of dentistry it is an emerging adjunct to mechanical debridement during endodontic and periodontal treatment. In the present study, we investigate the effect of photoactivated disinfection using riboflavin as a photosensitizer and blue LED light for activation, and compare it to photoactivated disinfection with the widely used combination of toluidine blue O and red light. Riboflavin is highly biocompatible and can be activated with LED lamps at hand in the dental office. To date, no reports are available on the antimicrobial effect of photoactivated disinfection using riboflavin/blue light on oral microorganisms. Planktonic cultures of eight organisms frequently isolated from periodontal and/or endodontic lesions (Aggregatibacter actinomycetemcomitans, Candida albicans, Enterococcus faecalis, Escherischia coli, Lactobacillus paracasei, Porphyromonas gingivalis, Prevotella intermedia and Propionibacterium acnes) were subjected to photoactivated disinfection with riboflavin/blue light and toluidine blue O/red light, and survival rates were determined by CFU counts. Within the limited irradiation time of one minute, photoactivated disinfection with riboflavin/blue light only resulted in minor reductions in CFU counts, whereas full kills were achieved for all organisms when using toluidine blue O/red light. The black pigmented anaerobes P. gingivalis and P. intermedia were eradicated completely by riboflavin/blue light, but also by blue light treatment alone, suggesting that endogenous chromophores acted as photosensitizers in these bacteria. On the basis of our results, riboflavin cannot be recommended as a photosensitizer used for photoactivated disinfection of periodontal or endodontic infections.

The increasing applicability of antifungal treatments, the limited range of available drug classes and the emergence of drug resistance in Candida spp. suggest the need for new treatment options. To explore the applicability of C. albicans photoinactivation, we examined nine structurally different imidazoacridinone derivatives as photosensitizing agents. The most effective derivatives showed a >10(4)-fold reduction of viable cell numbers. The fungicidal action of the three most active compounds was compared at different radiant powers (3.5 to 63 mW/cm2), and this analysis indicated that 7 mW/cm2 was the most efficient. The intracellular accumulation of these compounds in fungal cells correlated with the fungicidal activity of all 9 derivatives. The lack of effect of verapamil, an inhibitor targeting Candida ABC efflux pumps, suggests that these imidazoacridinones are not substrates for ABC transporters. Thus, unlike azoles, a major class of antifungals used against Candida, ABC transporter-mediated resistance is unlikely. Electron paramagnetic resonance (EPR)-spin trapping data suggested that the fungicidal light-induced action of these derivatives might depend on the production of superoxide anion. The highest generation rate of superoxide anion was observed for 1330H, 1610H, and 1611. Singlet oxygen production was also detected upon the irradiation of imidazoacridinone derivatives with UV laser light, with a low to moderate yield, depending on the type of compound. Thus, imidazoacridinone derivatives examined in the present study might act via mixed type I/type II photodynamic mechanism. The presented data indicate lack of direct correlation between the structures of studied imidazoacridinones, cell killing ability, and ROS production. However, we showed for the first time that for imidazoacridinones not only intracellular accumulation is necessary prerequisite of lethal photosensitization of C. albicans, but also localization within particular cellular structures. Our findings present IA derivatives as efficient antifungal photosensitizers with a potential to be used in local treatment of Candida infection.

Photodynamic therapy (PDT) is a promising method for localized and specific inactivation of fungi and bacteria. A nontoxic light-sensitive compound is taken up by cells, which are then exposed selectively to light, which activates toxicity of the compound. We investigated the potential of sublethal PDT using light-sensitive curcumin (CUR) in combination with blue (455 nm) light to promote reactive oxygen species (ROS) formation in the form of singlet oxygen and DNA damage of Candida albicans. Surprisingly, CUR-mediated PDT but also light alone caused significantly longer comet tails, an indication of DNA damage of C. albicans when compared with the negative control. The intracellular ROS production was also significantly higher for the group treated only with light. However, PDT compared to blue light alone significantly slowed DNA repair. Comet tails decreased during 30 min visualized as a 90% reduction in length in the absence of light for cells treated with light alone, while comet tails of cells treated with PDT only diminished in size about 45%. These results indicate that complex mechanisms may result in PDT in a way that should be considered when choosing the photosensitive compound and other aspects of the treatment design., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Superficial infections of the skin and mucous membranes caused by dermatophyte fungi are amongst the most common and challenging infections to treat. Previously we demonstrated the phototoxic effects of photodynamic therapy (PDT) towards Trichophyton rubrum, using a green laser to photoactivate Rose Bengal (RB). The aim of this study was to evaluate whether we could; (1) achieve a similar effect using an inexpensive light-emitting diode (LED) to photoactivate RB and (2) to evaluate whether our PDT regime could be combined with standard antifungal drug therapy and increase its effectiveness., Methods: We designed and built our own inexpensive green (530 nm) LED source and tested its efficacy as part our RB-PDT regime in vitro against T. rubrum. We also examined the potential benefits of incorporating PDT as part of combination therapy and whether the order in which this was done had an impact. First we subjected spore suspensions to sub-inhibitory concentrations of a number of antifungal agents (CLT, MCZ and TRB) for 72 hours followed by RB-PDT. Secondly we subjected spore suspensions to sub-inhibitory PDT followed by drug treatment and evaluated if there were any changes to the minimum inhibitory concentrations (MICs) of the drugs tested., Results: The optimal conditions for photoinactivation of T. rubrum using RB-PDT alone were 140 μM of RB and 24 J/cm2 of LED (equating to a 30-minute exposure). These parameters also caused a 100% reduction in the viability of the pathogenic yeast Candida albicans and the model fungus Saccharomyces cerevisiae. By combining our RB-PDT regime as an adjunct to antifungal drugs we were able to dramatically reduce the exposure times. Treatment of spore suspensions using a sub-inhibitory dose of clotrimazole (CLT) followed by RB-PDT, this order was critical, significantly reduced the exposure times required to achieve 100% inhibition of T. rubrum to 15 minutes as compared to RB-PDT alone., Conclusions: The combination of antifungal drug and RB-PDT represents an attractive alternative to the current antifungal therapies used to treat superficial fungal diseases. Our approach has the potential to reduce treatment times and drug dosages which can also reduce drug toxicity and improve patient compliance.

The pathogenic fungi Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans are an increasing cause of human mortality, especially in immunocompromised populations. During colonization and adaptation to various host environments, these fungi undergo morphogenetic alterations that allow for survival within the host. One key environmental cue driving morphological changes is external temperature. The Hsp90 chaperone protein provides one mechanism to link temperature with the signalling cascades that regulate morphogenesis, fungal development and virulence. Candida albicans is a model system for understanding the connections between morphogenesis and Hsp90. Due to the high degree of conservation in Hsp90, many of the connections in C. albicans may be extrapolated to other fungal pathogens or parasites. Examining the role of Hsp90 during development and morphogenesis in these three major fungal pathogens may provide insight into key aspects of adaptation to the host, leading to additional avenues for therapy., (© 2014 John Wiley & Sons Ltd.)

Aim: To compare the disinfection effect of Er,Cr:YSGG laser using radial firing tips with NaOCI in root canals infected with C. albicans and to evaluate the irradiation effect on the dentinal surfaces., Material and Methods: In total seventy-six mandibular premolar teeth were used. In order to standardize the incubation and sterilization procedure, eight teeth were used. Sixty-eight of the root canals were incubated with C. albicans suspension for 72 hours. The specimens were divided into 5 experimental groups. Two groups were constituted as Group 1 was irradiated with 1.5 W laser (n = 8) and group 2, which was irradiated with 2 W laser (n = 8). Two more groups were formed as Group 3 (2 W laser (n = 25) and Group 4 NaOCI (5%) (n = 25). Group 5 (n = 2) did not receive any treatment. Mann-Whitney U and Kruskal-Wallis H tests were used to compare the different laser output powers. Wilcoxon Signed Ranks Test was used in order to compare the Candida cfu/ml levels according to treatment protocols (P < 0.05)., Results: Both 1.5 W and 2 W laser resulted in a major reduction of C. albicans without a significant difference. The comparison of the dentin surfaces irradiated with Er,Cr:YSGG laser at two power settings resulted in similar morphological changes. However, NaOCI was found to be more effective in reduction of C. albicans than 2 W laser application., Conclusion: According to the results of the present study, the Er,Cr:YSGG laser with radial firing tips presented less antifungal effects on C. albicans in root canals of infected teeth than NaOCl solution.

Background: Candida spp. are recognized as a primary agent of severe fungal infection in immunocompromised patients, and are the fourth most common cause of bloodstream infections. Our study explores treatment with photodynamic therapy (PDT) as an innovative antimicrobial technology that employs a nontoxic dye, termed a photosensitizer (PS), followed by irradiation with harmless visible light. After photoactivation, the PS produces either singlet oxygen or other reactive oxygen species (ROS) that primarily react with the pathogen cell wall, promoting permeabilization of the membrane and cell death. The emergence of antifungal-resistant Candida strains has motivated the study of antimicrobial PDT (aPDT) as an alternative treatment of these infections. We employed the invertebrate wax moth Galleria mellonella as an in vivo model to study the effects of aPDT against C. albicans infection. The effects of aPDT combined with conventional antifungal drugs were also evaluated in G. mellonella., Results: We verified that methylene blue-mediated aPDT prolonged the survival of C. albicans infected G. mellonella larvae. The fungal burden of G. mellonella hemolymph was reduced after aPDT in infected larvae. A fluconazole-resistant C. albicans strain was used to test the combination of aPDT and fluconazole. Administration of fluconazole either before or after exposing the larvae to aPDT significantly prolonged the survival of the larvae compared to either treatment alone., Conclusions: G. mellonella is a useful in vivo model to evaluate aPDT as a treatment regimen for Candida infections. The data suggests that combined aPDT and antifungal therapy could be an alternative approach to antifungal-resistant Candida strains.

The objective of this study was to evaluate whether Candida albicans exhibits altered pathogenicity characteristics following sublethal antimicrobial photodynamic inactivation (APDI) and if such alterations are maintained in the daughter cells. C. albicans was exposed to sublethal APDI by using methylene blue (MB) as a photosensitizer (0.05 mM) combined with a GaAlAs diode laser (λ 660 nm, 75 mW/cm(2), 9 to 27 J/cm(2)). In vitro, we evaluated APDI effects on C. albicans growth, germ tube formation, sensitivity to oxidative and osmotic stress, cell wall integrity, and fluconazole susceptibility. In vivo, we evaluated C. albicans pathogenicity with a mouse model of systemic infection. Animal survival was evaluated daily. Sublethal MB-mediated APDI reduced the growth rate and the ability of C. albicans to form germ tubes compared to untreated cells (P < 0.05). Survival of mice systemically infected with C. albicans pretreated with APDI was significantly increased compared to mice infected with untreated yeast (P < 0.05). APDI increased C. albicans sensitivity to sodium dodecyl sulfate, caffeine, and hydrogen peroxide. The MIC for fluconazole for C. albicans was also reduced following sublethal MB-mediated APDI. However, none of those pathogenic parameters was altered in daughter cells of C. albicans submitted to APDI. These data suggest that APDI may inhibit virulence factors and reduce in vivo pathogenicity of C. albicans. The absence of alterations in daughter cells indicates that APDI effects are transitory. The MIC reduction for fluconazole following APDI suggests that this antifungal could be combined with APDI to treat C. albicans infections.

Temperature change is a ubiquitous environmental signal, which exerts powerful control over the development and virulence of microbial pathogens. For Candida albicans, the leading fungal pathogen of humans, temperature influences mating, phenotypic switching, resistance to antifungal drugs and the morphogenetic transition from yeast to filamentous growth. C. albicans morphogenesis is profoundly influenced by temperature, and most filament-inducing cues depend on a concurrent increase in temperature to 37°C before morphogenesis can occur, although the molecular mechanisms underpinning this temperature-dependent developmental transition remain largely unknown. We established that the thermally responsive molecular chaperone Hsp90 orchestrates temperature-dependent morphogenesis, via previously uncharacterized cellular circuitry, comprised of the cyclin-dependent kinase Pho85, the cyclin Pcl1 and the transcriptional regulator Hms1. Here we elaborate on Hsp90's pleiotropic effects on temperature-dependent morphogenetic circuitry, and highlight how changes in protein form and function in response to stress complements the diverse repertoire of mechanisms of microbial temperature sensing.

Superficial mycoses caused by dermatophyte fungi are among the most common infections worldwide, yet treatment is restricted by limited effective drugs available, drug toxicity, and emergence of drug resistance. The stilbene fluorescent brightener calcofluor white (CFW) inhibits fungi by binding chitin in the cell wall, disrupting cell wall integrity, and thus entails a different mechanism of inhibition than currently available antifungal drugs. To identify novel therapeutic options for the treatment of skin infections, we compared the sensitivity of representative strains of the dermatophyte Trichophyton rubrum and Candida albicans to CFW and a panel of fluorescent brighteners and phytoalexin compounds. We identified the structurally related stilbene fluorescent brighteners 71, 85, 113 and 134 as fungicidal to both T. rubrum and C. albicans to a similar degree as CFW, and the stilbene phytoalexins pinosylvan monomethyl ether and pterostilbene inhibited to a lesser degree, allowing us to develop a structure-activity relationship for fungal inhibition. Given the abilities of CFW to absorb UV(365 nm) and bind specifically to fungal cell walls, we tested whether CFW combined with UV(365 nm) irradiation would be synergistic to fungi and provide a novel photodynamic treatment option. However, while both treatments individually were cytocidal, UV(365 nm) irradiation reduced sensitivity to CFW, which we attribute to CFW photoinactivation. We also tested combination treatments of CFW with other fungal inhibitors and identified synergistic interactions between CFW and some ergosterol biosynthesis inhibitors in C. albicans. Therefore, our studies identify novel fungal inhibitors and drug interactions, offering promise for combination topical treatment regimes for superficial mycoses.

Unlabelled: Genetic diversity is often generated during adaptation to stress, and in eukaryotes some of this diversity is thought to arise via recombination and reassortment of alleles during meiosis. Candida albicans, the most prevalent pathogen of humans, has no known meiotic cycle, and yet it is a heterozygous diploid that undergoes mitotic recombination during somatic growth. It has been shown that clinical isolates as well as strains passaged once through a mammalian host undergo increased levels of recombination. Here, we tested the hypothesis that stress conditions increase rates of mitotic recombination in C. albicans, which is measured as loss of heterozygosity (LOH) at specific loci. We show that LOH rates are elevated during in vitro exposure to oxidative stress, heat stress, and antifungal drugs. In addition, an increase in stress severity correlated well with increased LOH rates. LOH events can arise through local recombination, through homozygosis of longer tracts of chromosome arms, or by whole-chromosome homozygosis. Chromosome arm homozygosis was most prevalent in cultures grown under conventional lab conditions. Importantly, exposure to different stress conditions affected the levels of different types of LOH events, with oxidative stress causing increased recombination, while fluconazole and high temperature caused increases in events involving whole chromosomes. Thus, C. albicans generates increased amounts and different types of genetic diversity in response to a range of stress conditions, a process that we term "stress-induced LOH" that arises either by elevating rates of recombination and/or by increasing rates of chromosome missegregation., Importance: Stress-induced mutagenesis fuels the evolution of bacterial pathogens and is mainly driven by genetic changes via mitotic recombination. Little is known about this process in other organisms. Candida albicans, an opportunistic fungal pathogen, causes infections that require adaptation to different host environmental niches. We measured the rates of LOH and the types of LOH events that appeared in the absence and in the presence of physiologically relevant stresses and found that stress causes a significant increase in the rates of LOH and that this increase is proportional to the degree of stress. Furthermore, the types of LOH events that arose differed in a stress-dependent manner, indicating that eukaryotic cells generate increased genetic diversity in response to a range of stress conditions. We propose that this "stress-induced LOH" facilitates the rapid adaptation of C. albicans, which does not undergo meiosis, to changing environments within the host.

The increased resistance of Candida albicans to antibiotic therapy indicates the need for alternative treatments for oral candidiasis. Photodynamic therapy (PDT) has been researched as an alternative tool to inactivate pathogenic microorganisms. It uses a combination of a photosensitizer and a visible light source. This study evaluated the susceptibility of C. albicans to PDT and compared the efficacy of 100 microg/mL methylene blue (MB) and toluidine blue (TB) as photosensitizers. The light source was Indium-Gallium-Aluminum Phosphide (InGaAIP) laser at 53 J/cm2. Suspensions of 108 cells/mL of C. albicans were subject to PDT for 5 minutes in 96-well plates, then decimal dilutions were plated on Sabouraud Dextrose agar After 48h incubation at 37 degreesC, the number of CFU/mL were obtained and submitted to statistical analysis using Kolmogorov-Smirnov, ANOVA (p<0.0001) and Tukey tests. The results showed that MB or laser irradiation alone did not have statistically significant antifungal activity compared to the positive control group (p> 0. 05). Conversely, the number of viable C. albicans cells was reduced significantly after PDT using MB or mainly TB associated to diode laser irradiation. The data proved the efficacy of PDT against C. albicans cells, regardless of the photosensitizer used.

The aim of this study was to evaluate the effect of specific parameters of low-level laser therapy (LLLT) on biofilms formed by Streptococcus mutans, Candida albicans or an association of both species. Single and dual-species biofilms--SSB and DSB--were exposed to laser doses of 5, 10 or 20 J/cm(2) from a near infrared InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm, 0.04 W). After irradiation, the analysis of biobilm viability (MTT assay), biofilm growth (cfu/mL) and cell morphology (SEM) showed that LLLT reduced cell viability as well as the growth of biofilms. The response of S. mutans (SSB) to irradiation was similar for all laser doses and the biofilm growth was dose dependent. However, when associated with C. albicans (DSB), S. mutans was resistant to LLLT. For C. albicans, the association with S. mutans (DSB) caused a significant decrease in biofilm growth in a dose-dependent fashion. The morphology of the microorganisms in the SSB was not altered by LLLT, while the association of microbial species (DSB) promoted a reduction in the formation of C. albicans hyphae. LLLT had an inhibitory effect on the microorganisms, and this capacity can be altered according to the interactions between different microbial species.

Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m(-2)) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log(10) after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log(10) after 6 h) was obtained only in the presence of riboflavin and 250 W m(-2) irradiance.

Photodynamic inactivation is a rapidly developing antimicrobial treatment that employs a nontoxic photoactivatable dye or photosensitizer in combination with harmless visible light to generate reactive oxygen species that are toxic to cells. Tetrapyrroles (e.g., porphyrins, chlorins, bacteriochlorins) are a class of photosensitizers that exhibit promising characteristics to serve as broad-spectrum antimicrobials. In order to bind to and efficiently penetrate into all classes of microbial cells, tetrapyrroles should have structures that contain (i) one or more cationic charge(s) or (ii) a basic group. In this report, we investigate the use of new stable synthetic bacteriochlorins that have a strong absorption band in the range 720 to 740 nm, which is in the near-infrared spectral region. Four bacteriochlorins with 2, 4, or 6 quaternized ammonium groups or 2 basic amine groups were compared for light-mediated killing against a gram-positive bacterium (Staphylococcus aureus), a gram-negative bacterium (Escherichia coli), and a dimorphic fungal yeast (Candida albicans). Selectivity was assessed by determining phototoxicity against human HeLa cancer cells under the same conditions. All four compounds were highly active (6 logs of killing at 1 microM or less) against S. aureus and showed selectivity for bacteria over human cells. Increasing the cationic charge increased activity against E. coli. Only the compound with basic groups was highly active against C. albicans. Supporting photochemical and theoretical characterization studies indicate that (i) the four bacteriochlorins have comparable photophysical features in homogeneous solution and (ii) the anticipated redox characteristics do not correlate with cell-killing ability. These results support the interpretation that the disparate biological activities observed stem from cellular binding and localization effects rather than intrinsic electronic properties. These findings further establish cationic bacteriochlorins as extremely active and selective near-infrared activated antimicrobial photosensitizers, and the results provide fundamental information on structure-activity relationships for antimicrobial photosensitizers.

The eukaryotic translation initiation factor 4E is an essential and highly conserved protein. As a part of the translational machinery, it plays a key role in the recruitment of mRNA via binding to its m(7)GpppN 5' terminal cap structure. The opportunistic human pathogen Candida albicans is the only known eukaryotic organism with the ability to survive defects in mRNA capping, which suggests unique features of its eIF4E protein. Here, we provide the first experimental evidence of the function of the C. albicans putative gene orf19.7626 as an eIF4E protein. We also show that Ca4E(Leu116) and Ca4E(Ser116) protein variants, both of which occur naturally in C. albicans due to the ambiguous decoding of the CUG(116) codon, display different sensitivities to elevated temperature. Our results clearly point to the importance of the S4-H4 loop for the function of the Ca4E translation initiation factor, and suggest the possible regulatory role of the codon-reading ambiguity within this loop in C. albicans. We proved Saccharomyces cerevisiae as a useful tool organism for studies of C. albicans translation initiation apparatus.

Purpose: To demonstrate the antimicrobial properties of riboflavin/UV-A (365 nm) against fungal pathogens., Methods: The antimicrobial properties of riboflavin/UV-A (365 nm), with or without previous treatment with amphotericin B, were tested on three groups of fungi selected from severe cases of keratomycosis: Candida albicans, Fusarium sp, and Aspergillus fumigatus. They were tested by using Kirby-Bauer discs with empty disc (control), riboflavin 0.1% alone (R), UV-A alone (UV-A), riboflavin 0.1% and additional UV-A exposure (R+UV-A), amphotericin B alone (A), amphotericin B and riboflavin 0.1% (A+R), amphotericin B and UV-A (A+UV-A), amphotericin B and riboflavin 0.1%, and additional UV-A exposure (A+R+UV-A). The mean growth inhibition zone (GIZ) was measured around the discs., Results: C. albicans, Fusarium sp, and A. fumigatus did not show any increased GIZ after treatment without previous amphotericin B medication. However, GIZ was significantly greater after pretreatment with amphotericin B and riboflavin/UV-A (A+R+UV-A) for C. albicans (P = 0.0005), Fusarium sp (P = 0.0023) and A. fumigatus (P = 0.0008) compared with A, A+R, and A+UV-A., Conclusions: Amphotericin B is believed to interact with fungi membrane sterols to produce aggregates that form transmembrane channels. Given that collagen is one of the principal components of the cornea, it is also probable that amphotericin B may diffuse easily after cross-linking. Previous treatment with amphotericin B allowed riboflavin/UV-A effectiveness against C. albicans, Fusarium sp, and A. fumigatus. This schema might be used in the future for the treatment of keratomycosis.

Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

Hsp90 is environmentally contingent molecular chaperone that influences the form and function of diverse signal transducers. Here we discuss our recent findings that Hsp90 regulates the morphogenetic transition from yeast to filamentous forms required for virulence of the most prevalent fungal pathogen of humans, Candida albicans, and does so via cAMP-PKA signalling. This transition is normally regulated by environmental cues that are contingent upon elevated temperature to relieve Hsp90-mediated repression of the morphogenetic program. Intriguingly, Hsp90 inhibition induces filamentation independent of the canonical PKA transcription factor Efg1, in striking similarity to a select set of morphogenetic stimuli. Further investigation will determine the downstream transcription factors through which Hsp90 regulates morphogenesis and the precise mechanism of Hsp90's interaction with the cAMP-PKA pathway. C. albicans is one of many fungal species that undergo a morphological transition in a temperature-dependent manner, thus Hsp90's capacity to govern this key developmental program may provide insight into morphogenesis of diverse organisms.

All organisms have evolved mechanisms that protect them against environmental stress. The major fungal pathogen of humans, Candida albicans, has evolved robust stress responses that protect it against human immune defences and promote its pathogenicity. However, C. albicans is unlikely to be exposed to heat shock as it is obligatorily associated with warm-blooded animals. Therefore, we examined the role of the heat shock transcription factor (Hsf1) in this pathogen. We show that C. albicans expresses an evolutionarily conserved Hsf1 (orf19.4775) that is phosphorylated in response to heat shock, induces transcription via the heat shock element (HSE), contributes to the global transcriptional response to heat shock, and is essential for viability. Why has Hsf1 been conserved in this obligate animal saprophyte? We reasoned that Hsf1 might contribute to medically relevant stress responses. However, this is not the case, as an Hsf1-specific HSE-lacZ reporter is not activated by oxidative, osmotic, weak acid or pH stress. Rather, Hsf1 is required for the expression of essential chaperones in the absence of heat shock (e.g. Hsp104, Hsp90, Hsp70). Furthermore, Hsf1 regulates the expression of HSE-containing genes in response to growth temperature in C. albicans. Therefore, the main role of Hsf1 in this pathogen might be the homeostatic modulation of chaperone levels in response to growth temperature, rather than the activation of acute responses to sudden thermal transitions.

We previously showed that Candida albicans orf19.4590, which we have renamed RFX2, expresses a protein that is reactive with antibodies in persons with candidiasis. In this study, we demonstrate that C. albicans RFX2 shares some functional redundancy with Saccharomyces cerevisiae RFX1. Complementation of an S. cerevisiae rfx1 mutant with C. albicans RFX2 partially restored UV susceptibility and the repression of DNA damage response genes. DNA damage- and UV-induced genes RAD6 and DDR48 were derepressed in a C. albicans rfx2 null mutant strain under basal conditions, and the mutant was significantly more resistant to UV irradiation, heat shock, and ethanol than wild-type strain SC5314. The rfx2 mutant was hyperfilamentous on solid media and constitutively expressed hypha-specific genes HWP1, ALS3, HYR1, ECE1, and CEK1. The mutant also demonstrated increased invasion of solid agar and significantly increased adherence to human buccal epithelial cells. During hematogenously disseminated candidiasis, mice infected with the mutant had a significantly delayed time to death compared to the wild type. During oropharyngeal candidiasis, mice infected with the mutant had significantly lower tissue burdens in the oral cavity and esophagus at 7 days and they were less likely to develop disseminated infections because of mucosal translocation. The data demonstrate that C. albicans Rfx2p regulates DNA damage responses, morphogenesis, and virulence.

Purpose: To demonstrate the antimicrobial properties of riboflavin/UVA (365 nm) against common pathogens., Methods: One group of bacteria (Pseudomonas aeruginosa [PA], Staphylococcus aureus [SA], and Staphylococcus epidermidis [SE]) was tested by using Kirby-Bauer discs with (1) empty disc (Control - C); (2) riboflavin 0.1% (B2); (3) riboflavin 0.1% previously activated by UVA (B2'); (4) UVA alone (UVA); (5) group 2+additional UVA exposure (UVA+B2); and (6) group 3+additional UVA exposure (UVA+B2'). In addition, another group of microbes was tested with the same approach: methicillin-resistant S. aureus (MRSA), multidrug-resistant P. aeruginosa (MDRPA), drug-resistant Streptococcus pneumoniae (DRSP), and Candida albicans (CA). The mean growth inhibition zone (GIZ) in square millimeters was measured around the discs. The mean standard deviation (MSD) was calculated to be 3.65 when alpha = 0.01. A mean deviation (MD) > MSD indicates a significant difference., Results: In the first group, the GIZ was significantly greater after UVA (MD = 14.30), UVA+B2 (MD = 39.61), and UVA+B2' (MD = 40.45) when compared with C, B2, and B2'. UVA alone was less effective than UVA+B2 (MD = 25.31) and UVA+B2' (MD = 26.15). The second group demonstrated increased GIZ in UVA (MD = 6.98), UVA+B2 (MD = 17.80), and UVA+B2' (MD = 21.15) when compared with C, B2, and B2'. UVA alone was less effective against the second group of bacteria than was UVA+B2 (MD = 10.82) and UVA+B2' (MD = 14.17). CA did not show any GIZ after treatment., Conclusions: Riboflavin/UVA was effective against SA, SE, PA, MRSA, MDRPA, and DRSP, but was ineffective on CA and has the potential for use in treatment of microbial keratitis in the future.

Candida albicans, clinically the most important human fungal pathogen, rapidly develops resistance to antifungal drugs. The acquisition of resistance has been linked to various types of genome changes. As part of an ongoing study of this problem, we investigated mutation, genome stability and drug resistance acquisition in C. albicans strains with deletions in the base excision repair (BER) genes NTG1, APN1 and OGG1, and in the nucleotide excision repair (NER) genes RAD2 and RAD10. The BER mutants did not exhibit any change in their susceptibility to DNA-damaging agents, but the NER mutants were extremely sensitive to UV-induced DNA damage. We did not observe any significant change in mutation, genome stability and antifungal drug sensitivity in the mutant strains we tested. However, we detected a number of intriguing phenotypic differences between strains bearing deletions in equivalent C. albicans and Saccharomyces cerevisiae BER and NER genes, which may be related to differences in the life cycles of these two fungi.

The aim of this study was to propose the use of red light-emitting diode (LED) as an alternative light source for methylene blue (MB) photosensitizing effect in photodynamic therapy (PDT). Its effectiveness was tested against Staphylococcus aureus (ATCC 26923), Escherichia coli (ATCC 26922), Candida albicans (ATCC 90028) and Artemia salina. The maximum absorption of the LED lamps was at a wavelength of 663 nm, at intensities of 2,4,6 and 12 J.cm-2 for 10, 20, 30 and 60 min of exposure, respectively. Assays with and without LED exposure were carried out in plates containing MB at concentrations of 7 to 140.8 (micro) M for microorganisms and 13.35 to 668.5 (micro) M for microorganisms or microcrustaceans. The LED exposure induced more than 93.05%, 93.7% and 93.33% of growth inhibition for concentrations of 42.2 (micro)M for S.aureus (D-value=12.05 min) and 35.2 (micro)M for E.coli (D-value=11.51 min) and C.albicans (D-value=12.18 min), respectively after 20 min of exposure. LED exposure for 1 h increased the cytotoxic effect of MB against A.salina from 27% to 75%. Red LED is a promising light device for PDT that can effectively inhibit bacteria, yeast and microcrustacean growth.

Candida albicans is a human fungal pathogen and has been extensively studied because of its clinical importance. Comprehensive gene analyses have, however, made little progress. This is because of the diploid and asexual characteristics of the fungus that hamper gene disruptions. In this study, we found that ultraviolet (UV) irradiation, as well as mutagen treatment, strongly stimulated loss of heterozygosity (LOH) in strains harboring artificially constructed heterozygosity. UV-induced LOH occurred more frequently in cells within the logarithmic phase of growth compared to those within the stationary phase of growth. This was observed at all loci tested on chromosome 7, except for a locus neighboring the centromere. C. albicans RAD52, whose orthologue in Saccharomyces cerevisiae was reported to be involved in DNA repair by homologous recombination, was shown to be required for UV-induced LOH. These results suggest that high efficiency LOH caused by UV irradiation could be a prominent tool for gene analyses in C. albicans.

Purpose: The purpose of the present study was to determine the degree of expression of virulence factors such as adherence, cell surface hydrophobicity (CSH) and production of proteinase by different morphological forms of Candida albicans causing oral candidiasis in human immunodeficiency virus (HIV)-infected individuals., Methods: C. albicans 3153A and two strains isolated from oral thrush in HIV infected individuals were induced to undergo phenotypic switching by exposure to UV light and the degree of expression of virulence factors by the different morphological forms was studied., Results: Three different morphological forms of C. albicans were obtained namely, star (S), wrinkled (W) and ring (R) types from the original smooth (O) variety. It was found that proteinase production was greatest with the W type followed by the R type and O type. The S type produced the least proteinase. Expression of cell surface hydrophobicity and adherence was greatest in the O type followed by the R and then the W type and finally the S type., Conclusions: The differential expression of virulence factors occurs with different phenotypic forms of C. albicans and this may provide a particular morphological type with a distinct advantage over other types in causing candidiasis.

The polymorphic fungus Candida albicans switches from yeast to filamentous growth in response to a range of genotoxic insults, including inhibition of DNA synthesis by hydroxyurea (HU) or aphidicolin (AC), depletion of the ribonucleotide-reductase subunit Rnr2p, and DNA damage induced by methylmethane sulfonate (MMS) or UV light (UV). Deleting RAD53, which encodes a downstream effector kinase for both the DNA-replication and DNA-damage checkpoint pathways, completely abolished the filamentous growth caused by all the genotoxins tested. Deleting RAD9, which encodes a signal transducer of the DNA-damage checkpoint, specifically blocked the filamentous growth induced by MMS or UV but not that induced by HU or AC. Deleting MRC1, the counterpart of RAD9 in the DNA-replication checkpoint, impaired DNA synthesis and caused cell elongation even in the absence of external genotoxic insults. Together, the results indicate that the DNA-replication/damage checkpoints are critically required for the induction of filamentous growth by genotoxic stress. In addition, either of two mutations in the FHA1 domain of Rad53p, G65A, and N104A, nearly completely blocked the filamentous-growth response but had no significant deleterious effect on cell-cycle arrest. These results suggest that the FHA domain, known for its ability to bind phosphopeptides, has an important role in mediating genotoxic-stress-induced filamentous growth and that such growth is a specific, Rad53p-regulated cellular response in C. albicans.

Tissue conditioners are susceptible to colonization by microorganisms. Therefore, the prevention of biofilm formation are important for oral hygiene. However, mechanical and chemical cleaning methods may cause clinical problems such as deformation or surface degradation of tissue conditioners. The objective of this study is to evaluate the antifungal effects of coating agents with a TiO2 photocatalyst. Photocatalytic antifungal effects on C. albicans biofilms and photodegradation effects of adsorbed protein were measured by colorimetric assays. Scanning electron microscopy was used to examine morphological changes in C. albicans. Viscosities of coating agents increased with incorporation of TiO2. However, both of coating agents with TiO2 were acceptable to the application by brush. The antifungal and protein degradation effects increased with the concentration of TiO2 in the coating agents. These effects also increased with radiation time. After 90 min radiation, the viability of C. albicans was reduced to 16.2+/-3.3 %. Scanning electron microscopy observation showed C. albicans remained on the coated surfaces even after 90 min radiation. These results suggest that coating agents with TiO2 photocatalyst can be effective for the maintenance of tissue conditioners when dentures are removed; during sleep.

Treatment of mucocutaneous and cutaneous Candida albicans infections with photosensitizing agents and light, termed photodynamic therapy (PDT), offers an alternative to conventional treatments. Initial studies using the clinically approved photosensitizer Photofrin demonstrated the susceptibility of C. albicans to its photodynamic effects. In the present study, we have further refined parameters for Photofrin-mediated photodynamic action against C. albicans and examined whether mechanisms commonly used by microorganisms to subvert either antimicrobial oxidative defenses or antimicrobial therapy, including biofilm formation, were operative. In buffer and defined medium, germ tubes preloaded with Photofrin retained their photosensitivity for up to 2 hours, indicating the absence of degradation or export of Photofrin by the organism. The addition of serum resulted in a gradual loss of photosensitivity over 2 hours. In contrast to an adaptive response by germ tubes to oxidative stress by hydrogen peroxide, there was no adaptive response to singlet oxygen-mediated stress by photodynamic action. C. albicans biofilms were sensitive to Photofrin-mediated phototoxicity in a dose-dependent manner. Finally, the metabolic activity of C. albicans biofilms following photodynamic insult was significantly lower than that of biofilms treated with amphotericin B for the same time period. These results demonstrate that several of the mechanisms microorganisms use to subvert either antimicrobial oxidative defenses or antimicrobial therapy are apparently not operative during Photofrin-mediated photodynamic treatment 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.

The growing resistance against antifungal agents has renewed the search for alternative treatment modalities, and antimicrobial photodynamic inactivation (PDI) is a potential candidate. The cationic porphyrin 5-phenyl-10,15,20-Tris(N-methyl-4-pyridyl)porphyrin chloride (TriP[4]) is a photosensitizer that in combination with light can inactivate bacteria, fungi, and viruses. For future improvement of the efficacy of PDI of clinically relevant fungi such as Candida albicans, we sought to understand the working mechanism by following the response of C. albicans exposed to PDI using fluorescence confocal microscopy and freeze-fracture electron microscopy. The following events were observed under dark conditions: TriP[4] binds to the cell envelope of C. albicans, and none or very little TriP[4] enters the cell. Upon illumination the cell membrane is damaged and eventually becomes permeable for TriP[4]. After lethal membrane damage, a massive influx of TriP[4] into the cell occurs. Only the vacuole membrane is resistant to PDI-induced damage once TriP[4] passes the plasma membrane. Increasing the incubation time of C. albicans with TriP[4] prior to illumination did not increase the influx of TriP[4] into the cell or the efficacy of PDI. After the replacement of 100% phosphate-buffered saline (PBS) by 10% PBS as the medium, C. albicans became permeable for TriP[4] during dark incubation and the efficacy of PDI increased dramatically. In conclusion, C. albicans can be successfully inactivated by the cationic porphyrin TriP[4], and the cytoplasmic membrane is the target organelle. TriP[4] influx occurred only after cell death.

Molecular evidence is provided in this paper to elucidate a long-standing intriguing phenomenon in fungal genetics: that many natural isolates of the constitutive diploid organism Candida albicans yield strain-specific, recessive mutants at a reproducible frequency that is as high as a few percent of the surviving cells after exposure to UV irradiation or other mutagens. Southern hybridization analysis and DNA sequence data indicated that C. albicans CA12, a clinical isolate, is heterozygous for the ADE2 gene, carrying one functional and one null allele. Sequence analysis of the null allele revealed the presence of a 1.3 kb deletion, which locates between two AATC repeats and spans the promoter and coding regions of the gene. The adenine auxotrophic mutants, which were readily isolated after UV irradiation of C. albicans CA12, were proved to be the segregants of mitotic recombination as they remained as diploid, not hemizygous or haploid, cells and were homozygous for ade2. Analysis of reciprocal products of the mitotic recombination detected that the process of loss of heterozygosity was mediated by mitotic crossing over (reciprocal exchange of genetic information) as well as gene conversion (non-reciprocal exchange of genetic information).

The basis of cerulenin resistance of Candida albicans strains 4918-2 and 4918-10 has been investigated. Parasexual genetic analyses established that cerulenin resistance to concentrations of at least 5 micrograms ml-1 is dominant in both strains. The results also showed that strain 4918-2 is heterozygous for resistance, while the change from resistance to sensitivity of strain 4918-10 is reversible. Experiments to define the mechanism(s) responsible for resistance focused on cerulenin uptake and fatty-acid synthase activity. Cerulenin uptake by strains 4918-2 and 4918-10 was 24% of that of the wild-type (strain 4918). Uptake was restored in UV-induced cerulenin-sensitive segregants of strains 4918-2 and 4918-10, and varied from 63% to 200% of parental values. Fatty-acid synthase from strains 4918-2 and 4918-10 was resistant to cerulenin as judged by differences in the inactivation of the enzyme by the agent. However, inactivation kinetics of fatty-acid synthase of cerulenin-sensitive segregants did not revert to the parental inactivation profile. Further investigation showed that nine out of ten segregants were resistant to cerulenin at concentrations between 1 and 4 micrograms ml-1 while strain 4918 was sensitive to cerulenin at all concentrations tested. Thus, the results suggest that alteration of fatty-acid synthase and changes in permeability contribute to total cerulenin resistance of each strain.

When cells of Candida albicans strain 3153A are treated with a low dose of ultraviolet irradiation, they switch at high frequency (10(-2)) between a number of switch phenotypes discriminated by colony morphology. Clones from switching lineages exhibit continuous reorganization of their ribosomal chromosomes (R chromosomes), while clones from lineages maintaining the original smooth phenotype exhibit no reorganization. R chromosome reorganization results in decreases as well as increases in size of the chromosomes, but changes are not reciprocal between R chromosome homologues.

In this study, we investigated whether mice given ultraviolet (UV)-B (280-320 nm) radiation in doses sufficient to alter cutaneous immune cells and impair the induction of contact hypersensitivity would also have impaired resistance to infectious agents administered at the site of UV irradiation. C3H mice were exposed to 400 J/m2 UVR from FS40 sunlamps on four consecutive days. Immediately after the last UV treatment, groups of mice were injected subcutaneously with Candida albicans, injected intradermally (ID) with Mycobacterium bovis bacillus Calmette-Guerin (BCG), or infected percutaneously with Schistosoma mansoni in UV-irradiated skin. The induction of the delayed hypersensitivity response to C. albicans and BCG, as assessed by footpad swelling, was unaffected by UV irradiation. However, the number of viable mycobacteria recovered from the lymphoid organs of BCG-infected mice was increased significantly in the UV-irradiated animals for a period of more than 2 months. Low-dose UV irradiation of the skin at the site of infection did not influence the number of S. mansoni parasites recoverable from the internal organs of mice that had been infected with cercariae percutaneously 6 weeks earlier. We conclude that the ability of UV radiation to impair the development of cell-mediated immunity to antigens introduced in a UV-irradiated site is not universal and depends on the particular antigen administered. We hypothesize that the involvement of epidermal Langerhans cells as the primary antigen-presenting cells in the induction of cell-mediated immunity may be the critical factor in determining whether a particular immune response will be affected by local UV irradiation.

Yeast cells of Candida albicans which had been attached to polylysine-coated microscope slides were induced to form buds or germ tubes in the presence of external electrical fields. The sites of budding and germ tube formation and the growth of germ tubes and hyphal branches were polarized preferentially towards the cathode. Buds were not converted to pseudohyphae or germ tubes by the field and the field had no effect on the positioning of nuclei or septa in the yeast cell or germ tube. Buds were less polarized than germ tubes at any given applied voltage. The polarization of buds reached a peak at an electrical field of 12 mV per cell. Polarization of germ tubes was biphasic, increasing rapidly with increasing field strengths up to 5 mV per cell, and then more slowly in stronger fields. An electrical field was only required for a fraction of the time taken for germ tubes to start to form, so cells retained a memory of experiencing an electrical field which influenced the selection of sites of evagination. Increasing the electrical field delayed the time of germ tube evagination and inhibited the rate of germ tube extension. Unlike previous findings with other filamentous fungi, germ tubes grew unidirectionally towards the cathode for extended periods and did not deviate to a perpendicular orientation. This result suggests that the septal pore of the filamentous form may have high electrical resistance and would act as an effective barrier to solute transport between intercalary compartments.