Sodium lignosulfonate causes cell membrane perturbation in the human fungal pathogen Candida albicans.

Underestimating fungal infections led to a gap in the development of antifungal medication. However, rising rates of morbidity and mortality with fungal infection have revealed an alarming rise in antifungal resistance also. Due to the eukaryotic properties of fungi and the close evolutionary similarity between fungal cells and human hosts, therapeutic targeting of Candida infections is troublesome, along with the development of resistance. The discovery of new antifungals is so far behind schedule that the antifungal pipeline is nearly empty. Previously, we have reported the activity and susceptibility of Sodium lignosulfonate (LIG) against C. albicans. In this work, we have established the mechanistic actions of LIG's activity. We performed flow cytometric analysis for membrane integrity, ergosterol binding assay, crystal violet assay, and membrane leakage assay to analyze quantitatively that the C. albicans membrane is being disrupted in response to LIG. Electron microscopic analysis with SEM and TEM confirmed changes in Candida cellular morphology and membrane perturbation respectively. These findings indicated that LIG causes cell membrane damage in C. albicans. This knowledge about LIG's mechanism of action against C. albicans could be used to explore it further as a lead antifungal molecule to develop it as a potent candidate for antifungal therapeutics in the future.

The Role of Ergosterol and Sphingolipids in the Localization and Activity of Candida albicans' Multidrug Transporter Cdr1p and Plasma Membrane ATPase Pma1p.

Opportunistic pathogen Candida albicans causes systemic infections named candidiasis. Due to the increasing number of multi-drug resistant clinical isolates of Candida sp., currently employed antifungals (e.g., azoles) are insufficient for combating fungal infection. One of the resistance mechanisms toward azoles is increased expression of plasma membrane (PM) transporters (e.g., Cdr1p), and such an effect was observed in C. albicans clinical isolates. At the same time, it has been proven that a decrease in PMs sphingolipids (SLs) content correlates with altered sensitivity to azoles and diminished Cdr1p levels. This indicates an important role for SL in maintaining the properties of PM and gaining resistance to antifungal agents. Here, we prove using a novel spot variation fluorescence correlation spectroscopy (svFCS) technique that CaCdr1p localizes in detergent resistant microdomains (DRMs). Immunoblot analysis confirmed the localization of CaCdr1p in DRMs fraction in both the C. albicans WT and erg11Δ/Δ strains after 14 and 24 h of culture. We also show that the C. albicanserg11Δ/Δ strain is more sensitive to the inhibitor of SLs synthesis; aureobasidin A (AbA). AbA treatment leads to a diminished amount of SLs in C. albicans WT and erg11Δ/Δ PM, while, for C. albicanserg11Δ/Δ, the general levels of mannose-inositol-P-ceramide and inositol-P-ceramide are significantly lower than for the C. albicans WT strain. Simultaneously, the level of ergosterol in the C. albicans WT strain after adding of AbA remains unchanged, compared to the control conditions. Analysis of PM permeabilization revealed that treatment with AbA correlates with the disruption of PM integrity in C. albicanserg11Δ/Δ but not in the C. albicans WT strain. Additionally, in the C. albicans WT strain, we observed lower activity of H+-ATPase, correlated with the delocalization of both CaCdr1p and CaPma1p.

A Deep Learning Approach to Capture the Essence of Candida albicans Morphologies.

We present deep learning-based approaches for exploring the complex array of morphologies exhibited by the opportunistic human pathogen Candida albicans. Our system, entitled Candescence, automatically detects C. albicans cells from differential image contrast microscopy and labels each detected cell with one of nine morphologies. This ranges from yeast white and opaque forms to hyphal and pseudohyphal filamentous morphologies. The software is based upon a fully convolutional one-stage (FCOS) object detector, a deep learning technique that uses an extensive set of images that we manually annotated with the location and morphology of each cell. We developed a novel cumulative curriculum-based learning strategy that stratifies our images by difficulty from simple yeast forms to complex filamentous architectures. Candescence achieves very good performance (~85% recall; 81% precision) on this difficult learning set, where some images contain hundreds of cells with substantial intermixing between the predicted classes. To capture the essence of each C. albicans morphology and how they intermix, we used a second technique from deep learning entitled generative adversarial networks. The resultant models allow us to identify and explore technical variables, developmental trajectories, and morphological switches. Importantly, the model allows us to quantitatively capture morphological plasticity observed with genetically modified strains or strains grown in different media and environments. We envision Candescence as a community meeting point for quantitative explorations of C. albicans morphology. IMPORTANCE The fungus Candida albicans can "shape shift" between 12 morphologies in response to environmental variables. The cytoprotective capacity provided by this polymorphism makes C. albicans a formidable pathogen to treat clinically. Microscopy images of C. albicans colonies can contain hundreds of cells in different morphological states. Manual annotation of images can be difficult, especially as a result of densely packed and filamentous colonies and of technical artifacts from the microscopy itself. Manual annotation is inherently subjective, depending on the experience and opinion of annotators. Here, we built a deep learning approach entitled Candescence to parse images in an automated, quantitative, and objective fashion: each cell in an image is located and labeled with its morphology. Candescence effectively replaces simple rules based on visual phenotypes (size, shape, and shading) with neural circuitry capable of capturing subtle but salient features in images that may be too complex for human annotators.

The Mnn10/Anp1-dependent N-linked outer chain glycan is dispensable for Candida albicans cell wall integrity.

Candida albicans cell wall glycoproteins, and in particular their mannose-rich glycans, are important for maintaining cellular integrity as well as host recognition, adhesion, and immunomodulation. The asparagine (N)-linked mannose outer chain of these glycoproteins is produced by Golgi mannosyltransferases (MTases). The outer chain is composed of a linear backbone of ∼50 α1,6-linked mannoses, which acts as a scaffold for addition of ∼150 or more mannoses in other linkages. Here, we describe the characterization of C. albicans OCH1, MNN9, VAN1, ANP1, MNN10, and MNN11, which encode the conserved Golgi MTases that sequentially catalyze the α1,6 mannose outer chain backbone. Candida albicans och1Δ/Δ, mnn9Δ/Δ, and van1Δ/Δ mutants block the earliest steps of backbone synthesis and like their Saccharomyces cerevisiae counterparts, have severe cell wall and growth phenotypes. Unexpectedly, and in stark contrast to S. cerevisiae, loss of Anp1, Mnn10, or Mnn11, which together synthesize most of the backbone, have no obvious deleterious phenotypes. These mutants were unaffected in cell morphology, growth, drug sensitivities, hyphal formation, and macrophage recognition. Analyses of secreted glycosylation reporters demonstrated that anp1Δ/Δ, mnn10Δ/Δ, and mnn11Δ/Δ strains accumulate glycoproteins with severely truncated N-glycan chains. This hypo-mannosylation did not elicit increased chitin deposition in the cell wall, which in other yeast and fungi is a key compensatory response to cell wall integrity breaches. Thus, C. albicans has evolved an alternate mechanism to adapt to cell wall weakness when N-linked mannan levels are reduced.

Vacuole and mitochondria patch (vCLAMP) and ER-mitochondria encounter structure (ERMES) maintain cell survival by protecting mitochondrial functions in Candida albicans.

Candida albicans is an important opportunistic fungus in the clinic. In recent years, with the widespread use of antibiotics, drug-resistant strains have been isolated in the clinic, so finding new drug targets has become an urgent problem to be solved. The vacuole and mitochondria patch (vCLAMP) and the ER-mitochondria encounter structure (ERMES) are new types of inner membrane junction systems in Saccharomyces cerevisiae. However, the functions in maintaining cell survival of the two structures have not yet been elucidated in C. albicans. In this study, VAM6 and MDM34 knockout mutants (vam6Δ/Δmet-MDM34) were constructed using an induction system regulated by the MET3 promoter. PI-positive assays showed that deletion of vCLAMP and ERMES led to abnormal growth of C. albicans. Furthermore, the vam6Δ/Δmet-MDM34 mutant exhibited obvious mitochondrial fragmentation, mtDNA damage, reduced ATP levels, and abnormal mitochondrial membrane potential, indicating its important role in maintaining the structures and functions of mitochondria. Moreover, deletion of vCLAMP and ERMES inhibited filamentous growth. Overall This study shows that vCLAMP and ERMES play important roles in maintaining the survival of C. albicans cells.

Coordination of fungal biofilm development by extracellular vesicle cargo.

The fungal pathogen Candida albicans can form biofilms that protect it from drugs and the immune system. The biofilm cells release extracellular vesicles (EVs) that promote extracellular matrix formation and resistance to antifungal drugs. Here, we define functions for numerous EV cargo proteins in biofilm matrix assembly and drug resistance, as well as in fungal cell adhesion and dissemination. We use a machine-learning analysis of cargo proteomic data from mutants with EV production defects to identify 63 candidate gene products for which we construct mutant and complemented strains for study. Among these, 17 mutants display reduced biofilm matrix accumulation and antifungal drug resistance. An additional subset of 8 cargo mutants exhibit defects in adhesion and/or dispersion. Representative cargo proteins are shown to function as EV cargo through the ability of exogenous wild-type EVs to complement mutant phenotypic defects. Most functionally assigned cargo proteins have roles in two or more of the biofilm phases. Our results support that EVs provide community coordination throughout biofilm development in C. albicans.

Rim101-upregulated Fets contribute to dark pigment formation in gray cells of Candida albicans.

Candida albicans has long been known to switch between white and opaque phases; however, a third cell type, referred to as the 'gray' phenotype, was recently characterized. The three phenotypes have different colonial morphologies, with white cells forming white-colored colonies and opaque and gray cells forming dark-colored colonies. We previously showed that Wor1-upregulated ferroxidases (Fets) function as pigment multicopper oxidases that regulate the production of dark-pigmented melanin in opaque cells. In this study, we demonstrated that Fets also contributed to dark pigment formation in gray colonies but in a Wor1-independent manner. Deletion of both WOR1 and EFG1 locked cells in the gray phenotype in some rich media. However, the efg1/efg1 wor1/wor1 mutant could switch between white and gray in minimal media depending on the ambient pH. Specifically, mutant cells exhibited the white phenotype at pH 4.5 but switched to gray at pH 7.5. Consistent with phenotype switching, Fets expressions and melanin production were also regulated by ambient pH. Ectopic expression of the Rim101-405 allele in the mutant enabled the pH restriction to be bypassed and promoted gray cell formation in acidic media. Our data suggest that Rim101-upregulated Fets contribute to dark pigment formation in the gray cells.

Proteinous Components of Neutrophil Extracellular Traps Are Arrested by the Cell Wall Proteins of Candida albicans during Fungal Infection, and Can Be Used in the Host Invasion.

One of defense mechanisms of the human immune system to counteract infection by the opportunistic fungal pathogen Candida albicans is the recruitment of neutrophils to the site of invasion, and the subsequent production of neutrophil extracellular traps (NETs) that efficiently capture and kill the invader cells. In the current study, we demonstrate that within these structures composed of chromatin and proteins, the latter play a pivotal role in the entrapment of the fungal pathogen. The proteinous components of NETs, such as the granular enzymes elastase, myeloperoxidase and lactotransferrin, as well as histones and cathelicidin-derived peptide LL-37, are involved in contact with the surface of C. albicans cells. The fungal partners in these interactions are a typical adhesin of the agglutinin-like sequence protein family Als3, and several atypical surface-exposed proteins of cytoplasmic origin, including enolase, triosephosphate isomerase and phosphoglycerate mutase. Importantly, the adhesion of both the elastase itself and the mixture of proteins originating from NETs on the C. albicans cell surface considerably increased the pathogen potency of human epithelial cell destruction compared with fungal cells without human proteins attached. Such an implementation of adsorbed NET-derived proteins by invading C. albicans cells might alter the effectiveness of the fungal pathogen entrapment and affect the further host colonization.

The Antifungal Action Mode of N-Phenacyldibromobenzimidazoles.

Our study aimed to characterise the action mode of N-phenacyldibromobenzimidazoles against C. albicans and C. neoformans. Firstly, we selected the non-cytotoxic most active benzimidazoles based on the structure-activity relationships showing that the group of 5,6-dibromobenzimidazole derivatives are less active against C. albicans vs. 4,6-dibromobenzimidazole analogues (5e-f and 5h). The substitution of chlorine atoms to the benzene ring of the N-phenacyl substituent extended the anti-C. albicans action (5e with 2,4-Cl2 or 5f with 3,4-Cl2). The excellent results for N-phenacyldibromobenzimidazole 5h against the C. albicans reference and clinical isolate showed IC50 = 8 µg/mL and %I = 100 ± 3, respectively. Compound 5h was fungicidal against the C. neoformans isolate. Compound 5h at 160-4 µg/mL caused irreversible damage of the fungal cell membrane and accidental cell death (ACD). We reported on chitinolytic activity of 5h, in accordance with the patterns observed for the following substrates: 4-nitrophenyl-N-acetyl-ß-d-glucosaminide and 4-nitrophenyl-ß-d-N,N',N″-triacetylchitothiose. Derivative 5h at 16 µg/mL: (1) it affected cell wall by inducing ß-d-glucanase, (2) it caused morphological distortions and (3) osmotic instability in the C. albicans biofilm-treated. Compound 5h exerted Candida-dependent inhibition of virulence factors.

Naringin-generated ROS promotes mitochondria-mediated apoptosis in Candida albicans.

Naringin is a flavonoid which has a therapeutic effect. However, the details of its antifungal mechanism have not yet been fully elucidated. This study focused on clarifying the relationship between naringin and Candida albicans, to understand its mode of antifungal action. In general, naringin is an antioxidant, but our results indicated that 1 mM naringin generates intracellular superoxide (O2- ) and hydroxyl radicals (OH- ). Reactive oxygen species (ROS) have a serious impact on Ca2+ signaling and the production of mitochondrial ROS. After exposure to enhanced O2- and OH- , mitochondrial Ca2+ overload and mitochondrial O2- generation were confirmed in C. albicans. It was verified that mitochondrial O2- transforms mitochondrial glutathione (GSH) to oxidized GSH (GSSG), leading to extreme oxidative stress in mitochondria. The previously observed Ca2+ accumulation and oxidative stress resulted in mitochondrial membrane potential (MMP) alteration and increased mitochondrial mass. In succession, cytochrome c release from the mitochondria to the cytosol was detected due to MMP loss. Cytochrome c promotes the initiation of apoptosis, and further experiments were performed to assess the apoptotic hallmarks. Metacaspases activation, chromosomal condensation, DNA fragmentation, and phosphatidylserine exposure were observed, indicating that naringin induces apoptosis in C. albicans. In conclusion, our findings manifested that naringin-generated O2- and OH- damage the mitochondria and that mitochondrial dysfunction-mediated apoptosis is novel antifungal mechanism of naringin.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells.

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Multivalent Presentations of Glycomimetic Inhibitor of the Adhesion of Fungal Pathogen Candida albicans to Human Buccal Epithelial Cells.

Candida albicans causes some of the most prevalent hospital-acquired fungal infections, particularly threatening for immunocompromised patients. C. albicans strongly adheres to the surface of epithelial cells so that subsequent colonization and biofilm formation can take place. Divalent galactoside glycomimetic 1 was found to be a potent inhibitor of the adhesion of C. albicans to buccal epithelial cells. In this work, we explore the effect of multivalent presentations of glycomimetic 1 on its ability to inhibit yeast adhesion and biofilm formation. Tetra-, hexa-, and hexadecavalent displays of compound 1 were built on RAFT cyclopeptide- and polylysine-based scaffolds with a highly efficient and modular synthesis. Biological evaluation revealed that the scaffold choice significantly influences the activity of the lower valency conjugates, with compound 16, constructed on a tetravalent polylysine scaffold, found to inhibit the adhesion of C. albicans to human buccal epithelial cells more effectively than the glycomimetic 1; however, the latter performed better in the biofilm reduction assays. Interestingly, the higher valency glycoconjugates did not outperform the anti-adhesion activity of the original compound 1, and no significant effect of the core scaffold could be appreciated. SEM images of C. albicans cells treated with compounds 1, 14, and 16 revealed significant differences in the aggregation patterns of the yeast cells.

Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters.

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.

Inhibition of Distinct Proline- or N-Acetylglucosamine-Induced Hyphal Formation Pathways by Proline Analogs in Candida albicans.

Candida albicans undergoes a yeast-to-hyphal transition that has been recognized as a virulence property as well as a turning point leading to biofilm formation associated with candidiasis. It is known that yeast-to-hyphal transition is induced under complex environmental conditions including temperature (above 35°C), pH (greater than 6.5), CO2, N-acetylglucosamine (GlcNAc), amino acids, RPMI-1640 synthetic culture medium, and blood serum. To identify the hyphal induction factor in the RPMI-1640 medium, we examined each component of RPMI-1640 and established a simple hyphal induction condition, that is, incubation in L-proline solution at 37°C. Incubation in GlcNAc solution alone, which is not contained in RPMI-1640, without any other materials was also identified as another simple hyphal induction condition. To inhibit hyphal formation, proline and GlcNAc analogs were examined. Among the proline analogs used, L-azetidine-2-carboxylic acid (AZC) inhibited hyphal induction under both induction conditions, but L-4-thiazolidinecarboxylic acid (T4C) specifically inhibited proline-induced hyphal formation only, while α-N-methyl-L-proline (mPro) selectively inhibited GlcNAc-induced hyphal formation. Hyphal formation in fetal bovine serum was also inhibited by AZC or T4C together with mPro without affecting the proliferation of yeast form. These results indicate that these proline analogs are ideal inhibitors of yeast-to-hyphal transition in C. albicans.

Spectroscopic and In Vitro Investigations of Boron(III) Complex with Meso-4-Methoxycarbonylpropylsubstituted Dipyrromethene for Fluorescence Bioimaging Applications.

This study focuses on the behavior of a new fluorescent marker for labeling individual biomolecules and staining cell organelles developed on a meso-substituted BODIPY platform. Boron(III) complex with meso-4-methoxycarbonylpropylsubstituted 3,3',5,5'-tetramethyl-2,2'-dipyrromethene has been synthesized and identified via visible, UV-, NMR- and MS-spectra X-ray. The behavior of fluorophore in solutions has been studied with various experimental techniques. It has been found that luminophore exhibits a high quantum yield (almost ~100-75%) in the blue-green region (513-520 nm) and has high photostability. In addition, biological analysis indicates that the fluorophore exhibits a tendency to effectively penetrate into cell membranes. On the other hand, the proposed BODIPY can be used to study the significant differences among a large number of pathogens of mycotic infections, as well as to visualize structural changes in the plasma membrane, which is necessary for the clearance of mammalian cells undergoing apoptotic cell death.

Mechanical force-induced morphology changes in a human fungal pathogen.

BACKGROUND: The initial step of a number of human or plant fungal infections requires active penetration of host tissue. For example, active penetration of intestinal epithelia by Candida albicans is critical for dissemination from the gut into the bloodstream. However, little is known about how this fungal pathogen copes with resistive forces upon host cell invasion. RESULTS: In the present study, we have used PDMS micro-fabrication to probe the ability of filamentous C. albicans cells to penetrate and grow invasively in substrates of different stiffness. We show that there is a threshold for penetration that corresponds to a stiffness of ~ 200 kPa and that invasive growth within a stiff substrate is characterized by dramatic filament buckling, along with a stiffness-dependent decrease in extension rate. We observed a striking alteration in cell morphology, i.e., reduced cell compartment length and increased diameter during invasive growth, that is not due to depolarization of active Cdc42, but rather occurs at a substantial distance from the site of growth as a result of mechanical compression. CONCLUSIONS: Our data reveal that in response to this compression, active Cdc42 levels are increased at the apex, whereas active Rho1 becomes depolarized, similar to that observed in membrane protrusions. Our results show that cell growth and morphology are altered during invasive growth, suggesting stiffness dictates the host cells that C. albicans can penetrate.

TETRALEC, Artificial Tetrameric Lectins: A Tool to Screen Ligand and Pathogen Interactions.

C-type lectin receptor (CLR)/carbohydrate recognition occurs through low affinity interactions. Nature compensates that weakness by multivalent display of the lectin carbohydrate recognition domain (CRD) at the cell surface. Mimicking these low affinity interactions in vitro is essential to better understand CLR/glycan interactions. Here, we present a strategy to create a generic construct with a tetrameric presentation of the CRD for any CLR, termed TETRALEC. We applied our strategy to a naturally occurring tetrameric CRD, DC-SIGNR, and compared the TETRALEC ligand binding capacity by synthetic N- and O-glycans microarray using three different DC-SIGNR constructs i) its natural tetrameric counterpart, ii) the monomeric CRD and iii) a dimeric Fc-CRD fusion. DC-SIGNR TETRALEC construct showed a similar binding profile to that of its natural tetrameric counterpart. However, differences observed in recognition of low affinity ligands underlined the importance of the CRD spatial arrangement. Moreover, we further extended the applications of DC-SIGNR TETRALEC to evaluate CLR/pathogens interactions. This construct was able to recognize heat-killed Candida albicans by flow cytometry and confocal microscopy, a so far unreported specificity of DC-SIGNR. In summary, the newly developed DC-SIGNR TETRALEC tool proved to be useful to unravel novel CLR/glycan interactions, an approach which could be applied to other CLRs.

Epigenetic cell fate in Candida albicans is controlled by transcription factor condensates acting at super-enhancer-like elements.

Cell identity in eukaryotes is controlled by transcriptional regulatory networks that define cell-type-specific gene expression. In the opportunistic fungal pathogen Candida albicans, transcriptional regulatory networks regulate epigenetic switching between two alternative cell states, 'white' and 'opaque', that exhibit distinct host interactions. In the present study, we reveal that the transcription factors (TFs) regulating cell identity contain prion-like domains (PrLDs) that enable liquid-liquid demixing and the formation of phase-separated condensates. Multiple white-opaque TFs can co-assemble into complex condensates as observed on single DNA molecules. Moreover, heterotypic interactions between PrLDs support the assembly of multifactorial condensates at a synthetic locus within live eukaryotic cells. Mutation of the Wor1 TF revealed that substitution of acidic residues in the PrLD blocked its ability to phase separate and co-recruit other TFs in live cells, as well as its function in C. albicans cell fate determination. Together, these studies reveal that PrLDs support the assembly of TF complexes that control fungal cell identity and highlight parallels with the 'super-enhancers' that regulate mammalian cell fate.

New anticandidal Cu(i) complexes with neocuproine and ketoconazole derived diphenyl(aminomethyl)phosphane: luminescence properties for detection in fungal cells.

The search for new antifungals is very important because the large genetic variation of pathogenic organisms has resulted in the development of increasingly effective defense mechanisms by microorganisms. Metal complexes as potential drugs are nowadays gaining interest, because they are characterized by accessible redox states of metal centers and a plethora of easily modifiable geometries. In this work we present two new copper(i) iodide or thiocyanide complexes with 2,9-dimethyl-1,10-phenanthroline (dmp) and a diphenylphosphane derivative of ketoconazole (KeP), where a ketoconazole acetyl group is replaced by the -CH2PPh2 unit, [CuI(dmp)KeP] (1-KeP) and [CuNCS(dmp)KeP] (2-KeP) - their synthesis and structural characteristics. The analysis of the intrinsic fluorescence of the ketoconazole moiety in the coordinated KeP molecule revealed that the copper(i) central atom does not act as a quencher and the observed decrease of fluorescence intensity is a result of a strong inner filter effect caused by the presence of the CuXdmp unit. Moreover, the complexes exhibit a remarkable MLCT (metal-ligand charge transfer) based phosphorescence with the emission maximum at 600-615 nm in aqueous media, which probably results from the formation of dimers and higher order oligomers in the most polar solutions. Both complexes proved to be promising antifungal agents towards Candida albicans, showing a relatively high efficiency towards the fluconazole resistant strains with -CDR1 and CDR2 or MDR1 efflux pump overexpression, which suggests that they overcome at least partially these defense mechanisms. Simulations of docking to the cytochrome P450 14α-demethylase (the azoles' primary molecular target) suggested that the compounds studied were rather incapable of competitively inhibiting this enzyme, unlike ketoconazole and the KeP ligand. On the other hand, the phosphorescence in aqueous solutions allowed recording the confocal micrographs of the complexes which showed that both of them are situated in spherical structures inside the cells, most likely in the vacuoles.

The effects of aloe emodin-mediated antimicrobial photodynamic therapy on drug-sensitive and resistant Candida albicans.

The extensive and repetitive use of antifungal drugs has led to the development of drug-resistant Candida albicans. Antimicrobial photodynamic therapy (aPDT) has received considerable attention as an emerging and promising approach to combat drug-resistant microbes. This study evaluated the photodynamic effects mediated by aloe emodin (AE), a natural compound isolated from Aloe vera and Rheum palmatum, on azole-sensitive and azole-resistant C. albicans in vitro. AE exhibited no significant dark toxicity, but in the presence of light, effectively inactivated C. albicans cells in a concentration-dependent manner. The uptake of AE by fungal cells was investigated by confocal laser scanning microscopy (CLSM), and the results showed that AE possessed stronger ability to enter into C. albicans cells following light irradiation. Transmission electron microscopy analysis suggested that AE-mediated aPDT could induce damage to the cell wall, cytoplasm, and nucleus. Damage to the surface of C. albicans was observed by scanning electron microscopy. These results suggest that AE is a potential PS for use in aPDT of drug-resistant C. albicans strains, and AE-mediated aPDT shows promise as an antifungal treatment.