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.

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.

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.

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.

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.

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.

Integrated proteomic and metabolomic analysis to study the effects of spaceflight on Candida albicans.

BACKGROUND: Candida albicans is an opportunistic pathogenic yeast, which could become pathogenic in various stressful environmental factors including the spaceflight environment. In this study, we aim to explore the phenotypic changes and possible mechanisms of C. albicans after exposure to spaceflight conditions. RESULTS: The effect of C. albicans after carried on the "SJ-10" satellite for 12 days was evaluated by proliferation, morphology, environmental resistance and virulence experiment. The result showed that the proliferation rate, biofilm formation, antioxidant capacity, cytotoxicity and filamentous morphology of C. albicans were increased in the spaceflight group compared to the control group. Proteomics and metabolomics technologies were used to analyze the profiles of proteins and metabolites in C. albicans under spaceflight conditions. Proteomic analysis identified 548 up-regulated proteins involved in the ribosome, DNA replication, base excision repair and sulfur metabolism in the spaceflight group. Moreover, 332 down-regulated proteins related to metabolic processes were observed. The metabolomic analysis found five differentially expressed metabolites. The combined analysis of proteomic and metabolomic revealed the accumulation of cysteine and methionine in C. albicans after spaceflight. CONCLUSIONS: Mechanisms that could explain the results in the phenotypic experiment of C. albicans were found through proteomic and metabolomic analysis. And our data provide an important basis for the assessment of the risk that C. albicans could cause under spaceflight environment.

Non-Toxic and Ultra-Small Biosilver Nanoclusters Trigger Apoptotic Cell Death in Fluconazole-Resistant Candida albicans via Ras Signaling.

Silver-based nanostructures are suitable for many biomedical applications, but to be useful therapeutic agents, the high toxicity of these nanomaterials must be eliminated. Here, we biosynthesize nontoxic and ultra-small silver nanoclusters (rsAg@NCs) using metabolites of usnioid lichen (a symbiotic association of algae and fungi) that exhibit excellent antimicrobial activity against fluconazole (FCZ)-resistant Candida albicans that is many times higher than chemically synthesized silver nanoparticles (AgNPs) and FCZ. The rsAg@NCs trigger apoptosis via reactive oxygen species accumulation that leads to the loss of mitochondrial membrane potential, DNA fragmentation, chromosomal condensation, and the activation of metacaspases. The proteomic analysis clearly demonstrates that rsAg@NCs exposure significantly alters protein expression. Most remarkable among the down-regulated proteins are those related to glycolysis, metabolism, free radical scavenging, anti-apoptosis, and mitochondrial function. In contrast, proteins involved in plasma membrane function, oxidative stress, cell death, and apoptosis were upregulated. Eventually, we also established that the apoptosis-inducing potential of rsAg@NCs is due to the activation of Ras signaling, which confirms their application in combating FCZ-resistant C. albicans infections.

Designing and enhancing the antifungal activity of corneal specific cell penetrating peptide using gelatin hydrogel delivery system.

BACKGROUND: Fungal keratitis is a major cause of corneal blindness accounting for more than one-third of microbiologically proven cases. The management of fungal keratitis is through topical or systemic antifungal medications alone or in combination with surgical treatment. Topical medications such as natamycin and voriconazole pose major challenges due to poor penetration across the corneal epithelium. To address the issue various carrier molecules like nanoparticles, lipid vesicles, and cell penetrating peptides were explored. But the major drawback such as non-specificity and lack of bioavailability remains. PURPOSE: In this study, we have attempted to design corneal specific cell penetrating peptide using subtractive proteomic approach from the published literature and tried to improve its bioavailability through gelatin hydrogel delivery system. MATERIAL AND METHODS: Using subtractive proteomic approach two peptides VRF005 and VRF007 were identified on the basis of solubility, cell permeability and amphipathicity. The peptides were modeled for three-dimensional structure and simulated for membrane penetration. The peptides were characterized using circular dichroism spectroscopy, dynamic light scattering and native polyacrylamide gel electrophoresis. Further uptake studies were performed on primary corneal epithelial cells and the stability was analyzed in corneal epithelial tissue lysates. Insilico prediction of peptides showed it to have antifungal activity which was further validated using colony forming assay and time killing kinetics. The duration of antifungal activity of peptide was improved using gelatin hydrogel through sustained delivery. RESULTS: VRF005 and VRF007 showed α-helical structure and was within the allowed region of Ramachandran plot. The simulation study showed their membrane penetration. The peptide uptake was found to be specific to corneal epithelial cells and also showed intracellular localization in Candida albicans and Fusarium solani. Peptides were found to be stable up to 2 hours when incubated with corneal epithelial tissue lysate. Dynamic light scattering, and native polyacrylamide gel electrophoresis revealed aggregation of peptides. VRF007 showed antifungal activity up to 24 hour whereas VRF005 showed activity up to 4 hours. Hence gelatin hydrogel-based delivery system was used to improve the activity. Actin staining of corneal epithelial cells showed that the cells were attached on gelatin hydrogel. CONCLUSION: We have designed corneal specific cell penetrating peptides using subtractive proteomic approach. Bioavailability and delivery of peptide was enhanced using gelatin hydrogel system.

Magnesium deprivation affects cellular circuitry involved in drug resistance and virulence in Candida albicans.

OBJECTIVES: Candida albicans has to struggle for the limited micronutrients present in the hostile host niche, including magnesium (Mg). The aim of this study was to examine the effect of Mg deprivation on drug resistance mechanisms and virulence traits of C. albicans. METHODS: The drug susceptibility of C. albicans strain SC5314 was determined by broth microdilution and spot assay. Efflux pump activity was measured using the substrate rhodamine 6G. Membrane intactness was studied by propidium iodide influx, and ergosterol levels were determined by the alcoholic KOH method. Metabolic flexibility was examined by studying the activity of glyoxylate cycle enzymes. Virulence factors were assessed by yeast-to-hyphae transition, biofilm formation and cell adherence. An in vivo study was also performed in a Caenorhabditis elegans infection model. RESULTS: Mg chelation leads to potentiation of membrane-targeting antifungals. The role of Mg on membrane homeostasis was explored and significant differences in ergosterol levels were found. Interestingly, it was also observed that Mg deprivation impedes the metabolic flexibility of C. albicans SC5314 by inhibiting glyoxylate cycle enzymes. Furthermore, Mg deprivation inhibited potential virulence traits, including morphological transition, biofilm formation and buccal epithelial cell adherence. All of the disrupted gene targets were validated by reverse transcription PCR. Lastly, enhanced survival of C. elegans infected with C. albicans SC5314 under Mg deprivation was observed. CONCLUSION: In view of the restricted growth of C. albicans in a Mg-deficient environment, approaches could be utilised to boost the effectiveness of existing antifungals thereby improving the management of fungal infections.

Face/Off: The Interchangeable Side of Candida Albicans.

Due to limited mobility, fungi, like most unicellular organisms, have evolved mechanisms to adapt to sudden chemical and/or physical variation in their environment. Candida albicans is recognized as a model organism to study eukaryotic responses to environmental changes, as this human commensal yeast but also opportunistic pathogen responds to numerous environmental cues through switching morphologies from yeast to hyphae growth. This mechanism is largely controlled by two major pathways: cAMP-PKA and MAPK, but each environmental signal is sensed by specific sensors. However, morphological switching is not the only response C. albicans exerts in response to environmental cues. Recently, fungal cell wall remodeling in response to host-derived environmental cues has been identified as a way for C. albicans to manipulate the innate immune system. The fungal cell wall is composed of a chitin skeleton linked to a network of ß-glucan, which anchors proteins and mannans to the fungal cell surface. As localized on the cell surface, these molecules drive interactions with the environment and other cells, particularly with host immune cells. C. albicans is recognized by immune cells such as neutrophils and macrophages via pathogen recognition receptors (PRRs) that bind different components of the cell wall. While ß-glucan and mannan are proinflammatory molecules, chitin can induce anti-inflammatory responses. Interestingly, C. albicans is able to regulate the exposure of these pathogen-associated molecular patterns (PAMPs) according to environmental cues resulting in a modulation of the host immune response. This review describes the mechanisms involved in C. albicans response to environmental changes and their effect on immune recognition.

Labeling of Phosphatidylinositol Lipid Products in Cells through Metabolic Engineering by Using a Clickable myo-Inositol Probe.

Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells.

Effects of Nd:YAG laser irradiation on the growth of Candida albicans and Streptococcus mutans: in vitro study.

The purpose of this study was to evaluate the effects of Nd:YAG laser with flat-top handpiece on the in vitro growth of Candida albicans and Streptococcus mutans. The incidence of C. albicans (opportunistic commensal) and S. mutans (facultatively anaerobic) infections is increasing, despite available treatments. Cultures of Streptococcus mutans and Candida albicans were irradiated using Nd:YAG laser (LightWalker, Fotona) with flat-top handpiece (Genova, LightWalker, Fotona) at the following parameters: group G1: 0.25 W, 10 Hz, 15 s, 3 J and group G2: 1 W, 10 Hz, 60s, 59 J. The results were evaluated directly and 24 h after irradiation using a quantitative culture method (estimation of colony-forming units in 1 ml of suspension, cfu/ml), and microscopic analysis with Janus green stain and compared with control group in which laser was not applied. C. albicans was reduced by 20 up to 54% for G1, and for G2 by 10 up to 60% directly after the application. The cfu/ml values for S. mutans decreased by 13% (p = 0.1771) for G1 and 89% (p < 0.0001) for G2. In both test groups 24 h after the application, the number of colony-forming units decreased by 15-46% for G1 and by 15-64% for G2. The arrested cell division, increasing the surface area and increasing the number of metabolically inactive cells, were observed in morphometric analysis. Macroscopic and microscopic analyses revealed a reduction in cell number and a significant decrease of cell metabolism after laser application for both C. albicans and S. mutans.

Propeptide genesis by Kex2-dependent cleavage of yeast wall protein 1 (Ywp1) of Candida albicans.

Candida albicans is a prevalent fungal resident and opportunistic pathogen of humans, exhibiting a variety of ovoid and filamentous morphologies. Anchored within the cell wall of the ovoid yeast form of C. albicans is an abundant glycoprotein termed yeast wall protein 1 (Ywp1). Ywp1 has an antiadhesive effect that may facilitate yeast cell dispersal; it also contributes to the masking of the glucan matrix of the yeast cell wall, potentially providing shielding from recognition by the human immune system. Mature Ywp1 consists of an O-glycosylated core of 378 amino acids associated with an N-glycosylated propeptide that originates from an N-terminal segment of Ywp1. A tribasic (-RRR-) sequence in the immature Ywp1 polypeptide is separated by 8 amino acids from a dibasic (-KR-) sequence that is a canonical site for cleavage by the intracellular endopeptidase Kex2, and cleavage occurs at both of these sites to generate an 11 kilodalton (kDa) propeptide that remains strongly associated with the mature core of Ywp1. Previous studies demonstrated an absence of the 11 kDa propeptide in strains lacking Kex2, but the presence of lesser amounts of a 12 kDa propeptide ostensibly (and paradoxically) arising from cleavage at the dibasic site. Subsequent studies of wild type strains, however, suggested that post-secretion cleavages were carried out in vitro by acid proteases in unbuffered cultures to generate the 12 kDa propeptide. Here, intact and Gfp-tagged Ywp1 are utilized to show that neither of the two multibasic sites is normally cleaved in the absence of Kex2, but that uncleaved Ywp1 is still N-glycosylated and subsequently anchored to the cell wall. This furthers our understanding of the multistep cleavage of this highly conserved sequence, as well as the possible contributions of the cleaved propeptide to the maturation and functioning of Ywp1.

Inhibition of adherence of the yeast Candida albicans to buccal epithelial cells by synthetic aromatic glycoconjugates.

The yeast Candida albicans is an opportunistic fungal pathogen which induces superficial and systemic infections in immunocompromised patients. Adherence to host tissue is critical to its ability to colonise and infect the host. The work presented here describes the synthesis of a small library of aromatic glycoconjugates (AGCs) and their evaluation as inhibitors of C. albicans adherence to exfoliated buccal epithelial cells (BECs). We identified a divalent galactoside, ligand 2a, capable of displacing over 50% of yeast cells already attached to the BECs. Fluorescence imaging indicates that 2a may bind to structural components of the fungal cell wall.

Role of homologous recombination genes RAD51, RAD52, and RAD59 in the repair of lesions caused by γ-radiation to cycling and G2/M-arrested cells of Candida albicans.

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.

Combating Drug-Resistant Fungi with Novel Imperfectly Amphipathic Palindromic Peptides.

Antimicrobial peptides are an important weapon against invading pathogens and are potential candidates as novel antibacterial agents, but their antifungal activities are not fully developed. In this study, a set of imperfectly amphipathic peptides was developed based on the imperfectly amphipathic palindromic structure R n(XRXXXRX)R n ( n = 1, 2; X represents L, I, F, or W), and the engineered peptides exhibited high antimicrobial activities against all fungi and bacteria tested (including fluconazole-resistant Candida albicans), with geometric mean (GM) MICs ranging from 2.2 to 6.62 µM. Of such peptides, 13 (I6) (RRIRIIIRIRR-NH2) that was Ile rich in its hydrophobic face had the highest antifungal activity (GMfungi = 1.64 µM) while showing low toxicity and high salt and serum tolerance. It also had dramatic LPS-neutralizing propensity and a potent membrane-disruptive mechanism against microbial cells. In summary, these findings were useful for short AMPs design to combat the growing threat of drug-resistant fungal and bacterial infections.

Antagonistic effect of Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC on hyphal development and adhesion of Candida albicans.

The morphological transition from yeast to a hyphal form, as well as the adhesion capability to the gastrointestinal tract, are implicated virulent determinant in Candida albicans and could be potential targets for prevention of the opportunistic pathogen. Based on this rationale, two yeast strains Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC along with reference strain Saccharomyces boulardii NCDC 363 were screened for the probiotic potential. Characters like pH, temperature, bile, simulated gastrointestinal juice tolerance tests, and Caco-2 cell line adhesion assay were determined in the present study. Further, the evaluation of its impact on C. albicans morphological transition and adhesion was assessed using microtitre germ tube test. In terms of probiotic characteristics, both the strains were tolerant to pH 2.5 and the presence of bile (0.3 to 0.6%) with an optimum growth temperature of 37°C. The strain KTP was also resistant to simulated gastric and intestinal juices as compared to control (13% and 41%, respectively) and NCDC 363 (55% and 35%, respectively). In contrast, both the yeasts had reduced adhesiveness to Caco-2 monolayer. Candida virulence in in vitro systems indicated that treatment of live probiotic yeast cells (108 ml) effectively reduced the filamentation and adhesion of C. albicans. The S. cerevisiae KTP had a profound effect on the hyphal development and adhesion when compared to the ApC and NCDC 363. The strain significantly reduced (P < .05) the hyphal growth in co-cultivated (93% and 94%, respectively) and pre-existing hyphae (54% and 68%) of strains C. albicans 183 and 1151. Isolates KTP and ApC also reduced the adhesion (≈ 22% and 41%, respectively) and transition of blastoconidia at two hours of incubation in abiotic surface. This study provides knowledge on the effect of potential probiotic yeasts such as Saccharomyces and non- Saccharomyces strains against virulence characteristic of Candida albicans.

Synthesis, characterization and evaluation of antimicrobial and cytotoxic activities of biogenic silver nanoparticles synthesized from Streptomyces xinghaiensis OF1 strain.

We report synthesis of silver nanoparticles (AgNPs) from Streptomyces xinghaiensis OF1 strain, which were characterised by UV-Vis and Fourier transform infrared spectroscopy, Zeta sizer, Nano tracking analyser, and Transmission electron microscopy. The antimicrobial activity of AgNPs alone, and in combination with antibiotics was evaluated against bacteria, namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis, and yeasts viz., Candida albicans and Malassezia furfur by using micro-dilution method. The minimum inhibitory concentration (MIC) and minimum biocidal concentration of AgNPs against bacterial and yeast strains were determined. Synergistic effect of AgNPs in combination with antibacterial and antifungal antibiotics was determined by FIC index. In addition, MTT assay was performed to study cytotoxicity of AgNPs alone and in combination with antibiotics against mouse fibroblasts and HeLa cell line. Biogenic AgNPs were stable, spherical, small, polydispersed and capped with organic compounds. The variable antimicrobial activity of AgNPs was observed against tested bacteria and yeasts. The lowest MIC (16 µg ml-1) of AgNPs was found against P. aeruginosa, followed by C. albicans and M. furfur (both 32 µg ml-1), B. subtilis and E. coli (both 64 µg ml-1), and then S. aureus and Klebsiella pneumoniae (256 µg ml-1). The high synergistic effect of antibiotics in combination with AgNPs against tested strains was found. The in vitro cytotoxicity of AgNPs against mouse fibroblasts and cancer HeLa cell lines revealed a dose dependent potential. The IC50 value of AgNPs was found in concentrations of 4 and 3.8 µg ml-1, respectively. Combination of AgNPs and antibiotics significantly decreased concentrations of both antimicrobials used and retained their high antibacterial and antifungal activity. The synthesis of AgNPs using S. xinghaiensis OF1 strain is an eco-friendly, cheap and nontoxic method. The antimicrobial activity of AgNPs could result from their small size. Remarkable synergistic effect of antibiotics and AgNPs offer their valuable potential in nanomedicine for clinical application as a combined therapy in the future.

Cytochrome c peroxidase regulates intracellular reactive oxygen species and methylglyoxal via enzyme activities of erythroascorbate peroxidase and glutathione-related enzymes in Candida albicans.

D-erythroascorbate peroxidase (EAPX1) deficiency causes glutathione deprivation, leading to the accumulation of methylglyoxal and reactive oxygen species (ROS), and especially, induction of cytochrome c peroxidase (Ccp1) in Candida albicans. Nevertheless, reciprocal effects between changes in Ccp1 activity and the antioxidative D-erythroascorbic acid- and glutathione-dependent redox status, which reflects methylglyoxal biosynthesis altering pathophysiology are unclear in eukaryotes. To elucidate the effect of CCP1 expression on EAPX1 and glutathione reductase (Glr1) activity-mediated D-erythroascorbic acid biosynthesis and redox homeostasis, the CCP1 gene was disrupted and overexpressed. First, we demonstrated both glutathione-independent and-dependent metabolite contents and their corresponding gene transcripts and enzyme activities (i.e., Ccp1, catalase-peroxidase [KatG], superoxide dismutase [Sod], Eapx1, and Glr1) in CCP1 mutants. Second, methylglyoxal-oxidizing alcohol dehydrogenase (Adh1) and methylglyoxal-reducing oxidoreductase activity on glycolytic methylglyoxal and pyruvate production and NAD(P)H content were determined in these mutants. Contrary to our expectation, CCP1 disruption (42.19±3.22nmolO2h-1mgwetcell-1) failed to affect cell respiration compared to the wild-type strain (41.62±7.11nmolO2h-1mgwetcell-1) under cyanide treatment, and in contrast to hydrogen peroxide (H2O2) treatment (21.74±1.03nmol O2h-1mgwetcell-1). Additionally, Ccp1 predominantly detoxified H2O2 rather than negligible scavenging activities towards methylglyoxal and other oxidants. CCP1 deficiency stimulated Sod and Adh1 activity but downregulated Glr1, Eapx1, catalase, and peroxidase activity while enhancing KatG, EAPX1, and GLR1 transcription by decreasing glutathione and D-erythroascorbic acid and increasing pyruvate. Noticeably, the ROS-accumulating CCP1-deficient mutant maintained steady-state levels of methylglyoxal, which was revealed to be regulated by methylglyoxal-oxidizing and -reducing activity with drastic changes in NAD(P)H. We confirmed and clarified our results by showing that CCP1/EAPX1 double disruptants underwent severe growth defects due to the D-erythroascorbic acid and glutathione depletion because of pyruvate overaccumulation. These observations were made in both budding and hyphal-growing CCP1 mutants. The revealed metabolic network involving Ccp1 and other redox regulators affected ROS and methylglyoxal through D-erythroascorbic acid and glutathione-dependent metabolites, thereby influencing dimorphism. This is the first report of the Ccp1-mediated D-erythroascorbic acid and glutathione biosynthesis accompanying methylglyoxal scavengers for full fungal virulence.