Allium sativum Extract Chemical Composition, Antioxidant Activity and Antifungal Effect against Meyerozyma guilliermondii and Rhodotorula mucilaginosa Causing Onychomycosis.

Onychomycosis is a major health problem due to its chronicity and resistance to therapy. Because some cases associate paronychia, any therapy must target the fungus and the inflammation. Medicinal plants represent an alternative for onychomycosis control. In the present work the antifungal and antioxidant activities of Alium sativum extract against Meyerozyma guilliermondii (Wick.) Kurtzman & M. Suzuki and Rhodotorula mucilaginosa (A. Jörg.) F.C. Harrison, isolated for the first time from a toenail onychomycosis case, were investigated. The fungal species were confirmed by DNA molecular analysis. A. sativum minimum inhibitory concentration (MIC) and ultrastructural effects were examined. At the MIC concentration (120 mg/mL) the micrographs indicated severe structural alterations with cell death. The antioxidant properties of the A. sativum extract were evaluated is a rat turpentine oil induced inflammation, and compared to an anti-inflammatory drug, diclofenac, and the main compound from the extract, allicin. A. sativum reduced serum total oxidative status, malondialdehyde and nitric oxide production, and increased total thiols. The effects were comparable to those of allicin and diclofenac. In conclusion, the garlic extract had antifungal effects against M. guilliermondii and R. mucilaginosa, and antioxidant effect in turpentine-induced inflammation. Together, the antifungal and antioxidant activities support that A. sativum is a potential alternative treatment in onychomycosis.

Antifungal effects of a 1,3,4-thiadiazole derivative determined by cytochemical and vibrational spectroscopic studies.

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 µg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and ß(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of ß(1→3) glucans and ß(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses.

Biochemical changes of polysaccharides and proteins within EPS under Pb(II) stress in Rhodotorula mucilaginosa.

Microorganisms have been widely applied to heavy metal adsorption due to their strong secretion of extracellular polymeric substances (EPS). This study explored the responses of Rhodotorula mucilaginosa (R1, a red yeast with substantial EPS supply) under Pb stress. The maximum sorption of Pb cations by R1 was ~650 mg/L. In particular, despite the declined microbial biomass, the total Pb sorption after incubation was actually elevated in the solution with high Pb concentration. At 0-1000 mg/L Pb(NO3)2 level, the longitudinal sizes of the yeast capsules increased from 2.04 to 2.90 µm. At 1500 mg/L, however, the survived yeast started to lose the membrane integrity of the cells. Meanwhile, the percentages of organic carbon contents of EPS decreased from 40% to 33% when the Pb(NO3)2 concentration raised to 2500 mg/L, confirming the incorporation of Pb2+ cations into the fungal EPS during the sorption. For the survived R1 cells, function of polysaccharides to resist Pb toxicity only worked at extremely high Pb(NO3)2 levels (>= 1500 mg/L). In contrast, proteins showed continuously enhanced ability to resist Pb toxicity, consistent with their increasing content (per cell) in the EPS. Moreover, ATR-IR spectra showed that the intensity of amide II peak at 1540 cm-1 was significantly increased, indicating elevated glutathione (GSH) in EPS. This suggested that GSH could be the critical Pb-binding component in EPS proteins. This study hence elucidated roles of polysaccharides and proteins in EPS under the toxicity caused by heavy metals.

Copper stress response in yeast Rhodotorula mucilaginosa AN5 isolated from sea ice, Antarctic.

Heavy metal pollution in Antarctic is serious by anthropogenic emissions and atmospheric transport. To dissect the heavy metal adaptation mechanisms of sea-ice organisms, a basidiomycetous yeast strain AN5 was isolated and its cellular changes were analyzed. Morphological, physiological, and biochemical characterization indicated that this yeast strain belonged to Rhodotorula mucilaginosa AN5. Heavy metal resistance pattern of Cd > Pb = Mn > Cu > Cr > Hg was observed. Scanning electron microscopic (SEM) results exhibited altered cell surface morphology under the influence of copper metal compared to that with control. The determination of physiological and biochemical changes manifested that progressive copper treatment significantly increased antioxidative reagents content and enzymes activity in the red yeast, which quench the active oxygen species to maintain the intercellular balance of redox state and ensure the cellular fission and growth. Comparative proteomic analysis revealed that, under 2 mM copper stress, 95 protein spots were tested reproducible changes of at least 10-fold in cells. Among 95 protein spots, 43 were elevated and 52 were decreased synthesis. After MALDI TOF MS/MS analysis, 51 differentially expressed proteins were identified successfully and classified into six functional groups, including carbohydrate and energy metabolism, nucleotide and protein metabolism, protein folding, antioxidant system, signaling, and unknown function proteins. Function analysis indicated that carbohydrate and energy metabolism-, nucleotide and protein metabolism-, and protein folding-related proteins played central role to the heavy metal resistance of Antarctic yeast. Generally, the results revealed that the yeast has a great capability to cope with heavy metal stress and activate the physiological and protein mechanisms, which allow more efficient recovery after copper stress. Our studies increase understanding of the molecular resistance mechanism of polar yeast to heavy metal, which will be benefitted for the sea-ice isolates to be a potential candidate for bioremediation of metal-contaminated environments.

Reversible naftifine-induced carotenoid depigmentation in Rhodotorula mucilaginosa (A. Jörg.) F.C. Harrison causing onychomycosis.

Rhodotorula mucilaginosa was isolated from a patient with onychomycosis, and identification was confirmed by morphological and cultural characteristics as well as by DNA molecular analysis. Antifungal agents naftifine (10 mg/mL, active substance in Exoderil) and bifonazole (10 mg/mL, active substance in Canespor) were tested in different concentrations to assess in vitro effects on fungal growth and carotenoid synthesis. The antifungal mechanisms of action of naftifine and bifonazole against R. mucilaginosa isolates were similar and affected the biosynthetic pathway of ergosterol. For the first time, this research demonstrates that naftifine affects the carotenoid biosynthetic pathway, producing depigmentation of R. mucilaginosa in solid and liquid media. Furthermore, depigmentation was a reversible process; naftifine-treated yeast cells that were depigmented resumed carotenoid production upon transfer to fresh media. Raman and UV-vis spectrophotometry in conjunction with chromatographic analysis detected changes in carotenoids in yeast cells, with torulene decreasing and B-carotene increasing after repigmentation. Transmission electron micrographs revealed critical ultrastructural modifications in the depigmented cells after naftifine treatment, i.e., a low-electron-density cell wall without visible mucilage or lamellate structure.

Dead biomass of Amazon yeast: A new insight into bioremediation and recovery of silver by intracellular synthesis of nanoparticles.

This investigation was undertaken to describe a natural process for the removal of silver and the simultaneous recovery of Ag/Ag2O nanoparticles by dead biomass of the yeast Rhodotorula mucilaginosa. The removal of silver ions from aqueous solution and the synthesis of Ag/Ag2O nanoparticles were analyzed based on physicochemical factors and equilibrium concentration, combined with transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and infrared spectroscopy (FTIR). A successful process for the synthesis of Ag/Ag2O nanoparticles was obtained, following the Langmuir isotherm model, showing a high biosorption capacity of silver (49.0 mg g-1). The nanoparticles were spherical, had an average size of 11.0 nm, were synthesized intracellularly and capped by yeast proteins. This sustainable protocol is an attractive platform for the industrial-scale production of silver nanoparticles and of a silver nanobiosorbent.

Polydopamine nanocoated whole-cell asymmetric biocatalysts.

Our whole-cell biocatalyst with a polydopamine nanocoating shows high catalytic activity (5 times better productivity than the native cell) and reusability (84% of the initial yield after 5 batches, 8 times higher than the native cell) in asymmetric reduction. It also integrates with titania, silica, and magnetic nanoparticles for multi-functionalization.

Application of high-content image analysis for quantitatively estimating lipid accumulation in oleaginous yeasts with potential for use in biodiesel production.

Biodiesel from oleaginous microorganisms is a viable substitute for a fossil fuel. Current methods for microorganism lipid productivity evaluation do not analyze lipid dynamics in single cells. Here, we described a high-content image analysis (HCA) as a promising strategy for screening oleaginous microorganisms for biodiesel production, while generating single-cell lipid dynamics data in large cell density. Rhodotorula slooffiae yeast were grown in standard (CTL) or lipid trigger medium (LTM), and lipid droplet (LD) accumulation was analyzed in deconvolved confocal microscopy images of cells stained with the lipophilic fluorescent Nile red (NR) dye using automated cell and LD segmentation. The 'vesicle segmentation' method yielded valid morphometric results for limited lipid accumulation in smaller LDs (CTL samples) and for high lipid accumulation in larger LDs (LTM samples), and detected LD localization changes. Thus, HCA can be used to analyze the lipid accumulation patterns likely to be encountered in screens for biodiesel production.

Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy.

Endophytes are microorganisms that colonize plant tissues internally without causing harm to the host. Despite the increasing number of studies on sweet orange pathogens and endophytes, yeast has not been described as a sweet orange endophyte. In the present study, endophytic yeasts were isolated from sweet orange plants and identified by sequencing of internal transcribed spacer (ITS) rRNA. Plants sampled from four different sites in the state of São Paulo, Brazil exhibited different levels of CVC (citrus variegated chlorosis) development. Three citrus endophytic yeasts (CEYs), chosen as representative examples of the isolates observed, were identified as Rhodotorula mucilaginosa, Pichia guilliermondii and Cryptococcus flavescens. These strains were inoculated into axenic Citrus sinensis seedlings. After 45 days, endophytes were re-isolated in populations ranging from 10(6) to 10(9) CFU/g of plant tissue, but, in spite of the high concentrations of yeast cells, no disease symptoms were observed. Colonized plant material was examined by scanning electron microscopy (SEM), and yeast cells were found mainly in the stomata and xylem of plants, reinforcing their endophytic nature. P. guilliermondii was isolated primarily from plants colonized by the causal agent of CVC, Xylella fastidiosa. The supernatant from a culture of P. guilliermondii increased the in vitro growth of X. fastidiosa, suggesting that the yeast could assist in the establishment of this pathogen in its host plant and, therefore, contribute to the development of disease symptoms.

Responses of Candida fukuyamaensis RCL-3 and Rhodotorula mucilaginosa RCL-11 to copper stress.

The effect of high Cu(II) concentrations on superoxide dismutase (SOD) and catalase (CAT) activity in Candida fukuyamaensis RCL-3 and Rhodotorula mucilaginosa RCL-11, previously isolated from a copper filter at a mine plant in Argentina, was studied. Addition of 0.1, 0.2 and 0.5 mM Cu(II) to the culture medium increased total SOD and CAT activity in both strains. Native polyacrylamide gel electrophoresis revealed two bands with SOD activity for C. fukuyamaensis RCL-3 and only one for R. mucilaginosa RCL-11; the three bands corresponded to MnSOD.Intracellular accumulation of copper and morphological changes was observed using electron microscopy. Dark bodies examined with transmission electron microscopy (TEM) after 48 h of incubation probably corresponded to copper deposits. The number of dark bodies in R. mucilaginosa RCL-11 grew with increasing incubation time, whereas in C. fukuyamaensis RCL-3 the amount decreased. Scanning electron micrographs (SEM) of C. fukuyamaensis RCL-3 did not reveal any differences compared with the control, but R. mucilaginosa RCL-11 cells were bigger than control ones. TEM confirmed absence of compartmentalization mechanisms in Cu(II) detoxification since electron-dense bodies were mainly found in the cytoplasm.

Identification, antifungal susceptibility and scanning electron microscopy of a keratinolytic strain of Rhodotorula mucilaginosa: a primary causative agent of onychomycosis.

Onychomycosis is a dermatological problem of high prevalence that mainly affects the hallux toenail. Onychomycosis caused by the yeast Rhodotorula mucilaginosa was identified using colony morphology, light microscopy, urease and carbohydrate metabolism in a 57-year-old immunocompetent patient from Rio de Janeiro, Brazil. High-resolution scanning electron microscopy of nail fragments, processed by a noncoating method, led to the observation with fine detail of the structures of both nail and fungus involved in the infection. Yeasts were mainly found inside grooves in the nail. Budding yeasts presented a spiral pattern of growth and blastoconidia were found in the nail groove region. Keratinase assays and keratin enzymography revealed that this isolate was highly capable of degrading keratin. Antifungal susceptibility tests showed that the fungus was susceptible to low concentrations of amphotericin B and 5-flucytosine and resistant to high concentrations of fluconazole, itraconazole, voriconazole and terbinafine. These findings showed data for the first time concerning the interaction of R. mucilaginosa in toenail infection and suggest that this emerging yeast should also be considered an opportunistic primary causative agent of onychomycosis.

Rhodotorula cycloclastica sp. nov., Rhodotorula retinophila sp. nov., and Rhodotorula terpenoidalis sp. nov., three limonene-utilizing yeasts isolated from soil.

During a search for yeasts that hydroxylate monoterpenes, four yeast strains were isolated from soil and plant residue in monoterpene-rich environments using enrichment techniques with cyclohexanedioic acid or cyclohexanedimethanol as sole carbon source. These strains were able to utilize (+)-limonene supplied as a vapor as only carbon source. The yeasts have a CoQ-10 system. Morphology and physiological properties of the strains did not fit any known yeast species. Recent analysis of the 26S D1/D2 and ITS-5.8S rDNA sequences of basidiomycetous yeasts showed that these strains represented three hitherto unknown species of Rhodotorula and fell in a cluster consisting of Rhodotorula philyla and the mycoparasitic fungus Colacogloea peniophorae. Descriptions of three new species Rhodotorula cycloclastica (type strain TVN 309=UOFS Y 2046=CBS 8448), Rhodotorula retinophila (type strain TVN 295=UOFS Y 2043=CBS 8446), Rhodotorula terpenoidalis (type strain TVN 310=UOFS Y 2042=CBS 8445) are proposed to accommodate these isolates.

Fluorescence and infrared spectrometric study of cell walls from Candida, kluyveromyces, Rhodotorula and schizosaccharomyces yeasts in relation with their chemical composition.

Composition, level, and arrangement of the structural polysaccharides determine biophysical properties of fungal cell walls. A small amount of a beta(1-->4) linear homopolymer of GlcNAc in the cell wall forms chitin. To study the components of the cell walls and to estimate the quantity of chitin for different strains, two spectroscopic methods were applied. Because chemical and enzymatic methods are destructive, long, and complex, fluorescence and infrared (IR) spectroscopies were applied on cell walls and on chitin enriched fractions. The results were compared to chemical assays. IR spectra allow identifying the structural types of polysaccharides in yeast walls. Fluorescence spectroscopy was not appropriated for a full and accurate quantitative determination of the polymers but revealed level variations similar to results obtained by chemical analytical methods. The infrared spectra, using a chemometric approach (PLS1), allowed a fairly good estimation of chitin in enriched fractions with respect to the chemical assays.

Continuous production of L-phenylalanine by Rhodotorula glutinis immobilized cells using a column reactor.

Studies have been conducted on L-phenylalanine (L-Phe) production and phenylalanine ammonia lyase (PAL) stabilization in the presence of several optimum effectors and reducing agents under bioconversion of transcinnamic acid (t-CA) conditions during repeated batch operations. L-Phe production was maximized and reuseability of PAL catalyst was extended to eight consecutive cycles (repeated batches) in the presence of optimum effectors (glutamic acid, polyethylene glycol and glycerol), thioglycolic acid and sparging with nitrogen gas. These best optimum bioconversion conditions desensitize the PAL catalyst to substantially elevated higher substrate t-CA concentrations and inhibit inactivation of PAL enzyme over longer reaction periods compared to the control. The fed batch mode operation of bioconversion of total t-CA (300 mM) to L-Phe was superior (65.2%, conversion), comparing with conventional batch and repeated batch (58.4%, conversion) operations after 120 h. Gamma irradiation process was employed to polymerize and crosslink polyvinyl alcohol (PVA) with N,N'-methylene-bisacrylamide (BIS) agent. The use of immobilized PAL biocatalyst containing cells in PVA-BIS copolymer gel carrier produced by radiation polymerization is obviously advantageous with regards to the yield of L-Phe which was increased in average 1.2-fold when compare to those obtained with free cells during optimum bioconversion process. When comparing the magnitudes of gamma irradiation effects on immobilized entrapped yeast cells in PVA-BIS copolymer gel carrier using scanning electron microscopy it was show that yeast cells were protected and capable to overcome these conditions and had normal shape and other features as free (unirradiated) intact yeast cells. Optimum conditions for continuous production of L-Phe by PVA-BIS copolymer carrier entrapped yeast cells in a packed bed column reactor in recycle fed-batch mode were investigated. Under these optimum conditions L-Phe accumulated to concentration 240.1 mM represts a total conversion yield of 80% (w/w) from (300 mM) t-CA after 84 h of reaction process, which was higher than that obtained after 120 h of reaction, 65.2% (w/w) from (300 mM) t-CA with free cells in fed-batch mode. The results also demonstrated that during about 4 weeks of repeated continuous recycle fed batch mode experiments (using immobilized cells in packed bed reactor), the final production of L-Phe concentrations decreased gradually in eight consecutive runs with no sign of breakage or disintegration of the carrier gel beads.

Expression of D-amino acid oxidase in Rhodotorula gracilis under induction conditions: a biochemical and cytochemical study.

D-amino acid oxidase is expressed to a high level in the yeast Rhodotorula gracilis (0.3% of total cell protein) through induction by D-alanine in a defined growth medium. Monospecific polyclonal antibodies against pure enzyme were obtained. Western blot analysis showed that the enzyme is synthesized as the mature polypeptide. The localization of the enzyme was investigated by immunoelectron microscopy using the postembedding immunogold technique and by submicroscopic enzyme cytochemistry. D-Amino acid oxidase was detected in peroxisomes, and quantitation of immunoelectron microscopic data indicated that the enzyme is exclusively confined to these organelles. Immunoelectron microscopic observations are in complete agreement with biochemical data showing that the enzyme is not expressed in the absence of D-alanine. Morphometric analysis demonstrated that induction of D-amino acid oxidase synthesis is associated with a 241% increase of peroxisome volume density and with a 31% increase of peroxisome size as compared to cells grown on non-inducing medium.

[Yeasts utilizing the surface-active substance Laurox-9]. / O drozhzhakh, utiliziruiushchikh poverkhnostno-aktivnoe veshchestvo lauroks-9.

Yeasts capable of utilizing the non-ionogenic surfactant Laurox-9 were identified as Cryptococcus humicolus (Daszewska) Golubev and Rhodotorula mucilaginosa (Jöerg) Harrison. IR spectrometry used for the quantitative assay of Laurox-9 showed that R. mucilaginosa utilized it as a sole carbon source. The first step of Laurox-9 metabolism was its hydrolytic cleavage yielding lauric acid and polyethyleneglycol. Optimal conditions were found for the electron-cytochemical localization of hydrolase involved in the primary degradation of Laurox-9 by yeasts. Its localization of the exocellular components was established.