Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide (R)-Tebuconazole in High Optical Purity Mediated by an Epoxide Hydrolase from Rhodotorula paludigensis.

Tebuconazole is a chiral triazole fungicide used globally in agriculture as a racemic mixture, but its enantiomers exhibit significant enantioselective dissimilarities in bioactivity and environmental behaviors. The steric hindrance caused by the tert-butyl group makes it a great challenge to synthesize tebuconazole enantiomers. Here, we designed a simple chemoenzymatic approach for the asymmetric synthesis of (R)-tebuconazole, which includes the biocatalytic resolution of racemic epoxy-precursor (2-tert-butyl-2-[2-(4-chlorophenyl)ethyl] oxirane, rac-1a) by Escherichia coli/Rpeh whole cells expressed epoxide hydrolase from Rhodotorula paludigensis (RpEH), followed by a one-step chemocatalytic synthesis of (R)-tebuconazole. It was observed that (S)-1a was preferentially hydrolyzed by E. coli/Rpeh, whereas (R)-1a was retained with a specific activity of 103.8 U/g wet cells and a moderate enantiomeric ratio (E value) of 13.4, which was remarkably improved to 43.8 after optimizing the reaction conditions. Additionally, a gram-scale resolution of 200 mM rac-1a was performed using 150 mg/mL E. coli/Rpeh wet cells, resulting in the retention of (R)-1a in a 97.0% ees, a 42.5% yields, and a 40.5 g/L/d space-time yield. Subsequently, the synthesis of highly optical purity (R)-tebuconazole (>99% ee) was easily achieved through the chemocatalytic ring-opening of the epoxy-precursor (R)-1a with 1,2,4-triazole. To elucidate insight into the enantioselectivity, molecular docking simulations revealed that the unique L-shaped substrate-binding pocket of RpEH plays a crucial role in the enantioselective recognition of bulky 2,2-disubstituted oxirane 1a.

Formation of Sb2O3 microcrystals by Rhodotorula mucilaginosa.

Antimony (Sb) pollution seriously endangers ecological environment and human health. Microbial induced mineralization can effectively convert metal ions into more stable and less soluble crystalline minerals by extracellular polymeric substance (EPS). In this study, an efficient Sb-resistant Rhodotorula mucilaginosa (R. mucilaginosa) was screened, which can resist 41 mM Sb(III) and directly transform Sb(III) into Sb2O3 microcrystals by EPS. The removal efficiency of R. mucilaginosa for 22 mM Sb(III) reached 70% by converting Sb(III) to Sb2O3. The components of supernatants as well as the effects of supernatants and pH on Sb(III) mineralization verified that inducible and non-inducible extracellular protein/polysaccharide biomacromolecules play important roles in the morphologies and sizes control of Sb2O3 formed by R. mucilaginosa respectively. Sb2O3 microcrystals with different morphologies and sizes can be prepared by the regulation of inducible and non-inducible extracellular biomacromolecules secreted by R. mucilaginosa. This is the first time to identify that R. mucilaginosa can remove Sb(III) by transforming Sb(III) into Sb2O3 microcrystals under the control of EPS. This study contributes to our understanding for Sb(III) biomineralization mechanisms and provides strategies for the remediation of Sb-contaminated environment.

The mitochondrial respiratory chain from Rhodotorula mucilaginosa, an extremophile yeast.

Rhodotorula mucilaginosa survives extreme conditions through several mechanisms, among them its carotenoid production and its branched mitochondrial respiratory chain (RC). Here, the branched RC composition was analyzed by biochemical and complexome profiling approaches. Expression of the different RC components varied depending on the growth phase and the carbon source present in the medium. R. mucilaginosa RC is constituted by all four orthodox respiratory complexes (CI to CIV) plus several alternative oxidoreductases, in particular two type-II NADH dehydrogenases (NDH2) and one alternative oxidase (AOX). Unlike others, in this yeast the activities of the orthodox and alternative respiratory complexes decreased in the stationary phase. We propose that the branched RC adaptability is an important factor for survival in extreme environmental conditions; thus, contributing to the exceptional resilience of R. mucilaginosa.

Fabrication and characterization of biaxially electrospun collagen/alginate nanofibers, improved with Rhodotorula mucilaginosa sp. GUMS16 produced exopolysaccharides for wound healing applications.

Fabrication of scaffolds with enhanced mechanical properties and desirable cellular compatibility is critical for numerous tissue engineering applications. This study was aimed at fabrication and characterization of a nanofiber skin substitute composed of collagen (Col)/sodium alginate (SA)/ polyethylene oxide (PEO)/Rhodotorula mucilaginosa sp. GUMS16 produced exopolysaccharides (EPS) were prepared using the biaxial electrospinning technique. This study used collagen extracted from the bovine tendon as a natural scaffold, sodium alginate as an absorber of excess wound fluids, and GUMS16 produced exopolysaccharides as an antioxidant. Collagen was characterized using FTIR and EDS analyses. The cross-linked nanofibers were characterized by SEM, FTIR, tensile, contact-angle, swelling test, MTT, and cell attachment techniques. The average diameter of Col nanofiber was 910 ± 89 nm. The Col and Col-SA/PEO non-woven mats' water contact angle measurement was 41.6o and 56.4o, Col/EPS1%, Col/EPS2%, Col-SA/PEO + EPS1%, and Col-SA/PEO + EPS2% were 61.4o, 58.3o, 38.5o, and 50.6o, respectively. Cell viability of more than 100% was shown in Col-SA/PEO + EPS nanofibers. Also, SEM images of cells on nanofiber scaffolds demonstrated that all nanofibers incorporated with GUMS16-produced EPS have good cell growth and proliferation. The acquired results expressed that the GUMS16-produced EPS can be considered a novel biomacromolecule in electrospun fibers that increase cell viability and proliferation.

Biosurfactant production by halophilic yeasts isolated from extreme environments in Botswana.

Nine morphologically distinct halophilic yeasts were isolated from Makgadikgadi and Sua pans, as pristine and extreme environments in Botswana. Screening for biosurfactant production showed that Rhodotorula mucilaginosa SP6 and Debaryomyces hansenii MK9 exhibited the highest biosurfactant activity using Xanthocercis zambesiaca seed powder as a novel and alternative inexpensive carbon substrate. Chemical characterization of the purified biosurfactants by Fourier Transform Infra-Red spectroscopy suggested that the biosurfactant from R. mucilaginosa SP6 was a rhamnolipid-type whereas the biosurfactant from D. hansenii MK9 was a sophorolipid-type. The two biosurfactants exhibited antimicrobial activities against eight pathogenic bacteria and fungal strains (Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Micrococcus luteus, Cryptococcus neoformans, Candida albicans and Aspergilus niger). The sophorolopid-type biosurfactant was found to be the most potent among the antimicrobial drug resistant strains tested. The findings open up prospects for the development of environmentally friendly antimicrobial drugs that use an inexpensive source of carbon to reduce the costs associated with the production of biosurfactants.

Harvesting of Rhodotorula glutinis via Polyaluminum Chloride or Cationic Polyacrylamide Using the Extended DLVO Theory.

Polyaluminum chloride (PAC) and cationic polyacrylamide (CPAM) play a crucial role for separating microorganisms from bulk media. However, the mechanism of adsorption between cells and flocculants remain to be further defined to improve the flocculation efficiency (FE) in extreme conditions. This study conducted the flocculation process of Rhodotorula glutinis induced by PAC and CPAM, firstly. The result demonstrated that CPAM possessed more efficient harvesting ability for R. glutinis compared to PAC. The difference of flocculation capacity was then thermodynamically explained by the extended DLVO (eDLVO) theory; it turned out that the poor harvesting efficiency of PAC was attributed to lacking of binding sites as well as low adsorption force within particles. Based on this, the FE of PAC to R. glutinis was mechanically enhanced to 99.84% from 32.89% with 0.2 g/L CPAM modification at an optimum pH of 9. Also, the paper will play a guiding role in the treatment of inorganic salt ions and organic matters in wastewater.

Conversion of Racemic Unnatural Amino Acids to Optically Pure Forms by a Coupled Enzymatic Reaction.

Genetic code expansion (GCE) technology is a useful tool for the site-specific modification of proteins. An unnatural amino acid (UAA) is one of the essential components of this technique, typically required at high concentration (1 mM or higher) in growth medium. The supply of UAAs is an important limitation to the application of GCE technology, as many UAAs are either expansive or commercially unavailable. In this study, two UAAs in a racemic mixture were converted into optically pure forms using two enzymes, the d-amino acid oxidase (RgDAAO) from Rhodotorula gracilis and the aminotransferase (TtAT) from Thermus thermophilus. In the coupled enzyme system, RgDAAO oxidizes the d-form of UAAs in a stereospecific manner and produces the corresponding α-keto acids, which are then converted into the l-form of UAAs by TtAT, resulting in the quantitative and stereospecific conversion of racemic UAAs to optically pure forms. The genetic incorporation of the optically pure UAAs into a target protein produced a better protein yield than the same experiments using the racemic mixtures of the UAAs. This method could not only be used for the preparation of optically pure UAAs from racemic mixtures, but also the broad substrate specificity of both enzymes would allow for its expansion to structurally diverse UAAs.

Production, purification, characterization, and biological properties of Rhodosporidium paludigenum polysaccharide.

The yield of marine red yeast polysaccharide (MRYP) obtained from Rhodosporidium paludigenum was increased by optimizing fermentation conditions, and the pure polysaccharide was extracted by column chromatography. The molecular weight of pure MRYP and the ratio of mannose to glucose in components of MRYP were determined. Antioxidant and antibacterial abilities of MRYP were investigated in vitro and in vivo. The optimal fermentation parameters were as follows: Medium 4, pH = 6.72, temperature = 30.18°C, blades speed = 461.36 r/min; the optimized yield reached 4323.90 mg/L, which was 1.31 times the original yield. The sequence of factors that affected the MRYP yield was the blades speed>pH>temperature. The main components of MRYP were MYH-1 and MYH-2. The molecular weights of MYH-1 and MYH-2 were 246.92 kDa and 21.88 kDa, respectively; they accounted for 53.60% and 28.75% of total polysaccharide. In MYH-1 and MYH-2, the proportion of glucose and mannose accounted for 46.94%, 38.46%, and 67.10%, 7.17%, respectively. In vitro, the ability of scavenging DPPH•, •OH, and [Formula: see text] radical was 32.26%, 24.34%, and 22.09%; the minimum inhibitory concentration (MIC) of MRYP was 480 µg/mg. In vivo, MRYP improved the lambs' body weight, antioxidant enzyme activity, and the number of probiotics, but it reduced the feed/gain (F/G) ratio and the number of pathogenic bacteria in 60-days-old lambs.

The characteristics of gelation of myofibrillar proteins combined with salt soluble Rhodotorula glutinis proteins by enzymatic crosslinking.

Some microbial single-cell proteins are capable of producing synergistic crosslinking interactions with edible proteins by rational regulation. Herein, we reported that salt soluble proteins (RGP) extracted from Rhodotorula glutinis in an alkaline and saline system may combine with myofibrillar proteins (MP) by transglutaminase (TG) polymerization to form improvable irreversible thermal co-gels. The combination of MP, RGP, and TG, namely restructured MP gels, led to significantly enhanced water holding capacity (WHC), up to 90.76 ± 1.88% (% of retained water) and textural properties (hardness, springiness, and adhesiveness) as well as decreases of 'gauche-gauche-gauche' SS bonds and α-helix conformations and increases of 'gauche-gauche-trans' SS bonds and ß-fold conformations, compared to MP and MP-RGP groups. Differential scanning calorimetry analysis validated that thermostability of myosins and actins from MP was reduced after using RGP, TG, and their combination, and unfolding and denaturation of myosin occurred easily during thermal co-gelation by TG and/or RGP.

Comparison of simple and rapid cell wall disruption methods for improving lipid extraction from yeast cells.

The present study examined the effect of six disruption methods of the cell wall (acid hydrolysis, ultrasonication, osmotic shock, pasteurization, homogenization with zirconia balls, and freezing/defrosting) on the efficiency of lipid extraction from yeast cells and the composition of fatty acids. Acid hydrolysis and sonication led to a significant increase in lipid extraction from Cyberlindnera jadinii ATCC 9950 and Rhodotorula glutinis LOCKR13 yeast cells. The amount of lipids extracted in these conditions increased for C. jadinii from 12.46 (biomass not subjected to any pretreatment) to 20.37 and 19.53 g/100 gd.w. after the application of acid hydrolysis and sonication, respectively, and for R. glutinis strain from 13.95 to 21.20 and 17.22 g/100 gd.w., respectively, for the same methods. Initial sonication of biomass led to a significant reduction in the percentage of unsaturated fatty acids. The largest differences in fatty acid composition were found for the sample homogenized with zirconium balls. This process resulted in the degradation of both oleic acid and linolenic acid. The obtained results revealed that the method that significantly increases lipid extraction and does not change the composition of fatty acids is acid hydrolysis with hydrochloric acid. In addition, it is easy, cheap, does not require specialized equipment, and therefore can be implemented in any laboratory.

Surfactant-assisted in situ transesterification of wet Rhodotorula glutinis biomass.

In situ transesterification of oleaginous microbes with short chain alcohol has been developed as a renewable process for the production of biodiesel. Dry biomass is often a requisite for the process to avoid the adverse effect of water on the productivity. As a consequence, large amount of energy consumption is required for prior biomass drying. In this study, the wet biomass of Rhodotorula glutinis, an oleaginous yeast, was used directly in in situ transesterification without biomass drying. The reaction conditions were optimized for the production of fatty acid methyl esters (FAME) and the effects of adding different surfactants were also studied. The highest FAME yield of 110% was achieved with a methanol loading of 1:100 at 90°C for 8 h as catalyzed by 0.36 M H2SO4, and the FAME content was 97%, which meets the 96.5% specified in both European biodiesel standards and Taiwanese biodiesel standards. The addition of 50 mM 3-(N,N-dimethylmyristylammonio)propanesulfonate (3-DMAPS, a zwitterionic surfactant) improved the FAME yield from 69% to 83%, which was obtained with a low methanol loading of 1:10 at 90°C for 10 h. Hence, the production of FAME with wet biomass under optimized reaction conditions was as effective as that with the dry form. This clearly indicates that using wet R. glutinis as the feedstock is feasible for the production of biodiesel by in situ transesterification.

Automated Pressurized Liquid Extraction of Microbial Lipids from Oleaginous Yeasts.

The lipids produced by oleaginous microbes are considered sustainable resources for biofuels. To facilitate controlled lipid production and lipid analysis, more efficient lipid extraction methods are required. This study describes the automated pressurized liquid extraction (APLE) method for lipid extraction from dried cells of the oleaginous yeast species Rhodosporidium toruloides and Cryptococcus curvatus. Cells were mixed with diatomite in a mortar, added to the sample chamber, and treated with a mixture of chloroform and methanol at 105 °C. More than 95% lipids were extracted. Analysis by using high-performance thin-layer chromatography showed that the neutral lipid contents in the obtained samples by APLE method were similar to those by the ball milling-assisted extraction method. The lipids had an essentially identical fatty acid composition compared with lipids extracted with the acid-heating extraction (AHE) method. This demonstrated that lipids can be efficiently extracted from oleaginous yeasts in less time and without harsh pretreatment procedures.

Antibacterial and Antibiofilm Activity of Biosynthesized Silver Nanoparticles Coated With Exopolysaccharides Obtained From Rhodotorula mucilaginosa.

Antibacterial resistance is one of the biggest threats to health and economy worldwide. In the present work, we evaluated the antibacterial and antibiofilm properties of silver nanoparticles produced by green synthesis with exopolysaccharides produced by Rhodotorula mucilaginosa UANL-001L, against pathogens of clinical importance. The extraction of exopolysaccharides produced by Rhodotorula mucilaginosa was performed according to a previously described method. Synthesis of the nanobiocomposite was performed by mixing silver nitrate, Acacia rigidula, and Rhodotorula mucilaginosa-produced exopolysaccharides. The newly synthesized nanobiocomposite was lyophilized and kept frozen for further analysis. The characterization of the nanobiocomposite was carried out by UV-visible spectroscopic, Fourier transform infrared analysis and the surface morphology was analyzed by Transmission Electron Microscopy. The antibacterial and antibiofilm activity was tested in 96-well plates for different concentrations of the nanobiocomposite against Escherichia. coli ATCC 11229, Staphylococcus aureus ATCC 6538, and Pseudomonas aeruginosa ATCC 27853. The Rhodotorula mucilaginosa- produced exopolysaccharides were useful in the silver nanoparticle synthesis and the resulting nanobiocomposite had antibacterial and antibiofilm activity against Gram-positive and Gram-negative bacteria at lower concentrations than previously reported. Due to these properties, the nanobiocomposite seems to be a promising biocide against pathogens of clinical relevance. In addition, the nanobiocomposite proved to be advantageous in the formulation of hybrid metal-polymer coatings for medical devices or industrial settings.

Production, characterization and biological activities of exopolysaccharides from a new cold-adapted yeast: Rhodotorula mucilaginosa sp. GUMS16.

Lately, it has been proved that yeast exopolysaccharides (EPS) are potentially applicable biopolymers, a fact that has led to incremental needs for their assessment. The current study is based on the biochemical and molecular level identification of the novel cold-adapted yeast Rhodotorula mucilaginosa sp. GUMS16. Possible antioxidant and antiproliferative activities, as well as extraction and characterization of the GUMS16-produced EPS, were assessed during the course of this study. The results indicated that the strain of GUMS16 is a cold-adapted yeast with growth capability at 4 °C and an approximate EPS production yield of 28.5 g/L which are characterized as highly branched beta-D-glucan having glucose and mannose residues (85:15 mol%, respectively) with an average molecular weight of 84 kDa. In comparison to hyaluronic acid, DPPH, and OH, the scavenging activity attributed to the GUMS16-produced EPS was higher alongside being dose-dependent. The biocompatibility profile of the EPS was well-recognized based on its zero-cytotoxicity rate on a normal cell model. Collectively, the favorable properties of the EPS accentuate their potential as biocompatible compound suitable for subsequent pharmaceutical and industrial applications.

Fatty acids profiles and estimation of the biodiesel quality parameters from Rhodotorula spp. from Antarctica.

OBJECTIVE: Oleaginous yeasts are a renewable and alternative source of oil for third-generation biodiesel. This work aimed to evaluate the effects of glucose concentration (30-100 g L-1) on growth, lipid synthesis, and fatty acids (FA) profile of three Rhodotorula spp. (R. glacialis R15, R. glutinis R4, and R. glutinis R48) isolated from Antarctica, and estimate the key quality parameters of the biodiesel produced by yeasts to confirm their potential as feedstocks for third-generation biodiesel synthesis. RESULTS: Yeasts accumulated 50-69.5% of lipids (w/w) under nitrogen-limitation and glucose-excess (C/N = 40-133). Glucose concentration increase influenced positively lipid accumulation (69.5% w/w) and FA profile of R. glacialis R15. Lipid accumulation (53% on average) of R. glutinis strains was not significantly affected by glucose concentration; content of saturated (~ 30%) and polyunsaturated FA (~ 29-30%) was slightly influenced. FA profiles of lipids synthesized by R15, R4, and R48 are similar to vegetable oils used in biodiesel industry with C16 and C18 FA (95-99%) as the major components, and contain mainly oleic (C18:1), palmitic (C16:0), and linoleic (C18:2) acids, which are suitable for biodiesel synthesis. Estimated fuel properties for biodiesel produced by R15, R4, and R48 satisfied all the criteria established by ASTM D6751 and EN 14214 with good cetane number, iodine value, and oxidation stability. An improvement in biodiesel quality of R15 was observed with the glucose increase. The best global properties of biodiesel from R4 were obtained with 30 g L-1 of glucose. CONCLUSIONS: Rhodotorula spp. from Antarctica are promising candidates for third-generation biodiesel synthesis.

Organic-solvent-free extraction of carotenoids from yeast Rhodotorula glutinis by application of ultrasound under pressure.

The extraction of Rhodotorula glutinis carotenoids by ultrasound under pressure (manosonication) in an aqueous medium has been demonstrated. The influence of treatment time, pressure, and ultrasound amplitude on R. glutinis inactivation and on the extraction of carotenoids was evaluated, and the obtained data were described mathematically. The extraction yields were lineal functions of those three parameters, whereas inactivation responded to a more complex equation. Under optimum treatment conditions, 82% of carotenoid content was recovered. Extraction of carotenoids in an aqueous medium was attributed to the capacity of ultrasound for cell disruption and emulsification. Cavitation caused the rupture of cell envelopes and the subsequent formation of small droplets of carotenoids surrounded by the phospholipids of the cytoplasmic membrane that would stabilize the emulsion. Analysis of the dispersed particle size of the extracts demonstrated that a fine, homogeneous emulsion was formed after treatment (average size: 230 nm; polydispersity <0.22). This research describes an innovative green process for extracting carotenoids from fresh biomass of R. glutinis in which only two unit operations are required: ultrasonic treatment, followed by a centrifugation step to discard cell debris. The extract obtained thanks to this procedure is rich in carotenoids (25 mg/L) and could be directly incorporated as a pigment in foods, beverages, and diet supplements; it can also be utilized as an ingredient in drugs or cosmetics.

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.

Cell Envelope Integrity and Capsule Characterization of Rhodotorula mucilaginosa Strains from Clinical and Environmental Sources.

Rhodotorula yeasts are pink, encapsulated basidiomycetes isolated from a variety of environments and clinical settings. They are increasingly linked with disease, particularly central venous catheter infections and meningitis, in immunocompromised patients. Eight clinical and eight environmental strains molecularly typed as Rhodotorula mucilaginosa were compared to six Cryptococcus neoformans strains for phenotypic variability. Growth on cell integrity-challenging media suggested that R. mucilaginosa cells possess differences in signaling pathways, cell wall composition, or assembly and that their membranes are more susceptible to perturbations than those of C. neoformans All 16 R. mucilaginosa strains produced urease, while none produced melanin with l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. India ink staining reveals that clinical R. mucilaginosa capsules are larger than environmental capsules but that both are generally smaller than C. neoformans capsules. All R. mucilaginosa strains were resistant to fluconazole. Only two clinical strains were susceptible to voriconazole; all of the environmental strains were resistant. We generated an anticapsular antibody (Rh1) to R. mucilaginosa; Rh1 did not bind C. neoformans control strains, was specific to Rhodotorula species, and bound to all tested Rhodotorula strains. Binding assays performed with wheat germ agglutinin (WGA), concanavalin A (ConA), calcofluor white (CFW), and eosin Y dye (EY) cell surface probes suggested that chitin may be more accessible in R. mucilaginosa but that the total abundance of chitooligomers is less than in C. neoformans This report describes a novel reagent that can be used to identify Rhodotorula species and lays the foundation for future cell envelope composition analysis.IMPORTANCE Currently, there is very little known about the phenotypic variability within species of Rhodotorula strains and the role of their capsule. Cryptococcus neoformans has been considered the only encapsulated human fungal pathogen, but as more individuals come to live in states of immunocompromised health, they are more susceptible to fungal infections, including those by RhodotorulaR. mucilaginosa species are some of those most commonly associated with clinical infections. We wanted to know if clinical and environmental strains of R. mucilaginosa demonstrated disparate capsule phenotypes. With limited antifungal options available and clinical Rhodotorula spp. often resistant to common antifungal drugs such as fluconazole, caspofungin (1, 2), and voriconazole (2), a better understanding of the fungal biology could inform the design and use of future antifungal drugs. The generation of an antibody specific to Rhodotorula fungi could be a useful diagnostic tool, and this work presents the first mention of such in the literature.

Toward a rapid method for the study of biodiversity in cold environments: the characterization of psychrophilic yeasts by MALDI-TOF mass spectrometry.

To investigate the potential of matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) as a platform to support biodiversity and phylogenetic studies of psychrophilic yeasts in cold environments, the technique was employed to rapidly characterize and distinguish three psychrophilic yeasts (Rhodotorula mucilaginosa, Naganishia vishniacii, and Dioszegia cryoxerica) from three mesophilic counterparts (Saccharomyces cerevisiae Cry Havoc, S. cerevisiae California V Ale, and S. pastorianus). A detailed workflow for providing reproducible mass spectral fingerprints of low molecular weight protein/peptide features specific to the organisms studied is presented. The potential of this approach as a tool in the study of biodiversity, systematics, and phylogeny of psychrophilic microorganisms is highlighted.