ABSTRACT
The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants.
Subject(s)
Cryptococcus/drug effects , Drug Resistance, Fungal , Flucytosine/pharmacology , Chromosomes, Fungal/genetics , Clone Cells , Cryptococcus/genetics , Cryptococcus/growth & development , Drug Resistance, Fungal/drug effects , Drug Resistance, Fungal/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Dosage , Gene Duplication , Gene Expression Regulation, Fungal/drug effects , Genes, Suppressor , Genetic Variation , Genome, Fungal , Intracellular Space/metabolism , Microbial Sensitivity Tests , Mutation/genetics , Reproducibility of Results , Uridine Diphosphate Glucuronic Acid/metabolismABSTRACT
The synergistic effects of carboxymethylcellulose (CMC) combined with Cryptococcus laurentii FRUC DJ1 were studied on controlling green mould resulting from Penicillium digitatum in grapefruit fruit. The results indicate that both C. laurentii and the CMC treatment suppressed P. digitatum conidia germination. In addition, C. laurentii growth in vitro was not affected by low CMC concentrations, nevertheless, the biofilm of C. laurentii was enhanced. Compared with the control fruit, the grapefruit had a lower green mould in all treatments. Significantly synergistic effects were caused by combining C. laurentii and CMC on minimum decay incidence and lesion diameter. Combined treatment induced defence enzyme activities, including chitinase, ß-1,3-glucanase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase, together with disease tolerance-associated total phenol. Also, this combination inhibited the pathogen growth by adhered to the hyphae and reduced its infection in fruit wounds. Moreover, the commercial quality parameters in the combined treatment of C. laurentii and CMC, including weight loss, total soluble solids, ascorbic acid, and titratable acidity, were superior to single treatment. The combination of C. laurentii and CMC can not only control postharvest decay but also maintain fruit qualities. Thus, it can be used in grapefruit for commercial purposes.
Subject(s)
Carboxymethylcellulose Sodium/pharmacology , Citrus paradisi/microbiology , Cryptococcus/physiology , Penicillium/pathogenicity , Plant Diseases/microbiology , Biofilms/drug effects , Biofilms/growth & development , Citrus paradisi/drug effects , Citrus paradisi/enzymology , Citrus paradisi/ultrastructure , Cryptococcus/growth & development , Penicillium/classification , Phenols/metabolismABSTRACT
Cryptococcosis is a fungal disease with worldwide distribution and wide array of clinical manifestations, caused by encapsulated basidiomycetous yeasts called Cryptococcus spp. It has traditionally been considered an opportunistic infection known to occur in immunocompromised hosts, particularly those who are infected with human immunodeficiency virus. However, this infection has also been reported in phenotypically 'normal' or otherwise clinically non-immunocompromised patients. The seemingly mysterious nature of this potentially fatal illness has always kept clinicians and diagnosticians in a dilemma. This case series reiterates this perspective.
Subject(s)
Cryptococcosis/diagnosis , Adult , Antifungal Agents/therapeutic use , Child , Cryptococcosis/drug therapy , Cryptococcosis/microbiology , Cryptococcus/growth & development , Cryptococcus/isolation & purification , Female , Humans , Male , Middle AgedABSTRACT
INTRODUCTION: Iron chelator has previously demonstrated fungicidal effects. This study aimed to investigate the antifungal activity of the iron chelators deferoxamine (DFO) and deferasirox (DSX) against Cryptococcus. MATERIALS AND METHODS: Cryptococcus neoformans and Cryptococcus gattii were used to determine the minimal inhibitory concentrations (MICs) of DFO and DSX, and the fractional inhibitory concentration index (FICI) of DFO and DSX when combined with amphotericin B (AMB). Expression of cryptococcal CFT1, CFT2, and CIR1 genes was determined using real-time polymerase chain reaction (PCR). RESULTS: Neither DFO nor DSX alone showed antifungal activity against Cryptococcus strains. When combined with AMB, the MICs of DFO and DSX decreased from>200µg/mL to 6.25 or 12.5µg/mL. The MIC of AMB decreased one-fold dilution in most strains when combined with iron chelators. The FICI of DFO+AMB and DSX+AMB was 0.5 and 1, respectively. C. neoformans showed significant growth retardation when incubated with a combination of sub-MIC concentrations of AMB and DFO; whereas, C. gattii demonstrated lesser growth retardation in DFO+AMB. No cryptococcal growth retardation was observed when DSX was combined with AMB. When C. neoformans was grown in DFO, the CFT1, CFT2, and CIR1 proteins were expressed 1.7, 2.0, and 0.9 times, respectively. When C. neoformans was grown in DSX, the CFT1, CFT2, and CIR1 genes were expressed 0.5, 0.6, and 0.3 times, respectively. CONCLUSION: Synergistic antifungal activity of combination DFO and AMB was observed in Cryptococcus. Relatively increased CFT1 and CFT2 expression may be associated with the effect of DFO that inhibits the growth of fungi.
Subject(s)
Cryptococcus/drug effects , Cryptococcus/growth & development , Cryptococcus/genetics , Iron Chelating Agents/pharmacology , Iron/metabolism , Amphotericin B/pharmacology , Antifungal Agents/pharmacology , Cryptococcosis/drug therapy , Cryptococcosis/microbiology , Cryptococcus/metabolism , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/genetics , Cryptococcus neoformans/growth & development , Cryptococcus neoformans/metabolism , Deferasirox/pharmacology , Deferoxamine/pharmacology , Drug Synergism , Fungal Capsules/drug effects , Fungal Capsules/genetics , Fungal Capsules/metabolism , Gene Expression Regulation, Fungal/drug effects , Humans , Invasive Fungal Infections/complications , Invasive Fungal Infections/drug therapy , Invasive Fungal Infections/microbiology , Iron Overload/complications , Iron Overload/drug therapy , Iron Overload/microbiology , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/genetics , Microbial Sensitivity Tests , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
The promising antimicrobial activity of essential oils (EOs) has led researchers to use them in combination with antimicrobial drugs in order to reduce drug toxicity, side effects, and resistance to single agents. Mentha x piperita, known worldwide as "Mentha of Pancalieri", is produced locally at Pancalieri (Turin, Italy). The EO from this Mentha species is considered as one of the best mint EOs in the world. In our research, we assessed the antifungal activity of "Mentha of Pancalieri" EO, either alone or in combination with azole drugs (fluconazole, itraconazole, ketoconazole) against a wide panel of yeast and dermatophyte clinical isolates. The EO was analyzed by GC-MS, and its antifungal properties were evaluated by minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) parameters, in accordance with the CLSI guidelines, with some modifications. The interaction of EO with azoles was evaluated through the chequerboard and isobologram methods. The results suggest that this EO exerts a fungicidal activity against yeasts and a fungistatic activity against dermatophytes. Interaction studies with azoles indicated mainly synergistic profiles between itraconazole and EO vs. Candida spp., Cryptococcus neoformans, and Trichophyton mentagrophytes. Thus, the "Mentha of Pancalieri" EO may act as a potential antifungal agent and could serve as a natural adjuvant for fungal infection treatment.
Subject(s)
Antifungal Agents/pharmacology , Azoles/pharmacology , Mentha piperita/chemistry , Monoterpenes/pharmacology , Oils, Volatile/chemistry , Animals , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Arthrodermataceae/drug effects , Arthrodermataceae/growth & development , Arthrodermataceae/isolation & purification , Candida/drug effects , Candida/growth & development , Candida/isolation & purification , Cryptococcus/drug effects , Cryptococcus/growth & development , Cryptococcus/isolation & purification , Drug Combinations , Drug Synergism , Fluconazole/pharmacology , Guidelines as Topic , Humans , Itraconazole/pharmacology , Ketoconazole/pharmacology , Microbial Sensitivity Tests , Monoterpenes/chemistry , Monoterpenes/isolation & purification , Oils, Volatile/isolation & purificationABSTRACT
Cryptococcosis is an opportunistic infection caused by the Cryptococcus species complex. An outbreak of cryptococcosis caused by Cryptococcus gattii (AFLP6/VGII) in North America has indicated the need for studies of this organism and its environmental niche. Difficulties in isolating the Cryptococcus spp. because of the overgrowth of filamentous fungi onto culture media and its low fungal population size under natural conditions limit studies of these pathogenic yeasts. We designed a selective medium that inhibits the growth of environmental filamentous fungi but does not inhibit that of Cryptococcus cells. After enrichment in acidified YPD media and inoculation onto selective media, Cryptococcus cells in brown-coloured colonies were isolated from environmental materials. This two-step method is useful for isolating environmental members of the Cryptococcus species complex, which is essential for further studies involving diversity and the microbe-environment relationship of this yeast.
Subject(s)
Cryptococcus/isolation & purification , Culture Media/chemistry , Environmental Microbiology , Microbiological Techniques , Cryptococcosis/microbiology , Cryptococcus/classification , Cryptococcus/growth & developmentABSTRACT
Poplar wood hemicellulose prehydrolysate was used for microbial oil production using an oleaginous microorganism Cryptococcus curvatus. Initially, the effect of substrate concentration and nitrogen content was investigated on synthetic media. Then poplar wood prehydrolysate without detoxification was used as substrate in the fermentation. The result showed that this strain is capable of consuming both hexose and pentose sugars, a challenge in fermentation of hemicellulosic streams. It was able to accumulate 36.98% of lipid and the fermentation resulted in 13.78 g/L of biomass and 5.13 g/L of lipid under optimum conditions after 164 h of fermentation. The lipid product obtained was characterized in terms of their fatty acid profiles. Overall, this study shows that it is possible to produce microbial oil from a sustainable renewable feedstock like poplar wood hemicellulose prehydrolysate. This robust strain used has the ability to grow on industrially produced hemicellulose which can help in the development of an integrated biorefinery, where all the three components of lignocellulosic biomass are utilized.
Subject(s)
Biomass , Cryptococcus/growth & development , Oils/metabolism , Polysaccharides , Wood/chemistry , Polysaccharides/chemistry , Polysaccharides/metabolismABSTRACT
Aim: This study aimed to evaluate the effects of proton pump inhibitors (PPIs) on growth and melanin production by Cryptococcus spp. Materials & methods: Minimum inhibitory concentrations (MICs) of omeprazole, esomeprazole, rabeprazole, pantoprazole and lansoprazole against Cryptococcus spp. were determined and the effect of PPIs on melanin production was evaluated, in the presence or absence of copper sulfate or glutathione. Results: PPIs showed MICs ranging from 125-1000 µg/ml and decreased melanization by Cryptococcus cells. Addition of copper sulfate or gluthatione restored melanogenesis of cells grown in the presence of PPIs. The presence of PPIs and glyphosate decreased copper sulfate toxicity (1 mM). Conclusion: PPIs inhibited melanogenesis of Cryptococcus spp., possibly by chelating copper or inhibiting copper ATPase transport.
Subject(s)
Antifungal Agents/pharmacology , Cryptococcus/drug effects , Cryptococcus/metabolism , Melanins/biosynthesis , Proton Pump Inhibitors/pharmacology , Adenosine Triphosphatases , Copper , Copper Sulfate/metabolism , Cryptococcus/growth & development , Culture Media/chemistry , Esomeprazole/pharmacology , Glutathione/metabolism , Glycine/analogs & derivatives , Humans , Lansoprazole/pharmacology , Microbial Sensitivity Tests , Omeprazole/pharmacology , Pantoprazole/pharmacology , Rabeprazole/pharmacology , GlyphosateABSTRACT
The efficiency of Cryptococcusalbidus was evaluated for its abilities to assimilate onion and apple hydrolysates as a medium for lipid production. Onion waste (OW) and apple waste (AW) were hydrolysed at an organic load of 2% total solids by indigenous microbes under mesophilic conditions. The indigenous microbes effectively hydrolysed both wastes giving the highest reducing sugar content of 4.8 g/L and 10.8 g/L with OW and AW hydrolysates, respectively. The microbiome analysis revealed that most of the indigenous microbes belonged to genus Bacillus and a significant population of α-proteobacteria and γ-proteobacteria were also present. Cell retention culture of C. albidus at a dilution rate of 0.01 h-1 resulted in a total dry cell weight (DCW) of 13.5 g/L with an intracellular lipid content of 20.0% at 168 h, corresponding to an enhancement of 3.48-folds and 2.37-folds in DCW and lipid concentration, respectively, as compared to batch fermentation.
Subject(s)
Biofuels , Carbohydrates/biosynthesis , Cryptococcus/growth & development , Lipids/biosynthesis , Medical Waste Disposal/methods , Hydrolysis , Lipids/chemistryABSTRACT
We found that a large inoculum of Cryptococcus gattii cells, when plated on Dulbecco's modified eagle's medium (DMEM) incorporated into agar, died within a few hours provided that DMEM agar plates had been stored in darkness for approximately 3 days after preparation. Standard conditions were developed for quantification of killing. The medium lost its fungicidal activity when exposed to visible light of wave length â¼400 nm. The amount of energy required was estimated at 5.8 × 104 joules @ 550 nm. Liquid DMEM conditioned by incubation over DMEM agar plates stored in darkness was fungicidal. We found that fungicidal activity was heat-stable (100°C). Dialysis tubing with MWC0 < 100 Daltons retained fungicidal activity. Neutral pH was required. Strains of Cryptococcus were uniformly sensitive, but some Candida species were resistant. Components of DMEM required for killing were pyridoxal and cystine. Micromolar amounts of iron shortened the time required for DMEM agar plates to become fungicidal when stored in the dark. Organic and inorganic compounds bearing reduced sulfur atoms at millimolar concentrations inhibited fungicidal activity. Our results point to a light-sensitive antifungal compound formed by reaction of pyridoxal with cystine possibly by Schiff base formation.
Subject(s)
Antifungal Agents/pharmacology , Cryptococcus/drug effects , Cryptococcus/radiation effects , Culture Media/chemistry , Light , Agar/chemistry , Cryptococcus/growth & development , Cystine/pharmacology , Hydrogen-Ion Concentration , Pyridoxal/pharmacology , Schiff Bases/chemistryABSTRACT
BACKGROUND: The CryptoDex trial showed that dexamethasone caused poorer clinical outcomes and slowed fungal clearance in human immunodeficiency virus-associated cryptococcal meningitis. We analyzed cerebrospinal fluid (CSF) cytokine concentrations from participants over the first week of treatment to investigate mechanisms of harm and test 2 hypotheses: (1) dexamethasone reduced proinflammatory cytokine concentrations, leading to poorer outcomes and (2) leukotriene A4 hydrolase (LTA4H) genotype influenced the clinical impact of dexamethasone, as observed in tuberculous meningitis. METHODS: We included participants from Vietnam, Thailand, and Uganda. Using the Luminex system, we measured CSF concentrations of the following: interferon γ, tumor necrosis factor (TNF) α, granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant 1, macrophage inflammatory protein 1α, and interleukin 6, 12p70, 8, 4, 10, and 17. We determined the LTA4H genotype based on the promoter region single-nucleotide polymorphism rs17525495. We assessed the impact of dexamethasone on cytokine concentration dynamics and the association between cytokine concentration dynamics and fungal clearance with mixed effect models. We measured the influence of LTA4H genotype on outcomes with Cox regression models. RESULTS: Dexamethasone increased the rate TNF-α concentration's decline in (-0.13 log2pg/mL/d (95% confidence interval, -.22 to -.06 log2pg/mL/d; P = .03), which was associated with slower fungal clearance (correlation, -0.62; 95% confidence interval, -.83 to -.26). LTA4H genotype had no statistically significant impact on outcome or response to dexamethasone therapy. Better clinical outcomes were associated with higher baseline concentrations of interferon γ. CONCLUSIONS: Dexamethasone may slow fungal clearance and worsen outcomes by increasing TNF-α concentration's rate of decline.
Subject(s)
Dexamethasone/adverse effects , Epoxide Hydrolases/genetics , Gene Expression/drug effects , Glucocorticoids/adverse effects , HIV Infections/drug therapy , Meningitis, Cryptococcal/drug therapy , Adaptor Proteins, Signal Transducing/cerebrospinal fluid , Adaptor Proteins, Signal Transducing/genetics , Chemokine CCL2/cerebrospinal fluid , Chemokine CCL2/genetics , Cryptococcus/drug effects , Cryptococcus/growth & development , Cryptococcus/pathogenicity , Epoxide Hydrolases/cerebrospinal fluid , Genotype , Granulocyte-Macrophage Colony-Stimulating Factor/cerebrospinal fluid , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , HIV/growth & development , HIV/pathogenicity , HIV Infections/complications , HIV Infections/immunology , HIV Infections/mortality , Humans , Interferon-gamma/cerebrospinal fluid , Interferon-gamma/genetics , Interleukins/cerebrospinal fluid , Interleukins/genetics , Meningitis, Cryptococcal/complications , Meningitis, Cryptococcal/immunology , Meningitis, Cryptococcal/mortality , Survival Analysis , Thailand , Treatment Outcome , Tumor Necrosis Factor-alpha/cerebrospinal fluid , Tumor Necrosis Factor-alpha/genetics , Uganda , VietnamABSTRACT
This study determined the ultraviolet-C (UV-C) dose necessary to reduce 90% population (DUV-C) of 17 spoilage yeasts and their composited inoculum in orange juice (pH 3.71, 11.60 °Brix, 0.55% citric acid, 2.46% w/v insoluble solids). Growth parameters of all test yeasts were first established to standardize the growth stage of the cells prior to harvesting and eventual UV-C challenge studies. Approximately 4-5 log CFU/ml cells in the mid-stationary growth phase (30.3 t0 39.9â¯h, 25⯰C) were suspended in 4â¯ml turbulent flowing juice and subjected to UV-C irradiation at an incident surface irradiance of 3.64-4.97â¯mW/cm2. The inactivation rates of each yeast and their composited inoculum were determined using 2 methods namely, the linear regression and Baranyi and Roberts (1994) model-fitting. Results showed that the yeasts exhibited either log-linear or biphasic inactivation behavior with downward concavity or inactivation lag. Regardless of the method of determination, Cryptococcus albidus (LJY1) exhibited the significantly greatest (pâ¯<â¯0.05) UV-C resistance with DUV-C values of 1924.31 and 2174.63â¯mJ/cm2. On the other hand, Candida parapsilosis was determined to be least resistant with a DUV-C values of 245.83 and 357.88â¯mJ/cm2. Majority of the DUV-C values determined from the model-fitting were greater than those calculated from linear regression. However, only those determined for the composited inoculum were significantly different. The results of this study address knowledge gaps pertinent to the UV-C resistance of less studied spoilage yeast, and help in better understanding the utility of this non-thermal food processing technology.
Subject(s)
Citrus sinensis/microbiology , Fruit and Vegetable Juices/microbiology , Fruit and Vegetable Juices/radiation effects , Microbial Viability/radiation effects , Ultraviolet Rays , Yeasts/radiation effects , Colony Count, Microbial , Cryptococcus/growth & development , Cryptococcus/radiation effects , Food Handling/methods , Food Microbiology/methods , Hot Temperature , Yeasts/growth & developmentABSTRACT
In the pursuit of new antifungal compounds, five coproduced lipopeptide variants (AF1 to AF5) from wild-type Bacillus subtilis RLID 12.1 were identified in our previous study. Out of five, AF4 was identified as a novel lead molecule belonging to the bacillomycin family showing less cytotoxicity at its respective minimum inhibitory concentrations (MIC) evaluated against 81 strains of Candida and Cryptococcus species (including clinical isolates); besides this, AF4 purified in the present study exhibited encouraging MIC values against 10 clinical mycelial fungi. Aiming for a selective production augmentation of AF4 lipopeptide variant, a new fermentation media comprising malt extract (1.01%), dextrose (0.55%), peptone (1.79%), MnSO4 (2 mM), and NaCl (0.5%) was formulated. Maximum production of 954.8 ± 10.8 mg/L was achieved with 44% selectivity at 30 °C compared to unoptimized conditions (186.4 ± 6.1 mg/L). Use of calcium alginate beads in the formulated media during the onset of lipopeptide production resulted in an augmentation in the selectivity of the most efficacious AF4 variant to about 72% presumably due to attenuation of other coproduced lipopeptide variants AF1 and AF2. Difference in yield of lipopeptides varied with bead size, bead preparation ratios, and sodium alginate concentrations. Use of Ca-alginate beads in the upstream production process of the lead AF4 variant may be considered as a novel strategy to address the potential challenge that may arise during the scale-up and downstream processing steps. Another significant finding derived from the study is that the proportion of bacillomycin variants of B. subtilis RLID 12.1 could be controlled by temperature and metal ions under static and shaking conditions.
Subject(s)
Alginates/chemistry , Antifungal Agents , Bacillus subtilis/chemistry , Candida/growth & development , Cryptococcus/growth & development , Peptides, Cyclic , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Antifungal Agents/pharmacology , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Peptides, Cyclic/chemistry , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/pharmacologyABSTRACT
Naturally fermented black table olives of the Gemlik variety are one of the most consumed fermented products in Turkey. The objective of this work was to identify yeast strains isolated during their natural fermentation by using Restriction Fragments Lengths Polymorphism-Polimerase Chain Reaction (RFLP-PCR) and DNA sequencing methods. The study also focused on determining the effect of regional differences on yeast microflora of naturally fermented Gemlik olives. A total of 47 yeast strains belonging to 12 different species which had been previously isolated from the natural brine of Akhisar and Iznik-Gemlik cv. olives were characterized by molecular methods. Forty-two of the tested strains could be identified by RFLP-PCR to species level. These yeast species were determined as Candida mycetangi, Candida hellenica, Candida membranaefaciens, Candida famata, Candida pelliculosa, Saccharomyces cerevisiae, and Zygosaccharomyces mrakii. Five strains were identified by DNA sequencing. These strains belonged to three different species: Aureobasidium pullulans, Kloeckera apiculate, and Cryptococcus saitoi. The most frequent species were C. famata and C. pelliculosa in both regions. PRACTICAL APPLICATION: This work studies the yeasts from Turkish table olives which could prove to be of importance to the food industry in that area. On the other hand, it compares identification by molecular and classical biochemical methods and offers an idea about the differences between the ecosystems of Gemlik olives in the Akhisar (AO) and Iznik (IO) regions. The study could be useful in characterizing a very important product and, in this way, could help to promote its marketing.
Subject(s)
DNA, Fungal/analysis , Fermentation , Food Microbiology , Olea/microbiology , Yeasts/growth & development , Base Sequence , Candida/genetics , Candida/growth & development , Candida/isolation & purification , Cryptococcus/genetics , Cryptococcus/growth & development , Cryptococcus/isolation & purification , Fruit/microbiology , Humans , Kloeckera/genetics , Kloeckera/growth & development , Kloeckera/isolation & purification , Pichia/isolation & purification , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/isolation & purification , Salts , Turkey , Yeasts/genetics , Yeasts/isolation & purification , Zygosaccharomyces/genetics , Zygosaccharomyces/growth & development , Zygosaccharomyces/isolation & purificationABSTRACT
Multiple species within the basidiomycete genus Cryptococcus cause cryptococcal disease. These species are estimated to affect nearly a quarter of a million people leading to â¼180,000 mortalities, annually. Sexual reproduction, which can occur between haploid yeasts of the same or opposite mating type, is a potentially important contributor to pathogenesis as recombination can generate novel genotypes and transgressive phenotypes. However, our quantitative understanding of recombination in this clinically important yeast is limited. Here, we describe genome-wide estimates of recombination rates in Cryptococcus deneoformans and compare recombination between progeny from α-α unisexual and a-α bisexual crosses. We find that offspring from bisexual crosses have modestly higher average rates of recombination than those derived from unisexual crosses. Recombination hot and cold spots across the C. deneoformans genome are also identified and are associated with increased GC content. Finally, we observed regions genome-wide with allele frequencies deviating from the expected parental ratio. These findings and observations advance our quantitative understanding of the genetic events that occur during sexual reproduction in C. deneoformans, and the impact that different forms of sexual reproduction are likely to have on genetic diversity in this important fungal pathogen.
Subject(s)
Cryptococcus/genetics , Homologous Recombination , Meiosis/genetics , Cryptococcus/growth & development , Genetic Linkage , Genome, FungalABSTRACT
The potential of lipid accumulation by oleaginous yeast Cryptococcus vishniaccii grown on amaranth seed aqueous extract (AAE) media was assessed. Maximum cell biomass productivity of 104 mg/L/h, lipid productivity of 54 mg/L/h, and lipid content of 52.31% were recorded on AAE when carbon to nitrogen (C:N) ratio increased from 134 to 147 after removal of ammonia nitrogen. The lipid droplet (LD) size (2.32 ± 0.38 µm) was visualized by fluorescence microscopy using Nile red stain indicating maximum accumulated triacylglycerol (TAG) at C:N 147. Fatty acid methyl ester (FAME) profile obtained after transesterification of extracted lipid revealed the presence of palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2). Data showed the presence of high monounsaturated fatty acid (MUFA) content (68.17%) depicting improved winter operating conditions of biodiesel. Various quality parameters of biodiesel were evaluated and compared to the American and European biodiesel standards specifications. Based on the lipid productivity, distribution of fatty acids, and evaluated properties obtained; the lipid accumulation by C. vishniaccii utilizing amaranth seeds as substrate could serve as a feasible feedstock for biodiesel production.
Subject(s)
Amaranthus/microbiology , Biofuels/analysis , Biotechnology/methods , Cryptococcus/metabolism , Seeds/microbiology , Amaranthus/chemistry , Biomass , Cryptococcus/growth & development , Fatty Acids/analysis , Lipid Droplets/metabolism , Plant Extracts/chemistry , Seeds/chemistryABSTRACT
Species within the human pathogenic Cryptococcus species complex are major threats to public health, causing approximately 1 million annual infections globally. Cryptococcus amylolentus is the most closely known related species of the pathogenic Cryptococcus species complex, and it is non-pathogenic. Additionally, while pathogenic Cryptococcus species have bipolar mating systems with a single large mating type (MAT) locus that represents a derived state in Basidiomycetes, C. amylolentus has a tetrapolar mating system with 2 MAT loci (P/R and HD) located on different chromosomes. Thus, studying C. amylolentus will shed light on the transition from tetrapolar to bipolar mating systems in the pathogenic Cryptococcus species, as well as its possible link with the origin and evolution of pathogenesis. In this study, we sequenced, assembled, and annotated the genomes of 2 C. amylolentus isolates, CBS6039 and CBS6273, which are sexual and interfertile. Genome comparison between the 2 C. amylolentus isolates identified the boundaries and the complete gene contents of the P/R and HD MAT loci. Bioinformatic and chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that, similar to those of the pathogenic Cryptococcus species, C. amylolentus has regional centromeres (CENs) that are enriched with species-specific transposable and repetitive DNA elements. Additionally, we found that while neither the P/R nor the HD locus is physically closely linked to its centromere in C. amylolentus, and the regions between the MAT loci and their respective centromeres show overall synteny between the 2 genomes, both MAT loci exhibit genetic linkage to their respective centromere during meiosis, suggesting the presence of recombinational suppressors and/or epistatic gene interactions in the MAT-CEN intervening regions. Furthermore, genomic comparisons between C. amylolentus and related pathogenic Cryptococcus species provide evidence that multiple chromosomal rearrangements mediated by intercentromeric recombination have occurred during descent of the 2 lineages from their common ancestor. Taken together, our findings support a model in which the evolution of the bipolar mating system was initiated by an ectopic recombination event mediated by similar repetitive centromeric DNA elements shared between chromosomes. This translocation brought the P/R and HD loci onto the same chromosome, and further chromosomal rearrangements then resulted in the 2 MAT loci becoming physically linked and eventually fusing to form the single contiguous MAT locus that is now extant in the pathogenic Cryptococcus species.
Subject(s)
Cryptococcus/cytology , Cryptococcus/genetics , Genes, Mating Type, Fungal , Genome, Fungal , Meiosis , Translocation, Genetic , Chromatin Immunoprecipitation , Computational Biology , Crossing Over, Genetic , Cryptococcus/growth & development , Cryptococcus/physiology , Cryptococcus neoformans/cytology , Cryptococcus neoformans/genetics , Cryptococcus neoformans/physiology , Epistasis, Genetic , Evolution, Molecular , Genetic Linkage , Genetic Loci , Genetic Structures , Linkage Disequilibrium , Molecular Sequence Annotation , Recombination, Genetic , Sequence Analysis, RNA , Species Specificity , SyntenyABSTRACT
Tailoring lipids from oleaginous yeasts to contain specific types of fatty acid is of considerable interest to food, fuel, and pharmaceutical industries. In this study, the essential oil obtained from Citrus sinesus L. has been used to alter the fatty acid composition of two common oleaginous yeasts, Rhodosporidium toruloides and Cryptococcus curvatus. With increasing levels of essential oil in the medium, the metabolic flux of the fatty acid biosynthesis pathway shifted towards saturated fatty acid production. Essential oil reduced the activities of elongase and ∆9 desaturase. This made the lipid obtained from both these yeasts rich in saturated fatty acids. At certain specific concentrations of the essential oil in the medium, the lipid obtained from R. toruloides and C. curvatus cultures was similar to mahuwa butter and palm oil, respectively. Limonene is the major constituents of orange essential oil. Its effect on one of the oleaginous yeasts, R. toruloides, was also studied separately. Effects similar to orange essential oil were obtained with limonene. Thus, we can conclude that limonene in orange essential oil brings about compositional change of microbial lipid produced in this organism.
Subject(s)
Citrus sinensis/chemistry , Cryptococcus/growth & development , Fatty Acids/biosynthesis , Oils, Volatile/pharmacology , Oils, Volatile/chemistryABSTRACT
The increasing demand of plant oils for biodiesel production has highlighted the need for alternative strategies based either on non-food crops or agro-industrial wastes that do not compete with food and feed production. In this context, the combined use of wastewater and oleaginous microorganisms could be a valuable production option. Ricotta cheese whey (RCW), one of the major byproducts of the dairy industry, is produced in very high and steadily increasing amounts and, due to its high organic load, its disposal is cost-prohibitive. In the present study, in order to assess the adequacy of RCW as a growth medium for lipid production, 18 strains of oleaginous yeasts were investigated in shaken flask for their growth and lipid-producing capabilities on this substrate. Among them, Cryptococcus curvatus NRRL Y-1511 and Cryptococcus laurentii UCD 68-201 adequately grew therein producing substantial amounts of lipids (6.8 and 5.1gL-1, respectively). A high similarity between the percent fatty acid methyl esters (FAME) composition of lipids from the former and the latter strain was found with a predominance of oleic acid (52.8 vs. 48.7%) and of total saturated fatty acids (37.9 vs. 40.8%). The subsequent scale transfer of the C. laurentii UCD 68-201 lipid production process on RCW to a 3-L STR led to significantly improved biomass and total lipid productions (14.4 and 9.9gL-1, respectively) with the biodiesel yield amounting to 32.6%. Although the C. laurentii FAME profile was modified upon process transfer, it resembled that of the Jatropha oil, a well established feedstock for biodiesel production. In conclusion, C. laurentii UCD 68-201, for which there is very limited amount of available information, turned out to be a very promising candidate for biodiesel production and wide margins of process improvement might be envisaged.
Subject(s)
Biofuels , Cryptococcus/growth & development , Whey , Biomass , Cheese , Fatty Acids/chemistry , Lipids/chemistryABSTRACT
BACKGROUND: Ebselen, an organoselenium compound and a clinically safe molecule has been reported to possess potent antifungal activity, but its antifungal mechanism of action and in vivo antifungal activity remain unclear. METHODS: The antifungal effect of ebselen was tested against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, and C. gattii clinical isolates. Chemogenomic profiling and biochemical assays were employed to identify the antifungal target of ebselen. Ebselen's antifungal activity in vivo was investigated in a Caenorhabditis elegans animal model. RESULTS: Ebselen exhibits potent antifungal activity against both Candida spp. and Cryptococcus spp., at concentrations ranging from 0.5 to 2µg/ml. Ebselen rapidly eradicates a high fungal inoculum within 2h of treatment. Investigation of the drug's antifungal mechanism of action indicates that ebselen depletes intracellular glutathione (GSH) levels, leading to increased production of reactive oxygen species (ROS), and thereby disturbs the redox homeostasis in fungal cells. Examination of ebselen's in vivo antifungal activity in two Caenorhabditis elegans models of infection demonstrate that ebselen is superior to conventional antifungal drugs (fluconazole, flucytosine and amphotericin) in reducing Candida and Cryptococcus fungal load. CONCLUSION: Ebselen possesses potent antifungal activity against clinically relevant isolates of both Candida and Cryptococcus by regulating GSH and ROS production. The potent in vivo antifungal activity of ebselen supports further investigation for repurposing it for use as an antifungal agent. GENERAL SIGNIFICANCE: The present study shows that ebselen targets glutathione and also support that glutathione as a potential target for antifungal drug development.