Antifungal drug resistance is a significant clinical problem, and antifungal agents that can evade resistance are urgently needed. In infective niches, resistant organisms often co-existed with sensitive ones, or a subpopulation of antibiotic-susceptible organisms may evolve into resistant ones during antibiotic treatment and eventually dominate the whole population. In this study, we established a co-culture assay in which an azole-resistant Candida albicans strain was mixed with a susceptible strain labeled with green fluorescent protein to mimic in vivo conditions and screen for antifungal drugs. Fluconazole was used as a positive control to verify the validity of this co-culture assay. Five natural molecules exhibited antifungal activity against both susceptible and resistant C. albicans. Two of these compounds, retigeric acid B (RAB) and riccardin D (RD), preferentially inhibited C. albicans strains in which the efflux pump MDR1 was activated. This selectivity was attributed to greater intracellular accumulation of the drugs in the resistant strains. Changes in sterol and lipid compositions were observed in the resistant strains compared to the susceptible strain, and might increase cell permeability to RAB and RD. In addition, RAB and RD interfered with the sterol pathway, further aggregating the decrease in ergosterol in the sterol synthesis pathway in the MDR1-activated strains. Our findings here provide an alternative for combating resistant pathogenic fungi.
ATP-Binding Cassette Transporters ; genetics ; metabolism ; Antifungal Agents ; chemistry ; metabolism ; pharmacology ; Azoles ; pharmacology ; Biosynthetic Pathways ; drug effects ; genetics ; Candida albicans ; chemistry ; drug effects ; metabolism ; Cell Membrane ; chemistry ; metabolism ; Coculture Techniques ; Drug Resistance, Fungal ; drug effects ; Ergosterol ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Lipids ; chemistry ; Molecular Structure ; Permeability ; Phenyl Ethers ; chemistry ; metabolism ; pharmacology ; Sterols ; chemistry ; metabolism ; Stilbenes ; chemistry ; metabolism ; pharmacology ; Triterpenes ; chemistry ; metabolism ; pharmacology

An extracellular lipase from Aureobasidium pullulans was obtained and purified with a specific activity of 17.7 U/mg of protein using ultrafiltration and a DEAE-Sepharose Fast Flow column. Characterization of the lipase indicated that it is a novel finding from the species A. pullulans. The molecular weight of the lipase was 39.5 kDa, determined by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited its optimum activity at 40 °C and pH of 7. It also showed a remarkable stability in some organic solutions (30%, v/v) including n-propanol, isopropanol, dimethyl sulfoxide (DMSO), and hexane. The catalytic activity of the lipase was enhanced by Ca²⁺ and was slightly inhibited by Mn²⁺ and Zn²⁺ at a concentration of 10 mmol/L. The lipase was activated by the anionic surfactant SDS and the non-ionic surfactants Tween 20, Tween 80, and Triton X-100, but it was drastically inhibited by the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). Furthermore, the lipase was able to hydrolyze a wide variety of edible oils, such as peanut oil, corn oil, sunflower seed oil, sesame oil, and olive oil. Our study indicated that the lipase we obtained is a potential biocatalyst for industrial use.
Ascomycota/enzymology* ; Calcium ; Catalysis ; Corn Oil/metabolism* ; Detergents/chemistry* ; Enzyme Stability ; Fungal Proteins/chemistry* ; Glucans/chemistry* ; Hexanes/chemistry* ; Hydrogen-Ion Concentration ; Hydrolysis ; Industrial Microbiology ; Lipase/chemistry* ; Manganese/chemistry* ; Olive Oil/metabolism* ; Peanut Oil/metabolism* ; Sesame Oil/metabolism* ; Substrate Specificity ; Sunflower Oil/metabolism* ; Surface-Active Agents ; Temperature ; Zinc/chemistry*

OBJECTIVE: To evaluate the efficacy of cis-2-dodecenoic acid (BDSF) in the treatment and prevention of vaginal candidiasis in vivo. METHODS: The activities of different concentrations of BDSF against the virulence factors of Candida albicans (C. albicans) were determined in vitro. An experimental mouse model of Candida vaginitis was treated with 250 μmol/L BDSF. Treatment efficiency was evaluated in accordance with vaginal fungal burden and inflammation symptoms. RESULTS: In vitro experiments indicated that BDSF attenuated the adhesion and damage of C. albicans to epithelial cells by decreasing phospholipase secretion and blocking filament formation. Treatment with 30 μmol/L BDSF reduced the adhesion and damage of C. albicans to epithelial cells by 36.9% and 42.3%, respectively. Treatment with 200 μmol/L BDSF completely inhibited phospholipase activity. In vivo mouse experiments demonstrated that BDSF could effectively eliminate vaginal infection and relieve inflammatory symptoms. Four days of treatment with 250 μmol/L BDSF reduced vaginal fungal loads by 6-fold and depressed inflammation. Moreover, BDSF treatment decreased the expression levels of the inflammatory chemokine-associated genes MCP-1 and IGFBP3 by 2.5- and 2-fold, respectively. CONCLUSION BDSF is a novel alternative drug that can efficiently control vaginal candidiasis by inhibiting the virulence factors of C. albicans.
Animals ; Candida albicans ; drug effects ; metabolism ; pathogenicity ; physiology ; Candidiasis, Vulvovaginal ; drug therapy ; genetics ; immunology ; microbiology ; Chemokine CCL2 ; genetics ; immunology ; Disease Models, Animal ; Fatty Acids, Monounsaturated ; administration & dosage ; Female ; Fungal Proteins ; genetics ; metabolism ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; genetics ; immunology ; Mice ; Virulence ; drug effects ; Virulence Factors ; genetics ; metabolism

In this study, we analyzed the physical interactions of the dominant negative isoform of MoYpt7. Our results show that MoYpt7 interacts with MoGdi1. The dominant negative isoform of MoYpt7 (dominant negative isoform, N125I) is essential for colony morphology, conidiation, and pathogenicity in the rice blast fungus. These results further demonstrate the biological functions of MoYpt7 in Magnaporthe oryzae.
DNA Mutational Analysis ; Fungal Proteins/metabolism* ; Gene Expression Regulation, Fungal ; Genes, Fungal ; Green Fluorescent Proteins/metabolism* ; Magnaporthe/genetics* ; Microscopy, Fluorescence ; Mutation ; Oryza/microbiology* ; Phenotype ; Plant Diseases/microbiology* ; Protein Isoforms

Gene Expression Regulation, Fungal ; Gene Silencing ; Heterochromatin ; metabolism ; Histone Chaperones ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Transcriptional Elongation Factors ; genetics ; metabolism

Effective expression of pIFN-α in recombinant Pichia pastoris was conducted in a 5 L fermentor. Ethanol accumulation during the late glycerol feeding period inhibited heterologous protein expression. Comparative transcriptome analysis was thus performed to compare the gene transcription profiles of Pichia pastoris KM71H in high and low ethanol concentration environments. The results showed that during the glycerol cultivation stage, 545 genes (265 up-regulated and 280 down-regulated) were differentially expressed with ethanol stress. These genes were mainly involved in protein synthesis, energy metabolism, cell cycle and peroxisome metabolism. During the methanol induction stage, 294 genes (171 up-regulated and 123 down-regulated) were differentially expressed, which were mainly related to methanol metabolism, amino acid metabolism and protein synthesis. Ethanol stress increased protein misfolding and reduced structural integrity of ribosome and mitochondria during cultivation stage, and led to the failure of endoplasmic reticulum stress removal and damaged amino acid metabolism during induction stage in Pichia pastoris.
Amino Acids ; metabolism ; Bioreactors ; Endoplasmic Reticulum Stress ; Energy Metabolism ; Ethanol ; chemistry ; Gene Expression Profiling ; Gene Expression Regulation, Fungal ; Glycerol ; Methanol ; Pichia ; metabolism ; Protein Biosynthesis ; drug effects ; Protein Folding ; Recombinant Proteins ; biosynthesis ; Transcriptome

BACKGROUND: Next-generation sequencing (NGS) can detect many more microorganisms of a microbiome than traditional methods. This study aimed to analyze the vaginal microbiomes of Korean women by using NGS that included bacteria and other microorganisms. The NGS results were compared with the results of other assays, and NGS was evaluated for its feasibility for predicting vaginitis. METHODS: In total, 89 vaginal swab specimens were collected. Microscopic examinations of Gram staining and microbiological cultures were conducted on 67 specimens. NGS was performed with GS junior system on all of the vaginal specimens for the 16S rRNA, internal transcribed spacer (ITS), and Tvk genes to detect bacteria, fungi, and Trichomonas vaginalis. In addition, DNA probe assays of the Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis were performed. Various predictors of diversity that were obtained from the NGS data were analyzed to predict vaginitis. RESULTS: ITS sequences were obtained in most of the specimens (56.2%). The compositions of the intermediate and vaginitis Nugent score groups were similar to each other but differed from the composition of the normal score group. The fraction of the Lactobacillus spp. showed the highest area under the curve value (0.8559) in ROC curve analysis. The NGS and DNA probe assay results showed good agreement (range, 86.2-89.7%). CONCLUSIONS: Fungi as well as bacteria should be considered for the investigation of vaginal microbiome. The intermediate and vaginitis Nugent score groups were indistinguishable in NGS. NGS is a promising diagnostic tool of the vaginal microbiome and vaginitis, although some problems need to be resolved.
Area Under Curve ; Bacteria/*genetics/isolation & purification ; Bacterial Proteins/genetics ; Candida/*genetics/isolation & purification ; Female ; Fungal Proteins/genetics ; Gardnerella vaginalis/genetics/isolation & purification ; High-Throughput Nucleotide Sequencing ; Humans ; *Microbiota ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; ROC Curve ; Sequence Analysis, DNA ; Trichomonas vaginalis/genetics/isolation & purification ; Vagina/*microbiology ; Vaginitis/*diagnosis/microbiology

Anidulafungin is an effective antifungal medicine, which can inhibit activities of candida in vitro and in vivo. Echinocandin B (ECB) is the key precursor of Anidulafungin, thus the price and market prospect of Anidulafungin is directly due to the fermentation titer of ECB. In this study, Aspergillus nidulans was used for ECB fermentation, and the influence of adding microparticles on ECB fermentation was studied, such as talcum powder, Al2O3, and glass beads. The particle size and concentration were the key factors for mycelium morphology and ECB production, and ECB production could reach 1 262.9 mg/L and 1 344.1 mg/L by adding talcum powder of 20 g/L (d50 = 14.2 μm) and 7 glass beads (6 mm), an increase by 33.2% and 41.7%, respectively. The results indicated that the mycelium morphology of filamentous microorganisms and the product yield of fermentation could be improved by adding microparticles remarkably, and it provide an important method for the fermentative optimization of filamentous microorganisms.
Antifungal Agents ; metabolism ; Aspergillus nidulans ; metabolism ; Echinocandins ; biosynthesis ; Fermentation ; Fungal Proteins ; biosynthesis ; Industrial Microbiology ; methods

The main commercial production of fructooligosaccharides (FOS) comes from enzymatic transformation using sucrose as substrate by microbial enzyme fructosyltransferase. A fructosyltransferase genomic DNA was isolated from Aspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to GenBank (KF699529). The cDNA of the fructosyltransferase, containing an open reading frame of 1 887 bp, was further cloned by RT-PCR. The fructosyltransferase gene from Aspergillus niger was functionally expressed both in Escherichia coli and Pichia pastoris GS 115. The highest activity value for the construction with the α-factor signal peptide reached 431 U/mL after 3 days of incubation. The recombinant enzyme is extensively glycosylated, and the active form is probably represented by a homodimer with an apparent molecular mass of 200 kDa as judged from mobility in seminative PAGE gels. The extracellular recombinant enzyme converted sucrose mostly to FOS, mainly 1-kestose and nystose, liberating glucose. FOS reached a maximal value and represented about 58% of total sugars present in the reaction mixture after 4 h reaction. The results suggest that the availability of recombinant Pichia pastoris as a new source of a FOS-producing enzyme might result of biotechnology interest for industrial application.
Aspergillus niger ; enzymology ; genetics ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Fungal Proteins ; genetics ; metabolism ; Glycosylation ; Hexosyltransferases ; genetics ; metabolism ; Molecular Sequence Data ; Molecular Weight ; Pichia ; Sucrose ; metabolism ; Trisaccharides ; metabolism

OBJECTIVETo investigate the effects of butyl alcohol extract of baitouweng decoction (BAEB) on the fungal cell surface hydrophobicity (CSH), filamentation and biofilm formation of Candida tropicalis.

METHODGradual dilution method was used to determine the MIC. XTT assay was applied to determine the SMIC80. Time-Kill assay was employed to draw the Time-Kill curve. The water-hydrocarbon two-phase assay was used to measure the cell surface hydrophobicity. Scanning electron microscopy (SEM) was applied to observe the morphological changes of the biofilm. Confocal laser scanning microscopy (CLSM) was applied to determine the thickness of the biofilm. The quantification real-time PCR (qRT-PCR) was used to detect expression changes of releated genes (UME6, ALST3 and NRG1). result: The MICs of BAEB against C. tropicalis strains are determined as 64-128 mg x L(-1). The SMIC80 s of BAEB against the biofilm of Candida tropicalis strains are determined as 256-512 mg x L(-1). Time-Kill curve results indicate that BAEB has a promise fungicidal effect at 256 and 512 mg x L(-1). SEM results shows that 512 mg x L(-1) BAEB can inhibit the formation of C. tropicalis biofilm on Silicone catheter, and the morphology of biofilm is also affected by BAEB. The thickness of C. tropicalis biofilm is reduced by BAEB according to CLSM results. Furthermore, qRT-PCR results indicate that expression of UME6 and ALST3 are significantly down-regulated by BAEB 256,512 mg x L(-1), and NRG1 is not affected by BAEB.

CONCLUSIONBAEB inhibits effectively the CSH, filamentation and biofilm formation of VVC strains of C. tropicalis.

Antifungal Agents ; chemistry ; pharmacology ; Biofilms ; drug effects ; Candida tropicalis ; drug effects ; genetics ; physiology ; Candidiasis ; microbiology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Gene Expression Regulation, Fungal ; drug effects ; Humans ; Virulence Factors ; genetics ; metabolism