OBJECTIVETo observe the different mRNA levels of Candida albicans ALS gene family between planktonic and biofilm-grown cells.

METHODSATCC 90038 and a wild strain of Candida albicans, biofilm models in vitro were formed on glass slides. After 48 hours' incubation, the biofilm-grown cells were harvested. Half-quantification of ALS1 and ALS4 mRNA was based on the amplification by one-step RT-PCR.

RESULTSThe amounts of ALS1 and ALS4 mRNA of the wild strain in biofilm increased comparing with planktonic cells, while ATCC 90038 didn't.

CONCLUSIONThe members of ALS gene family may play important roles in the course of Candida albicans biofilm formation.

Biofilms ; Candida albicans ; Fungal Proteins ; Humans ; Mouth ; microbiology ; RNA, Messenger

OBJECTIVETo establish real-time PCR and loop-mediated isothermal amplification (LAMP) systems for detecting Cryptococcus neoformans CAP10 gene.

METHODSSpecific primers were designed targeting CAP10 gene of Cryptococcus neoformans, and the plasmid was constructed. After optimization of the reaction condition, the plasmid was quantitatively detected using real-time PCR and LAMP, and the detection sensitivity and specificity were evaluated. Clinical samples were also detected using the two methods.

RESULTSThe detection sensitivity of real-time PCR and LAMP was 6.8×10(1) and 6.8×10(3) copies, respectively. Real-time PCR yielded a higher positivity rate than LAMP. Both of the two methods showed a high detection specificity and produced negative results in the detection of Neisseria meningitidis, Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus niger and Escherichia coli.

CONCLUSIONReal-time PCR is highly sensitive and specific for detecting Cryptococcus neoformans CAP10 gene but requires sophisticated equipment. LAMP, though with a relatively lower sensitivity, is simple to operate without the need of special equipment, and the result can be conveniently observed. Both of the two methods are suitable for detecting Cryptococcus neoformans and evaluating the treatment outcomes.

Cryptococcus neoformans ; genetics ; Fungal Proteins ; genetics ; Nucleic Acid Amplification Techniques ; methods ; Plasmids ; RNA, Fungal ; genetics ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

BACKGROUND: Traditionally, mating types of dermatophytes had been identified by mating experiments. It took a long time and there were many limitations. Recently, we can figure out the fungal mating types using molecular mating type analysis by detecting mating type (MAT) genes. The mating type (+) specific gene of the high-mobility-group (HMG) DNA binding domain and the mating type (-) specific gene of alpha-box were found in Arthroderma simii and A. vanbreuseghemii. OBJECTIVE: We applied this molecular mating type analysis to strains of Trichophyton interdigitale, T. rubrum, Microsporum canis in Korea and compared these results with previous reports. METHODS: Thirty-four strains of T. interdigitale (12 granular types, 9 powdery types, 8 purple-red types, 5 cottony types), 5 strains of T. rubrum, and 5 strains of M. canis were examined. We analyzed ribosomal RNA internal transcribed space 1, 4 sequencing of T. interdigitale subtypes and investigated the mating type of dermatophytes using alpha-box gene and HMG gene primers. RESULTS: Among 12 strains of granular type of T. interdigitale, 9 strains were type (-) and other 3 strains were type (+). All of them were zoophilic. All strains of powdery, purple-red and cottony types of T. interdigitale were type (+) and anthropophilic. In T. rubrum and M. canis, all strains were type (-). These results were matched with previously reported studies. CONCLUSION: The molecular mating type analysis of dermatophytes was quicker method than conventional mating experiments. Moreover, MAT genes are highly conserved even in apparently asexual fungi. The results were well matched with previous reports with traditional mating tests.
Arthrodermataceae* ; DNA ; Fungi ; Genes, Mating Type, Fungal ; Korea ; Microsporum ; RNA, Ribosomal ; Trichophyton

Animals ; DNA, Fungal ; Phosphorylation ; RNA Polymerase II ; Sequence Homology, Nucleic Acid ; T-Box Domain Proteins ; genetics

In this study, we used traditional morphological and molecular identification methods to preliminarily identify two strains of dermatophytes. The two strains were observed under the microscope. And then the dermatophytes were cultured on Sabouraud's dextrose agar (SDA). The 18S rRNA regions of the two dermatophyte strains were amplified by polymerase chain reaction (PCR), and the PCR products were sequenced and compared with GenBank data. BLAST tools and DNAMAN software were used to analyze the sequences. To further determine highly homologous sequences, a phylogenetic tree was constructed using the Neighbor-Joining method. The two strains of dermatophytes were identified by traditional morphological identification as Epidermophyton floccosum and Microsporum ferrugineum. The 18S rRNA sequence analyses showed high similarities to Cladosporium cladosporioides isolate C115LM-UFPR and Ascomycete sp. LB68A1A2. Epidermophyton and Cladosporium belong to dermatophyte, while Microsporum ferrugineum and Ascomycete belong to microsporum. The two novel strains of dermatophytes were therefore identified as Cladosporium cladosporioides isolate C115LM-UFPR (JN650537, Cladosporium) and Ascomycete sp. LB68A1A2 (AY770409, Ascomycete sp).
Arthrodermataceae ; cytology ; genetics ; isolation & purification ; Humans ; Hyphae ; cytology ; RNA, Fungal ; genetics ; RNA, Ribosomal, 18S ; genetics ; Skin ; microbiology

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1x10(6) ml(-1), the percentages of conidium germination and appressorium formation were (97.98+/-0.67)% and (97.88+/-0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 microg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.
DNA, Complementary ; genetics ; isolation & purification ; DNA, Fungal ; genetics ; isolation & purification ; Gene Library ; Genes, Fungal ; Magnaporthe ; genetics ; growth & development ; pathogenicity ; Oryza ; microbiology ; Plant Diseases ; microbiology ; RNA, Fungal ; genetics ; isolation & purification

OBJECTIVETo investigate the DNA genome and RNA expression in 5-flurocytosine-resistant strains of Candida albicans from vaginal candidasis.

METHODSSixteen strains of Candida albicans were selected from clinically diagnosed revul-vaginal candidasis. Eight 5-flurocytosine-sensitive isolates and 8 resistant isolates were examined by France Media FUNGUS sensitive test. DNA genome was detected with random amplification polymorph DNA. RNA expression was detected with random amplification polymorph RNA method.

RESULTSThere were no distinct differences between 5-flurocytosine-sensitive and resistant Candida albicans in DNA genome, while RNA expression showed significant differences between 5-flurocytosine-resistant and sensitive strains.

CONCLUSIONClinical 5-flurocytosine-resistant strains of Candida albicans from revul-vaginal candidasis may be related to phenotype changes.

Adolescent ; Adult ; Antifungal Agents ; pharmacology ; Candida albicans ; drug effects ; genetics ; Candidiasis, Vulvovaginal ; microbiology ; DNA, Fungal ; analysis ; Drug Resistance, Fungal ; Female ; Flucytosine ; pharmacology ; Humans ; Middle Aged ; RNA, Fungal ; analysis ; Random Amplified Polymorphic DNA Technique

Knowledge of simulated microgravity (SMG)-induced changes in the pathogenicity of microorganisms is important for success of long-term spaceflight. In a previous study using the high aspect ratio vessel bioreactor, we showed that the yeast species Saccharomyces cerevisiae underwent a significant phenotypic response when grown in modeled microgravity, which was reflected in the analysis of gene expression profiles. In this study, we establish that Candida albicans responds to SMG in a similar fashion, demonstrating that there is a conserved response among yeast to this environmental stress. We also report that the growth of C. albicans in SMG results in a morphogenic switch that is consistent with enhanced pathogenicity. Specifically, we observed an increase in filamentous forms of the organism and accompanying changes in the expression of two genes associated with the yeast-hyphal transition. The morphological response may have significant implications for astronauts' safety, as the fungal pathogen may become more virulent during spaceflight.
Candida albicans ; cytology ; genetics ; growth & development ; pathogenicity ; Candidiasis ; immunology ; Cell Polarity ; Cells, Cultured ; Fungal Proteins ; genetics ; Gene Expression Regulation, Fungal ; Humans ; Microscopy, Fluorescence ; RNA, Fungal ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Saccharomyces cerevisiae ; cytology ; genetics ; growth & development ; Virulence ; Weightlessness Simulation

Four dsRNA bands were extracted from Pleurotus ostreatus TD300 by the dsRNA isolation technique with sizes of 8.2 kb, 2.5 kb, 2.1 kb, and 1.1 kb, respectively. Four virus-eliminated methods, i. e. hyphal tips cut (HTC), protoplast regeneration (PR), single spore hybridization (SSH), and frozen and lyophilized (FL), were applied to prepare virus-eliminated strains, and one virus-eliminated strain was selected for each virus-elimination method. The virus-eliminated strains were named as HTC8, PR15, FL01, and SSH11, respectively. There were low concentration of 8.2 kb dsRNA remained in HTC8, as well as low concentration of 8.2 kb and 2.5 kb dsRNA remained in FL01. However, no dsRNA remained in PR15 and SSH11. The hyphal growth rate and laccase activity of the virus-eliminated strains increased, especially HTC8 and PR15, whose hyphal growth rate was higher by 22.73% and 18.18%, and laccase activities higher by 145.83% and 134.38% than that of the original strain, respectively. The conclusion is that hyphal tips cut and protoplast regeneration are suitable to prepare virus-eliminated strains of edible fungi.
Food Microbiology ; Freeze Drying ; Hybridization, Genetic ; Hyphae ; virology ; Pleurotus ; cytology ; genetics ; growth & development ; virology ; Protoplasts ; virology ; RNA, Double-Stranded ; analysis ; isolation & purification ; RNA, Fungal ; analysis ; isolation & purification ; Spores, Fungal ; genetics ; virology ; Viruses ; isolation & purification

Filamentous fungi are important industrial microorganisms. The focus on its metabolic engineering is to optimize the metabolic pathway with gene expression regulation technology to meet with the industrial production needs. Antisense RNA technology due to its simplicity compared with the gene knock-out technology has great perspectives in filamentous fungal metabolic control. It is an efficient method for regulating gene expression and a key tool for metabolic engineering. In this article, we addressed the mechanism of antisense RNA technology and its applications in filamentous fungal metabolic engineering. Additionally, future perspectives were discussed.
Fungi ; genetics ; Gene Expression Regulation, Fungal ; Genetic Engineering ; methods ; Industrial Microbiology ; methods ; Metabolism ; RNA, Antisense ; genetics ; metabolism