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1.
J. appl. oral sci ; 28: e20190578, 2020. tab, graf
Article in English | LILACS, BBO | ID: biblio-1101256

ABSTRACT

Abstract Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.


Subject(s)
Humans , Tooth Root/microbiology , Candida albicans/genetics , DNA, Fungal/genetics , Root Caries/microbiology , Biofilms/growth & development , Candida albicans/isolation & purification , Candida albicans/growth & development , Gene Expression , Gene Expression Regulation, Fungal , Up-Regulation , Sequence Analysis, RNA , Transcriptome , Morphogenesis
2.
Article in Chinese | WPRIM | ID: wpr-771507

ABSTRACT

The aim of this paper was to investigate the inhibitory effect of extract of Coptidis Rhizoma(ECR) on invasion of Candida albicans hyphae in vitro.XTT reduction method was used to evaluate the metabolic activity of C.albicans.The colony edge growth of C.albicans was observed by solid medium.The growth of C.albicans hyphae was determined on semi-solid medium.The morphology and viability changes of C.albicans hyphae were assessed by scanning electron microscope and fluorescence microscope.qRT-PCR method was used to detect the ALS3 and SSA1 expression of C.albicans invasin genes.The results showed that the metabolic viability by XTT method detected that the activity of C.albicans was gradually decreased under the intervention of 64,128 and 256 mg·L-1 of ECR respectively.128,256 mg·L-1 of ECR significantly inhibited colony folds and wrinkles on solid medium and the hyphal invasion in semi-solid medium.Scanning electron microscopy and fluorescence microscopy showed that 128,256 mg·L-1 of ECR could inhibit the formation of C.albicans hyphae.qRT-PCR results showed that the expression of invasin gene ALS3 and SSA1 was down-regulated,and especially 256 mg·L-1 of ECR could down-regulate the two genes expression by 4.8,1.68 times respectively.This study showed that ECR can affect the invasiveness of C.albicans by inhibiting the growth of hyphae and the expression of invasin.


Subject(s)
Adenosine Triphosphatases , Genetics , Candida albicans , Drugs, Chinese Herbal , Pharmacology , Fungal Proteins , Genetics , Gene Expression Regulation, Fungal , HSP70 Heat-Shock Proteins , Genetics , Hyphae , Microscopy, Electron, Scanning
3.
Article in Chinese | WPRIM | ID: wpr-774597

ABSTRACT

This study aimed to investigate the effect of butyl alcohol extract of Baitouweng Decoction( BAEB) on Candida albicans biofilms based on pH signal pathway. The morphology of biofilms of the pH mutants was observed by scanning electron microscope. The biofilm thickness of the pH mutants was measured by CLSM. The biofilm activity of the pH mutants was analyzed by microplate reader.The biofilm damage of the pH mutants was detected by flow cytometry. The expression of pH mutant biofilm-related genes was detected by qRT-PCR. The results showed that the deletion of PHR1 gene resulted in the defect of biofilm,but there were more substrates for PHR1 complementation. BAEB had no significant effect on the two strains. RIM101 gene deletion or complementation did not cause significant structural damage,but after BAEB treatment,the biofilms of both strains were significantly inhibited. For the biofilm thickness,PHR1 deletion or complementation caused the thickness to decrease,after BAEB treatment,the thickness of the two strains did not change significantly. However,RIM101 gene deletion or complementation had little effect on the thickness,and the thickness of the two strains became thinner after adding BAEB. For biofilm activity,PHR1 deletion or complementation and RIM101 deletion resulted in decreased activity,RIM101 complementation did not change significantly; BAEB significantly inhibited biofilm activity of PHR1 deletion,PHR1 complemetation,RIM101 deletion and RIM101 complemetation strains. For the biofilm damage,PHR1 gene deletion or complementation,RIM101 gene deletion or complementation all showed different degrees of damage; after adding BAEB,the damage rate of PHR1 deletion or complementation was not significantly different,but the damage rate of RIM101 deletion or complementation was significantly increased. Except to the up-regulation of HSP90 gene expression,ALS3,SUN41,HWP1,UME6 and PGA10 genes of PHR1 deletion,PHR1 complementation,RIM101 deletion,and RIM101 complementation strains showed a downward expression trend. In a word,this study showed that mutations in PHR1 and RIM101 genes in the pH signaling pathway could enhance the sensitivity of the strains to the antifungal drug BAEB,thus inhibiting the biofilm formation and related genes expression in C. albicans.


Subject(s)
1-Butanol , Biofilms , Candida albicans , Drugs, Chinese Herbal , Pharmacology , Fungal Proteins , Gene Expression Regulation, Fungal , Hydrogen-Ion Concentration , Plant Extracts , Pharmacology , Signal Transduction
4.
Braz. j. microbiol ; 49(supl.1): 160-165, 2018. tab, graf
Article in English | LILACS | ID: biblio-974323

ABSTRACT

Abstract Sclareol is an important intermediate for ambroxide synthesis industries. Hyphozyma roseonigra ATCC 20624 was the only reported strain capable of degrading sclareol to the main product of sclareol glycol, which is the precursor of ambroxide. To date, knowledge is lacking about the effects of sclareol on cells and the proteins involved in sclareol metabolism. Comparative proteomic analyses were conducted on the strain H. roseonigra ATCC 20624 by using sclareol or glucose as the sole carbon source. A total of 79 up-regulated protein spots with a >2.0-fold difference in abundance on 2-D gels under sclareol stress conditions were collected for further identification. Seventy spots were successfully identified and finally integrated into 30 proteins. The up-regulated proteins under sclareol stress are involved in carbon metabolism; and nitrogen metabolism; and replication, transcription, and translation processes. Eighteen up-regulated spots were identified as aldehyde dehydrogenases, which indicating that aldehyde dehydrogenases might play an important role in sclareol metabolism. Overall, this study may lay the fundamentals for further cell engineering to improve sclareol glycol production.


Subject(s)
Ascomycota/metabolism , Fungal Proteins/metabolism , Diterpenes/metabolism , Ascomycota/genetics , Ascomycota/chemistry , Fungal Proteins/chemistry , Carbon/metabolism , Electrophoresis, Gel, Two-Dimensional , Gene Expression Regulation, Fungal , Proteomics , Glucose/metabolism
5.
Braz. j. microbiol ; 47(2): 468-479, Apr.-June 2016. tab, graf
Article in English | LILACS | ID: lil-780832

ABSTRACT

Abstract Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.


Subject(s)
Trichoderma/enzymology , Fungal Proteins/metabolism , Polyketide Synthases/metabolism , Plant Diseases/microbiology , Trichoderma/classification , Trichoderma/genetics , Fungal Proteins/genetics , Fungal Proteins/chemistry , Molecular Sequence Data , Gene Expression Regulation, Fungal , Sequence Alignment , Amino Acid Sequence , Mycelium/enzymology , Mycelium/genetics , Polyketide Synthases/genetics , Polyketide Synthases/chemistry
6.
Chinese Journal of Biotechnology ; (12): 584-598, 2016.
Article in Chinese | WPRIM | ID: wpr-337439

ABSTRACT

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.


Subject(s)
Amino Acids , Metabolism , Bioreactors , Endoplasmic Reticulum Stress , Energy Metabolism , Ethanol , Chemistry , Gene Expression Profiling , Gene Expression Regulation, Fungal , Glycerol , Methanol , Pichia , Metabolism , Protein Biosynthesis , Protein Folding , Recombinant Proteins , Transcriptome
7.
Chinese Journal of Biotechnology ; (12): 901-911, 2016.
Article in Chinese | WPRIM | ID: wpr-242288

ABSTRACT

Chromosomal integration of heterologous genes or pathways is preferred over the use of episomal plasmids for its inherently stability and thus more desirable in the industrial setting. However, the position of integration of heterologous genes in the genome influences the expression levels. In combination of high throughput transformation of the Yeast Knock-out Collection (YKO) and FACS analysis, the position effect on heterologous reporter gene gfp was identified across the whole genome in yeast. In total 428 high-expressed sites and 444 low-expressed sites were spotted, providing massive data to analyze patterns and reasons for region dependency of gene expression on the genome-wide scale.


Subject(s)
Gene Expression Regulation, Fungal , Gene Knock-In Techniques , Genes, Reporter , Genome, Fungal , Saccharomyces cerevisiae , Genetics
8.
Protein & Cell ; (12): 288-296, 2015.
Article in English | WPRIM | ID: wpr-757593

ABSTRACT

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.


Subject(s)
Alanine , Chemistry , Metabolism , Amino Acid Motifs , Aspartic Acid , Chemistry , Metabolism , Autophagy , Genetics , Autophagy-Related Proteins , Carrier Proteins , Chemistry , Metabolism , Gene Expression Regulation, Fungal , Membrane Proteins , Chemistry , Metabolism , Models, Molecular , Molecular Sequence Data , Nitrogen , Phagosomes , Chemistry , Metabolism , Phosphorylation , Protein Transport , Saccharomyces cerevisiae , Genetics , Metabolism , Saccharomyces cerevisiae Proteins , Chemistry , Genetics , Metabolism , Serine , Chemistry , Metabolism , Signal Transduction , Sirolimus , Pharmacology
9.
Article in Chinese | WPRIM | ID: wpr-337922

ABSTRACT

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHOD</b>Gradual 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.</p><p><b>CONCLUSION</b>BAEB inhibits effectively the CSH, filamentation and biofilm formation of VVC strains of C. tropicalis.</p>


Subject(s)
Antifungal Agents , Chemistry , Pharmacology , Biofilms , Candida tropicalis , Genetics , Physiology , Candidiasis , Microbiology , Drugs, Chinese Herbal , Chemistry , Pharmacology , Fungal Proteins , Genetics , Metabolism , Gene Expression Regulation, Fungal , Humans , Virulence Factors , Genetics , Metabolism
10.
Rev. chil. infectol ; 31(5): 511-517, oct. 2014. ilus, graf, tab
Article in Spanish | LILACS | ID: lil-730266

ABSTRACT

Introduction: The commensal yeast Candida albicans, can cause superficial or systemic candidiasis in susceptible hosts. In Chile, azole antifungals are the most widely used drugs in the treatment of candidiasis. In a previous study performed at our center, 2.1 and 1.6% of clinical isolates of C. albicans were found to be resistant to fluconazole and voriconazole, respectively. Objective: To characterize the resistance mechanisms involved in azoles resistance in Chilean clinical isolates. Methodology: Eight resistant, nine susceptible-dose dependent (SDD) and 10 susceptible strains (n: 27) were selected according to the Clinical Laboratory Standards Institute (CLSI) M27-S3 criteria, from vaginal and urine samples. Mutations in the 408-488 region of the ERG11 gene were studied by sequencing, and the relative expression of ERG11 gene and efflux pump genes CDR1, CDR2 and MDR1, was evaluated by quantitative real-time PCR (q-PCR). Results: No mutations were detected in the ERG11 gene and its overexpression was found only in 12.5% of the resistant strains (1/8). The most prevalent mechanism of resistance was the over-expression of efflux pumps (62.5%; 5/8). Conclusion: The study of the expression of efflux pumps by q-PCR could be a useful diagnostic tool for early detection of azole resistance in C. albicans.


Introducción: Candida albicans es una levadura comensal capaz de causar una infección oportunista en hospederos susceptibles denominada candidiasis, que puede ser superficial o sistémica. En Chile, los antifúngicos más utilizados para el tratamiento de las candidiasis son los azoles. En un estudio previo en nuestro centro, se detectó que 2,1 y 1,6% de cepas clínicas de C. albicans fueron resistentes a fluconazol y voriconazol, respectivamente. Objetivo: Caracterizar los mecanismos de resistencia involucrados en la resistencia a azoles en cepas clínicas chilenas. Metodología: Según los criterios del Clinical Laboratory Standards Institute (CLSI) M27-S3, se seleccionaron ocho cepas resistentes, nueve cepas susceptibles dosis dependiente (SDD) y 10 cepas sensibles (n: 27), aisladas de flujo vaginal y orina. Se evaluó la presencia de mutaciones en la región 408-488 del gen ERG11 por secuenciación y la expresión relativa del gen ERG11 y de los genes de bombas de eflujo CDR1, CDR2 y MDR1 por RPC en tiempo real cuantitativa (q-PCR). Resultados: No se encontraron mutaciones en el gen ERG11 y la sobre-expresión de éste sólo se presentó en 12,5% de las cepas resistentes (1/8). El mecanismo prevalente en la cepas resistentes fue la sobre-expresión de bombas de eflujo encontrándose en 62,5% de las cepas resistentes (5/8). Conclusión: El estudio de la expresión bombas de eflujo por q-PCR podría ser una herramienta diagnóstica útil para la detección temprana de resistencia a azoles en C. albicans.


Subject(s)
Female , Humans , Antifungal Agents/pharmacology , Candida albicans/drug effects , Fluconazole/pharmacology , Voriconazole/pharmacology , Chile , Candida albicans/genetics , Candida albicans/isolation & purification , Drug Resistance, Fungal , Gene Expression Regulation, Fungal , Genes, Fungal/genetics , Real-Time Polymerase Chain Reaction , RNA, Fungal/genetics
11.
Braz. j. microbiol ; 45(3): 873-883, July-Sept. 2014. ilus, graf, tab
Article in English | LILACS | ID: lil-727016

ABSTRACT

The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and β-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of β-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and β-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent.


Subject(s)
Gene Expression Regulation, Fungal/drug effects , Glucose/metabolism , Hexokinase/metabolism , Penicillins/biosynthesis , Penicillium chrysogenum/genetics , Penicillium chrysogenum/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Hexokinase/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Transformation, Genetic , beta-Galactosidase/biosynthesis
12.
Acta Pharmaceutica Sinica ; (12): 273-276, 2014.
Article in Chinese | WPRIM | ID: wpr-297980

ABSTRACT

This study is to investigate the effect of Euphorbia humifusa effective fraction (EHEF) on the CYP51 enzyme activity, the lanosterol content and the MEP, SUB gene expression of Trichophyton rubrum. Trichophyton rubrum was treated by EHEF for 7 days at 26 degrees C. The activity of CYP51 enzyme of Trichophyton rubrum in the cell membrane was determined by using ELISA kit, and the lanosterol content was investigated by using high performance liquid chromatography (HPLC), and the MEP, SUB gene expression of Trichophyton rubrum was detected with the reverse transcription polymerase chain reaction (RT-PCR) method. Results showed that EHEF can decrease the membrane CYP51 enzyme activity, and it also can accumulate the fungal lanosterol in a dose-dependent manner, and it also can decrease the gene expression of MEP and SUB. The antifungal mechanism of EHEF may be related to the inhibition on CYP51 enzyme activity, and to the effects on fungal cell membrane ergosterol biosynthesis. It may also play an antifungal effect by inhibiting the MEP, SUB gene expression of fungal proteases.


Subject(s)
Antifungal Agents , Pharmacology , Cell Membrane , Metabolism , Drugs, Chinese Herbal , Pharmacology , Enzyme Activation , Euphorbia , Chemistry , Gene Expression Regulation, Fungal , Lanosterol , Metabolism , Metalloproteases , Metabolism , Plants, Medicinal , Chemistry , Sterol 14-Demethylase , Metabolism , Subtilisins , Metabolism , Trichophyton , Genetics , Metabolism
13.
Article in Chinese | WPRIM | ID: wpr-341807

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of ethyl acetate extract of Huanglian Jiedu decoction (EAHD) on hyphae development of Candida albicans.</p><p><b>METHOD</b>Inverted microscope, fluorescence microscope, SEM were applied to inspect the Morphological change of C. albicans treated by EAHD at different concentrations. Solid agar plate was utilized to evaluate the colony morphology. Quantitative Real-ime PCR(qRT-PCR) was adopted to observe the expression of hyphae-specific genes such as HWP1, ALS3, UME6, CSH1, SUN41, CaPDE2.</p><p><b>RESULT</b>EAHD with concentration of 312 and 1 250 mg . L-1 could inhibit formation of hyphae and colony morphology. The expression of HWP1, ALS3, UME6, CSH1 were downregulated 4. 13, 3. 64, 2. 46, 2. 75 folds ,while the expression of SUN41 were upregulated 7. 26 folds, CaPDE2 keep unchanged.</p><p><b>CONCLUSION</b>EAHD could inhibit formation of hyphae and colony morphologies of C. albicans through downregulating HWP1, ALS3, UME6 and CSH1.</p>


Subject(s)
Acetates , Biofilms , Candida albicans , Cell Biology , Genetics , Down-Regulation , Drugs, Chinese Herbal , Pharmacology , Fungal Proteins , Genetics , Gene Expression Regulation, Fungal , Hyphae , Medicine, Chinese Traditional , Microscopy, Fluorescence , Reverse Transcriptase Polymerase Chain Reaction
14.
Article in Chinese | WPRIM | ID: wpr-244568

ABSTRACT

Along with the increase in fungal infections, Candida albicans prevention and control become the focus of anti-fungal infection at present. This study aims to discuss the effect monomer andrographolide (AG) on C. albicans biofilm dispersion. In the experiment, micro-well plates and medical catheter pieces were used to establish the C. albicans biofilm model. It was discovered by XTT assay and flat band method that 1 000, 500, 250 mg x L(-1) AG could impact the activity of C. albicans biofilm dispersion cells. The morphological structures of residual biofilms on catheter pieces were observed with scanning electron microscopy, which showed that 1 000, 500, 250 mg x L(-1) AG could induce C. albicans biofilm dispersion in a dose-dependent manner, and the dispersed cells were dominated by the yeast phase. According to the real-time fluorescence quantification PCR (qRT-PCR) test, AG could up-regulate HSP90 expression and down-regulate UME6 and PES1 expressions. This study demonstrates that AG could induce C. albicans biofilm dispersion to some extent.


Subject(s)
Anti-Inflammatory Agents , Pharmacology , Biofilms , Candida albicans , Genetics , Physiology , Diterpenes , Pharmacology , Dose-Response Relationship, Drug , Fungal Proteins , Genetics , Gene Expression Regulation, Fungal , HSP90 Heat-Shock Proteins , Genetics , Microscopy, Electron, Scanning , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
15.
Acta Pharmaceutica Sinica ; (12): 1520-1527, 2014.
Article in Chinese | WPRIM | ID: wpr-299103

ABSTRACT

The medicinal fungi, which are of great importance in traditional medicine, are facing the problems of wild resources scarcity and low concentration of bioactive compounds. Velvet family and LaeA global regulator play a vital role in secondary metabolism and developmental programs, which are found in a wide variety of fungi ranging from Chytridiomycota to Basidiomycota. This review elaborates the structures and functions between Velvet family and LaeA protein. The Velvet family which shares the Velvet protein domain, including VeA (Velvet), VelB (Velvet like B), VosA (viability of spores A) and VelC (Velvet like C), acts on the regulation function is secondary metabolism and developmental programs such as asexual and sexual development. Furthermore, the function is affected by environmental factors such as light and temperature. LaeA protein which owns S-adenosylmethionine-dependent methyltransferase domain, coordinately regulates development and secondary metabolism by regulating and modifying the Velvet proteins. The regulation of LaeA is mediated by light receptor proteins. Therefore, clarifying the mechanism of Velvet and LaeA proteins in medicinal fungi will pave the way for nurturing medicinal fungi and improving production of bioactive compounds.


Subject(s)
Fungal Proteins , Metabolism , Fungi , Chemistry , Gene Expression Regulation, Fungal , Genes, Regulator , Protein Structure, Tertiary , Secondary Metabolism , Structure-Activity Relationship
16.
Acta Pharmaceutica Sinica ; (12): 206-210, 2013.
Article in Chinese | WPRIM | ID: wpr-235682

ABSTRACT

Cytochrome P450 (CYP450) is a key element in the Ganoderma triterpenoid biosynthetic pathway. The catalytic reaction process for CYP450 requires NADPH / NADH for electron transfer. After searching the genome dataset of Ganoderma lucidum, the unique sequence encoding CYP450 and NADPH were discovered, separately. The open reading frames of GLCYP450 and GLNADPH were cloned separately using RT-PCR strategy from G lucidum. The appropriate restriction enzyme cutting sites were introduced at the 5' and 3' ends of gene sequence. The genes of GLCYP450 and GLNADPH were recombined into the yeast expression vector pESC-URA, leading to the formation of the yeast expression plasmid pESC-GLNADPH-GLCYP450. This study provides a foundation for researching Ganoderma triterpene biosynthesis using the approach of synthetic biology.


Subject(s)
Amino Acid Sequence , Cloning, Molecular , Cytochrome P-450 Enzyme System , Genetics , DNA, Complementary , Genetics , Metabolism , Gene Expression Regulation, Fungal , Genetic Vectors , NADP , Genetics , Open Reading Frames , Plasmids , Reishi , Genetics , Metabolism , Synthetic Biology , Triterpenes , Metabolism , Yeasts , Genetics , Metabolism
17.
Acta Pharmaceutica Sinica ; (12): 228-235, 2013.
Article in Chinese | WPRIM | ID: wpr-235679

ABSTRACT

Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S. cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S. cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S. cerevisiae. The well-characterized DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S. cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of DsRed-Monomer gene. The successful construction of pGBT9Red lays the foundation for further analysis of promoter activity and screening of promoter element libraries.


Subject(s)
Amino Acid Sequence , Base Sequence , Genetics , Cloning, Molecular , DNA Primers , Gene Expression Regulation, Fungal , Genetic Vectors , Luminescent Proteins , Genetics , Metabolism , Plasmids , Genetics , Promoter Regions, Genetic , Genetics , Recombinant Proteins , Genetics , Saccharomyces cerevisiae , Genetics , Metabolism , Synthetic Biology , Transformation, Genetic
18.
Biol. Res ; 44(4): 351-355, 2011. ilus, tab
Article in English | LILACS | ID: lil-626734

ABSTRACT

Transcription factor Ace1 from Saccharomyces cerevisiae regulates the expression of target genes when the copper concentration reaches 200 ìÌ levels. We are studying the ortholog of Ace1 from fungus Phanerochaete chrysosporium PcACE1, isolated by complementation in yeast. In this report we show the localization of the transactivation region of PcACE1. Different PcACE1 fragments were ligated in frame to the GAL4 DNA-binding domain by site-directed mutagenesis in a suitable yeast expression vector. Transformation of an appropriate Saccharomyces cerevisiae strain was used as host. This strain contains the fusion GAL1:lacZ in its genome under the control of promoter sequences recognized by GAL4. Finally, we measured â-galactosidase activity in each yeast clone. The activation of the reporter gene is proportional to the transactivation capacity of the transcription factor PcACE1. The results obtained indicate that PcACE1 transactivation domain is located in the carboxy terminal half and contains an array of cysteines in the form of Cys-X-Cys and Cys-X2-Cys and a 60% of Ser. Therefore, these results show that this type of Cys motif can function as transcription activating domain not only in transcription factors that respond to minimal copper concentrations but also in those that respond to high copper concentrations. This is the first transactivation domain reported in a basidiomycete fungus.


Subject(s)
Gene Expression Regulation, Fungal/genetics , Phanerochaete/genetics , Transcription Factors/genetics , Transcription, Genetic/genetics , Transcriptional Activation/genetics , Base Sequence , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction
19.
Chinese Journal of Biotechnology ; (12): 917-925, 2011.
Article in Chinese | WPRIM | ID: wpr-324486

ABSTRACT

In Candida albicans, adaptation to environmental pH is relevant to its pathogenicity. Calcium signaling pathway involves in many stress responses and often accompany with Ca2+ fluctuation. We constructed CCH1 and MID1 mutant strains and studied their effect on calcium influx and further investigated the regulation by Crz1p transcription factor. We used PCR-directed gene disruption to construct cch1delta/delta and mid1delta/delta null mutant. By using a flow cytometry-based method we monitored the free cytosolic Ca2+ levels under alkaline stress. Moreover, we constructed pPHO89-LacZ plasmids and by beta-Galactosidase assays, we analyzed the changes of LacZ activities after gene disruption. The results showed that alkaline stress induced calcium burst reduced obviously in cch1delta/delta and mid1delta/delta mutant strains, also for LacZ activities, and fully abolished in crz1delta/delta mutant strain. Finally, by realtime PCR, we confirmed the regulation role of Crz1p in CCH1 and MID1 genes but in a calcineurin independent way. Studies on the effect of calcium pathway on response to alkaline stress will provide an important theoretical basis for Candida albicans infection-oriented treatment and new drug targets.


Subject(s)
Calcium Channels , Metabolism , Candida albicans , Genetics , Metabolism , Physiology , Fungal Proteins , Genetics , Physiology , Gene Expression Regulation, Fungal , Hydrogen-Ion Concentration , Signal Transduction , Stress, Physiological , Transcription Factors , Metabolism , Physiology
20.
Chinese Journal of Biotechnology ; (12): 1142-1148, 2011.
Article in Chinese | WPRIM | ID: wpr-324478

ABSTRACT

Secondary metabolites of filamentous fungi are important sources of new drugs, and their biosynthetic processes are regulated by numerous factors. Recent studies indicate that many filamentous fungal secondary metabolites are regulated by epigenetic modifications, which not only affect the titers of secondary metabolites, but also activate the cryptic gene clusters. This review summarizes recent advances of epigenetic application in filamentous fungal secondary metabolite biosynthesis, especially the types of fungal epigenetic modification and epigenetic remodeling of the fungal secondary metabolism. The application of epigenetic theory in filamentous fungi is becoming a new strategy for fungal strain improvement and a powerful method to obtain novel natural products.


Subject(s)
Acetylation , DNA Methylation , DNA-Binding Proteins , Genetics , Metabolism , Epigenesis, Genetic , Epigenomics , Methods , Fungal Proteins , Genetics , Fungi , Genetics , Metabolism , Gene Expression Regulation, Fungal , Histones , Metabolism , Metabolic Networks and Pathways , Genetics
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