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- Saccharomyces cerevisiae Can Secrete Sapp1p Proteinase of Candida parapsilosis But Cannot Use It for Efficient Nitrogen Acquisition
- Zuzana Vinterová , Václava Bauerová , Ji&# , Hana Sychrová , Olga Hru&# , Iva Pichová
- J. Microbiol. 2013;51(3):336-344. Published online June 28, 2013
- DOI: https://doi.org/10.1007/s12275-013-2422-4
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- 2 Citations
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Abstract
- Secreted aspartic proteinase Sapp1p of Candida parapsilosis represents one of the factors contributing to the pathogenicity of the fungus. The proteinase is synthesized as an inactive pre-pro-enzyme, but only processed Sapp1p is secreted into extracellular space. We constructed a plasmid containing the SAPP1 coding sequence under control of the ScGAL1 promoter and used it for proteinase expression in a Saccharomyces cerevisiae kex2Δ mutant. Because Sapp1p maturation depends on cleavage by Kex2p proteinase, the kex2Δ mutant secreted only the pro-form of Sapp1p. Characterization of this secreted proteinase form revealed that the Sapp1p signal peptide consists of 23 amino acids. Additionally, we prepared a plasmid with the SAPP1 coding sequence under control of its authentic CpSAPP1 promoter, which contains two GATAA motifs. While in C. parapsilosis SAPP1 expression is repressed by good low molecular weight nitrogen sources (e.g., ammonium ions), S. cerevisiae cells harboring this plasmid secreted a low concentration of active proteinase regardless of the type of nitrogen source used. Quantitative real-time PCR analysis of a set of genes related to nitrogen metabolism and uptake (GAT1, GLN3, STP2, GAP1, OPT1, and PTR2) obtained from S. cerevisiae cells transformed with either plasmid encoding SAPP1 under control of its own promoter or empty vector and cultivated in media containing various nitrogen sources also suggested that SAPP1 expression can be connected with the S. cerevisiae regulatory network. However, this regulation occurs in a different manner than in C. parapsilosis.
- NOTE] Antifungal Activity of Extracellular Hydrolases Produced by Autolysing Aspergillus nidulans Cultures
- Melinda Szilágyi , Fruzsina Anton , Katalin Forgács , Jae-Hyuk Yu , István Pócsi , Tamás Emri
- J. Microbiol. 2012;50(5):849-854. Published online November 4, 2012
- DOI: https://doi.org/10.1007/s12275-012-2001-0
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- 8 Citations
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Abstract
- Carbon-starving Aspergillus nidulans cultures produce high activities of versatile hydrolytic enzymes and, among these, ChiB endochitinase and EngA β-1,3-endoglucanase showed significant antifungal activity against various fungal species. Double deletion of engA and chiB diminished the antifungal activity of the fermentation broths and increased conidiogenesis and long-term viability of A. nidulans, but decreased the growth rate on culture media containing weak carbon sources. Production of ChiB and EngA can influence fungal communities either directly due to their antifungal properties or indirectly through their effects on vegetative growth. Our data suggest saprophytic fungi as promising future candidates to develop novel biocontrol technologies.
- Neovastat(Æ-941) inhibits the airway inflammation and hyperresponsiveness in a murine model of asthma
- Sook-Young Lee , Soon-Young Paik , Su-Mi Chung ,
- J. Microbiol. 2005;43(1):11-16.
- DOI: https://doi.org/2145 [pii]
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Abstract
- Matrix metalloproteinase (MMP)-9 plays an important role in the pathogenesis of bronchial asthma. Neovastat, having significant antitumor and antimetastatic properties, is classified as a naturally occurring multifunctional antiangiogenic agent. We evaluated the therapeutic effect of Neovastat on airway inflammation in a mouse model of asthma. BALB/c mice were immunized subcutaneously with ovalbumin (OVA) on days 0, 7, 14, and 21 and challenged with inhaled OVA on days 26, 29, and 31. Neovastat was administrated by gavage (5 mg/kg body weight) three times with 12 h intervals, beginning 30 min before OVA inhalation. On day 32, mice were challenged with inhaled methacholine, and enhanced pause (Penh) was measured as an index of airway hyperresponsiveness. The severity of airway inflammation was determined by differential cell count of bronchoalveolar lavage (BAL) fluid. The MMP-9 concentration in BAL fluid samples was measured by ELISA, and MMP-9 activity was measured by zymography. The untreated asthma group showed an increased inflammatory cell count in BAL fluid and Penh value compared with the normal control group. Mice treated with Neovastat had significantly reduced Penh values and inflammatory cell counts in BAL fluid compared with untreated asthmatic mice. Furthermore, mice treated with Neovastat showed significantly reduced MMP-9 concentrations and activity in BAL fluid. These results demonstrate that Neovastat might have new therapeutic potential for airway asthmatic inflammation. <br><br><br>
- Purification and characterization of extracellular aspartic proteinase of candida albicans
- Na, Byoung-Kuk , Lee, Seong Il , Kim, Sin Ok , Park, Young Kil , Bai, Gill Han , Kim, Sang Jae , Song, Chul Yong
- J. Microbiol. 1997;35(2):109-116.
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Abstract
- An extracellular proteinase of Candida albicans was purified by a combination of 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephacryl S-200 HR molecular sieve chromatography. Its mlecular weight was approximately 41 kDa on SDS-PAGE and isoelectric point was 4.4. The enzyme was inhibited by pepstain A. Optimum enzyme activity ranged from pH 2.0 to 3.5 with its maximum at pH 2.5 and a temperature of 45℃. The addition of divalent cations, Ca^2+, Zn^2+ and Mg^2+, resulted in no significant inhibition of enzymatic activity. However, some inhibitory effects were observed by Fe^2+, Ag^2+ and Cu^2+. With BSA as substrate, an apparent K_m was determined to be 7 × 10^7 M and K_I, using pepstatin A as an inhibitor, was 8.05 × 10^8 M. N-terminal amino acid sequence was QAVPVTLXNEQ. Degradation of BSA and fibronectin was shown but not collagen, hemoglobin, immunoglobulin G, or lysozyme. The enzyme preferred peptides with Glu and Leu at the P₁position, but the enzyme activity was highly reduced when the P₂position was Phe or Pro. This enzyme showed antigenicity against sera of patients with candidiasis.
- Intracellular Posttranslational Modification of Aspartyl Proteinase of Candida albicans and the Role of the Glycan Region of the Enzyme
- Byoung-Kuk Na , Chul-Yong Song
- J. Microbiol. 2000;38(4):218-223.
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Abstract
- Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAP1) of Candida albicans. Three intracellular forms of SAP1 were detected by immunoblotting using monoclonal antibody (MAb) CAP1. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAP1 and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAP1. These results show that SAP1 is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor form undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretory vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAP1 was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in part, be related to enzyme stability and activity.
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