Pathogenic yeasts Candida albicans and Candida parapsilosis possess a ß-type carbonic anhydrase Nce103p, which is involved in CO2 hydration and signaling. C. albicans lacking Nce103p cannot survive in low CO2 concentrations, e.g., in atmospheric growth conditions. Candida carbonic anhydrases are orthologous to the Saccharomyces cerevisiae enzyme, which had originally been detected as a substrate of a non-classical export pathway. However, experimental evidence on localization of C. albicans and C. parapsilosis carbonic anhydrases has not been reported to date. Immunogold labeling and electron microscopy used in the present study showed that carbonic anhydrases are localized in the cell wall and plasmatic membrane of both Candida species. This localization was confirmed by Western blot and mass spectrometry analyses of isolated cell wall and plasma membrane fractions. Further analysis of C. albicans and C. parapsilosis subcellular fractions revealed presence of carbonic anhydrases also in the cytosolic and mitochondrial fractions of Candida cells cultivated in shaken liquid cultures, under the atmospheric conditions.

• Klíčová slova
• Candida albicans, Candida parapsilosis, Nce103p, carbonic anhydrase, cell wall, electron microscopy, localization, mass spectrometry,
• MeSH
• buněčná membrána enzymologie   MeSH
• buněčná stěna enzymologie   MeSH
• Candida albicans enzymologie   růst a vývoj   MeSH
• Candida parapsilosis enzymologie   růst a vývoj   MeSH
• cytosol enzymologie   MeSH
• elektronová mikroskopie MeSH
• fungální proteiny metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• karboanhydrasy metabolismus   MeSH
• mitochondrie enzymologie   MeSH
• techniky vsádkové kultivace MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fungální proteiny MeSH
• karboanhydrasy MeSH

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.

• MeSH
• Candida enzymologie   genetika   MeSH
• dusík metabolismus   MeSH
• endopeptidasy metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• Saccharomyces cerevisiae enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dusík MeSH
• endopeptidasy MeSH