M Eugenia Sepúlveda-González1, Berenice Parra-Ortega2, Yuliana Betancourt-Cervantes2, César Hernández-Rodríguez2, Juan Xicohtencatl-Cortes3, Lourdes Villa-Tanaca4. 1. Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional, Delegación Miguel Hidalgo, México, D.F., Mexico; Laboratorio de Investigación en Bacteriología Intestinal, Unidad de Hemato-Oncología e Investigación, Hospital Infantil de México Federico Gómez, Delegación Cuauhtémoc, México, D.F., Mexico. 2. Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional, Delegación Miguel Hidalgo, México, D.F., Mexico. 3. Laboratorio de Investigación en Bacteriología Intestinal, Unidad de Hemato-Oncología e Investigación, Hospital Infantil de México Federico Gómez, Delegación Cuauhtémoc, México, D.F., Mexico. Electronic address: juanxico@yahoo.com. 4. Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional, Delegación Miguel Hidalgo, México, D.F., Mexico. Electronic address: lourdesvilla@hotmail.com.
Abstract
BACKGROUND: The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. AIMS: The present paper is the first report on proteolytic activity in the C. glabrata vacuole. METHODS: Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. RESULTS: Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. CONCLUSIONS: The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen.
BACKGROUND: The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. AIMS: The present paper is the first report on proteolytic activity in the C. glabrata vacuole. METHODS: Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. RESULTS: Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. CONCLUSIONS: The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen.