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Role of Autophagic Markers During Clearance of Aspergillus Fumigatus from Epithelial Cells
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A3864 - Role of Autophagic Markers During Clearance of Aspergillus Fumigatus from Epithelial Cells
Author Block: M. Goyal1, M. Singh1, A. Chakrabarti2, A. K. Ghosh2; 1Pediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh, India, 2Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Rationale: Aspergillus fumigatus is an air-borne fungal pathogen responsible for a range of pulmonary diseases depending on immune status of the host. The precise mechanism that makes Aspergillus an important pathogen in lungs is not yet fully understood. In the present study, an attempt has been be made to investigate the contribution of autophagic pathway to the clearance of A.fumigatus from airway epithelial cells. Methods: Germinated spores/conidia of clinical strains of A.fumigatus NCCPF no. 770231 and NCCPF no. 770233 isolated from patients with ABPA were used in this study to infect alveolar (A549) epithelial cell line. Kinetic analysis of internalization of A.fumigatus spores in epithelial cells and various markers of autophagic machinery were investigated using microscopic studies (transmission, scanning and fluorescent) in a time dependent manner. The invasion index was calculated by phagocytosis and nystatin protection assays. Results: Kinetic analysis of internalization by TEM studies have evidently shown that conidia reside in single-membrane autophagosomes in contrast to standard double membrane autophagosomes. At late stages of infection, few degraded intracelluar conidia were observed inside the cytoplasm of cell with no apparent changes (cell destruction) in cell morphology of pneumocytes. However, germination of conidia was not noticed until 8h infection period. The abundance of autophagic vacuoles was also confirmed by lysotracker, acridine orange, MDC staining. The number of conidia recovered showed nearby twofold increase from 2h to 4h postinfection with a steady increase till 6h. However, a gradual decrease was observed in invasion index 8h postinfection. Our results are in concordance with a recent study which reported a novel autophagic pathway termed LC3 associated phagocytosis (LAP) functioning in the clearance of A.fumigatus. However, further experiments with specific autophagic markers will help to shed more light upon its autophagic machinery. Conclusion: This study helps toward understanding the potential impact of how Aspergillus fumigatus affects the killing and clearance of infection by cells of innate immune system which certainly help to further explore the role of host pathogen interactions in the infection process.
Author Block: M. Goyal1, M. Singh1, A. Chakrabarti2, A. K. Ghosh2; 1Pediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh, India, 2Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Rationale: Aspergillus fumigatus is an air-borne fungal pathogen responsible for a range of pulmonary diseases depending on immune status of the host. The precise mechanism that makes Aspergillus an important pathogen in lungs is not yet fully understood. In the present study, an attempt has been be made to investigate the contribution of autophagic pathway to the clearance of A.fumigatus from airway epithelial cells. Methods: Germinated spores/conidia of clinical strains of A.fumigatus NCCPF no. 770231 and NCCPF no. 770233 isolated from patients with ABPA were used in this study to infect alveolar (A549) epithelial cell line. Kinetic analysis of internalization of A.fumigatus spores in epithelial cells and various markers of autophagic machinery were investigated using microscopic studies (transmission, scanning and fluorescent) in a time dependent manner. The invasion index was calculated by phagocytosis and nystatin protection assays. Results: Kinetic analysis of internalization by TEM studies have evidently shown that conidia reside in single-membrane autophagosomes in contrast to standard double membrane autophagosomes. At late stages of infection, few degraded intracelluar conidia were observed inside the cytoplasm of cell with no apparent changes (cell destruction) in cell morphology of pneumocytes. However, germination of conidia was not noticed until 8h infection period. The abundance of autophagic vacuoles was also confirmed by lysotracker, acridine orange, MDC staining. The number of conidia recovered showed nearby twofold increase from 2h to 4h postinfection with a steady increase till 6h. However, a gradual decrease was observed in invasion index 8h postinfection. Our results are in concordance with a recent study which reported a novel autophagic pathway termed LC3 associated phagocytosis (LAP) functioning in the clearance of A.fumigatus. However, further experiments with specific autophagic markers will help to shed more light upon its autophagic machinery. Conclusion: This study helps toward understanding the potential impact of how Aspergillus fumigatus affects the killing and clearance of infection by cells of innate immune system which certainly help to further explore the role of host pathogen interactions in the infection process.
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