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Development of a Dual Organism Model for Aspergillus Fumigatus Spores and Human Airway Epithelial Cells at the Single Cell-Spore Level
Description
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A3865 - Development of a Dual Organism Model for Aspergillus Fumigatus Spores and Human Airway Epithelial Cells at the Single Cell-Spore Level
Author Block: W. Tam1, A. Toor1, G. K. Singhera1, B. Sahin1, B. Whalen1, A. M. Barlow1, L. Culibrk1, D. R. Dorscheid1, M. M. Moore2, S. J. Tebbutt1; 1University of British Columbia, Vancouver, BC, Canada, 2Simon Fraser University, Burnaby, BC, Canada.
Rationale
Aspergillus fumigatus is a saprotrophic filamentous fungus capable of causing a number of infectious respiratory diseases in susceptible individuals. The initial stages of infection by A. fumigatus are characterized by inhalation of fungal conidia (spores) and their subsequent interaction with the airway epithelium. Little is known regarding the specific mechanisms of interaction between A. fumigatus spores and the airway cells. We therefore developed a single cell-single spore experimental model to allow novel imaging and molecular approaches to further study this important dual organism interaction. We hypothesized that single cell-single spore interaction events could be detected and captured via fluorescence-activated cell sorting (FACS) for subsequent analyses including microscopic imaging and molecular measurement.
Methods
The human bronchial epithelial cell line (1HAE) was grown to confluence in 24-well plates with Dulbecco's Modified Eagle's Medium (DMEM) plus 10% of fetal bovine serum (FBS). A. fumigatus spores that were genetically modified to express green fluorescence protein (GFP) were applied to these cells (MOI = 10 spores/cell) and incubated at 37°C for 3 hours. Unbound spores were washed off with PBS, then the cell-spore co-cultures were trypsinized and stained with DAPI (nuclear stain), prior to sorting into 384-well plates via FACS. A cells-only control sample was used to define the baseline fluorescence intensity for setting up different gates in flow cytometry. A standardized gating protocol was set later, with one gate for the lower 25% of the GFP-positive population and one gate for 30%-50% of the same population. The sorted cells with their attached/internalized spores were observed under microscopy.
Results
Cells-only and cells treated with spores could be differentiated as distinct populations based on GFP fluorescence. Potential internalization events of single spores were observed in single 1HAE cells, using appropriate gating in FACS, approximately 50% of the time. Attachment of spores to the cell surface was also observed.
Conclusion
We have successfully developed a single host cell-single spore model. Single spore internalization events were enriched and imaged by microscopy, include time-lapse microscopy. Our future goal is to obtain transcriptomic and proteomic data from single cells infected with a single spore.
Author Block: W. Tam1, A. Toor1, G. K. Singhera1, B. Sahin1, B. Whalen1, A. M. Barlow1, L. Culibrk1, D. R. Dorscheid1, M. M. Moore2, S. J. Tebbutt1; 1University of British Columbia, Vancouver, BC, Canada, 2Simon Fraser University, Burnaby, BC, Canada.
Rationale
Aspergillus fumigatus is a saprotrophic filamentous fungus capable of causing a number of infectious respiratory diseases in susceptible individuals. The initial stages of infection by A. fumigatus are characterized by inhalation of fungal conidia (spores) and their subsequent interaction with the airway epithelium. Little is known regarding the specific mechanisms of interaction between A. fumigatus spores and the airway cells. We therefore developed a single cell-single spore experimental model to allow novel imaging and molecular approaches to further study this important dual organism interaction. We hypothesized that single cell-single spore interaction events could be detected and captured via fluorescence-activated cell sorting (FACS) for subsequent analyses including microscopic imaging and molecular measurement.
Methods
The human bronchial epithelial cell line (1HAE) was grown to confluence in 24-well plates with Dulbecco's Modified Eagle's Medium (DMEM) plus 10% of fetal bovine serum (FBS). A. fumigatus spores that were genetically modified to express green fluorescence protein (GFP) were applied to these cells (MOI = 10 spores/cell) and incubated at 37°C for 3 hours. Unbound spores were washed off with PBS, then the cell-spore co-cultures were trypsinized and stained with DAPI (nuclear stain), prior to sorting into 384-well plates via FACS. A cells-only control sample was used to define the baseline fluorescence intensity for setting up different gates in flow cytometry. A standardized gating protocol was set later, with one gate for the lower 25% of the GFP-positive population and one gate for 30%-50% of the same population. The sorted cells with their attached/internalized spores were observed under microscopy.
Results
Cells-only and cells treated with spores could be differentiated as distinct populations based on GFP fluorescence. Potential internalization events of single spores were observed in single 1HAE cells, using appropriate gating in FACS, approximately 50% of the time. Attachment of spores to the cell surface was also observed.
Conclusion
We have successfully developed a single host cell-single spore model. Single spore internalization events were enriched and imaged by microscopy, include time-lapse microscopy. Our future goal is to obtain transcriptomic and proteomic data from single cells infected with a single spore.
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