Development of High Throughput Assay for the Detection of Amyloid Fibrils in Pseudomonas aeruginosa Cell Cultures.
Faculty Mentor
Benjamin Lundgren
Presentation Type
Poster
Start Date
May 2025
End Date
May 2025
Location
PUB NCR
Primary Discipline of Presentation
Chemistry and Biochemistry
Abstract
Pseudomonas aeruginosa is well known for its formation of biofilms, a large extracellular network of interconnected polymeric substances that allows this bacterium to adhere to other cells, tissues, and provides resistance towards antibiotics. It is hypothesized that a key structural component of biofilms are functional amyloid fibrils. These functional amyloid fibrils are created intracellularly, excreted, and anchored to the outside of the cell membrane. In order to detect when amyloid fibrils are being produced by P. aeruginosa, a microplate spectroscopy assay was developed to measure fluorescence changes of a dye upon its binding to amyloid fibrils. The dye exhibits a characteristic fluorescence emission shift that occurs when Congo Red (CR) and Thioflavin T (ThT) dyes bind to amyloid ß-sheet motifs. The high throughput nature of this assay will allow us to quickly screen multiple environmental conditions, growth conditions, and cell types affecting amyloid production. From this study we observed that CR yielded a more robust response and lower background fluorescence than ThT, and that CR is more suitable for measuring functional amyloids in situ. We found that the formation of biofilms interfered with accurate measurements of amyloid fibrils, and that future developments need to be specific for the identification of amyloid fibrils versus generic biofilm components. Fluorescent measurement of functional amyloids will be used in future experiments to measure how amyloid expression is regulated on the genetic level.
Recommended Citation
Staaleson, Sean, "Development of High Throughput Assay for the Detection of Amyloid Fibrils in Pseudomonas aeruginosa Cell Cultures." (2025). 2025 Symposium. 29.
https://dc.ewu.edu/srcw_2025/ps_2025/p2_2025/29
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Development of High Throughput Assay for the Detection of Amyloid Fibrils in Pseudomonas aeruginosa Cell Cultures.
PUB NCR
Pseudomonas aeruginosa is well known for its formation of biofilms, a large extracellular network of interconnected polymeric substances that allows this bacterium to adhere to other cells, tissues, and provides resistance towards antibiotics. It is hypothesized that a key structural component of biofilms are functional amyloid fibrils. These functional amyloid fibrils are created intracellularly, excreted, and anchored to the outside of the cell membrane. In order to detect when amyloid fibrils are being produced by P. aeruginosa, a microplate spectroscopy assay was developed to measure fluorescence changes of a dye upon its binding to amyloid fibrils. The dye exhibits a characteristic fluorescence emission shift that occurs when Congo Red (CR) and Thioflavin T (ThT) dyes bind to amyloid ß-sheet motifs. The high throughput nature of this assay will allow us to quickly screen multiple environmental conditions, growth conditions, and cell types affecting amyloid production. From this study we observed that CR yielded a more robust response and lower background fluorescence than ThT, and that CR is more suitable for measuring functional amyloids in situ. We found that the formation of biofilms interfered with accurate measurements of amyloid fibrils, and that future developments need to be specific for the identification of amyloid fibrils versus generic biofilm components. Fluorescent measurement of functional amyloids will be used in future experiments to measure how amyloid expression is regulated on the genetic level.
