Transient binding facilitates super-resolution imaging of functional amyloid fibrils on living bacteria
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Curli, which are the major proteinaceous component of the Escherichia coli biofilm extracellular matrix, help protect cells against environmental stressors including dehydration and antibiotics. Composed of the amyloid proteins CsgA and CsgB, curli self-assemble as these protomers are secreted into the extracellular space. The mechanisms of curli assembly and their functional roles within the extracellular matrix are incompletely understood. High-resolution imaging tools compatible with live-cell conditions provide a critical means to investigate the assembly and function of curli in their native context.
In this study, we use super-resolution imaging to visualize curli fibrils on living bacterial cells. Transient amyloid binding of the fluorogenic dye Nile blue facilitates two complementary super-resolution fluorescence microscopy approaches: single-molecule localization microscopy and super-resolution optical fluctuation imaging. Additionally, imaging fluorescence correlation spectroscopy was used to measure the characteristic relaxation times associated with Nile blue binding to CsgA fibrils. Together, these approaches offer a framework for imaging-based biophysical characterization of curli structures on living cells.
Importance/impact statement
Escherichia coli and other enteric bacteria secrete amyloid proteins that self-assemble into fibrillar structures called curli, forming a key component of biofilm extracellular matrices. Bacterial biofilms confer resilience to harsh environments with broad implications for human health. In this study, we extend transient amyloid binding to the novel application of super-resolution fluorescence microscopy of curli on living cells, offering promising approaches to gain structural and mechanistic insights.
