Searching for novel functional amyloids in biofilms

The identification of novel amyloids is key to understand the many roles of amyloids in biofilm structure and function. Unfortunately, isolation of amyloids is a challenging and not always successful process. However, by taking advantage of the structural stability and the polymeric nature of the amyloids, compared to most other proteins, a new method has been developed to screen for potential amyloids. Utilizing formic acid’s ability to depolymerize an amyloid fibril, the samples were freeze-dried and dissolved in increasing concentrations of formic acid (0-100%) and then analyzed for a characteristic increase in protein abundance at a higher percentage of formic acid, where they are dissolved. The proteins were identified using label-free quantitative LC-MS/MS, and plots of all identified proteins were studied for potential amyloid patterns. The method has successfully been evaluated on two established Gram-negative amyloid systems and used to find a novel amyloid candidate in the potential pathogenic Aeromonas sp. Proof of concept for the application to complex microbial biofilms was done using activated sludge, where amyloid producing E. coli was added and subsequently traced. In addition, another potential novel amyloid candidate was found in the activated sludge biofilm.