In vitro and in situ structural characterization of the biofilm-associated functional amyloids in Pseudomonas aeruginosa bacteria

Bacterial resistance to antibiotics poses a critical global threat and is partly due to the formation of stable biofilms which shield bacterial cells from antibiotics. Cystic fibrosis in lugs and some hospital-acquired infections caused by Pseudomonas aeruginosa are closely linked to the formation of biofilms and resistance mechanisms, which include the presence of amyloid fibrils. Amyloids are a class of proteins known for their ability to self-assemble into highly structured fibrils. Their pivotal role as major virulence factors in microbial pathogens makes them leading targets for developing novel antimicrobial treatments. However, our understanding of microbial amyloids is limited, especially in a physiological context. This gap is particularly pronounced for the Functional Amyloids in Pseudomonas (Faps). Detailed insights into their structures and how they interact within biofilms are still elusive, mostly due to the limits of imaging technologies. To address this knowledge gap, in the FAPbulous project, I will use a multidisciplinary approach by combining structural biology, biophysics and genetic engineering techniques to (1) determine the in vitro structures of Faps amyloid fibrils, (2) characterize their molecular mechanism within biofilms and (3) assess the effects of inhibitors known to prevent Faps fibrillation on biofilm formation and resilience. This highly innovative project will be achieved by using the recent advances in cryo-electron and super resolution fluorescence microscopies to gain unprecedented insights into the structural dynamics of Faps within medically relevant biofilms and unveil their detailed mechanism of action, paving the way for novel antimicrobial strategies. The interdisciplinarity of FAPbulous will massively improve my research profile, endowing me with the scientific and soft skills necessary to perform future multidisciplinary research at the interface of structural biology, host:pathogen interactions and protein aggregation.