Structural and functional investigations of bacterial fibrillar adhesins

PROJECT SUMMARY The ability of bacteria to adhere to each other and both biotic and abiotic surfaces is key to their ability to cause infection and persist in the environment. Bacteria can adhere using proteinaceous adhesins, including fibrillar adhesins. Key characteristics of fibrillar adhesins include: (i) they are extracellular, surface-associated proteins, (ii) they possess an adhesive domain as well as a repetitive stalk domain, and (iii) they are either a monomer or homotrimer (i.e., identical, coiled-coil) of a high molecular weight protein. Fibrillar adhesins are widely abundant in bacteria with at least 26% of all UniProt bacterial reference proteomes containing predicted fibrillar adhesin- like proteins. They provide a range of functionalities to the bacterial surface including adherence to host tissues, and in many instances, fibrillar adhesins are key to structuring biofilms, which are aggregated bacterial communities that cause chronic, difficult-to-treat infections. Thus, strategies to block fibrillar adhesin-mediated bacterial adhesion and biofilm formation are desirable therapeutic targets. However, despite the wide-abundance and prominent role in infection that fibrillar adhesins play, we have limited understanding of fibrillar adhesins because their massive size and repetitive sequences have prevented structural and molecular biophysical studies of full-length, intact proteins. Instead, structures and biomolecular interactions of well-behaving domains of some fibrillar adhesins have been determined, and in a handful of cases, the domain structures have been stitched together with a heavy reliance on homology modeling. The lack of structural insight into fibrillar adhesins is problematic because protein structure is directly related to function. As it currently stands, the field lacks key understanding of a widely used mechanism of bacterial attachment, and we are missing out on opportunities to rationally design therapeutics to prevent bacterial attachment to abiotic and biotic surfaces and biofilm formation. To address this knowledge gap, we will develop novel approaches to elucidate the structure and interactions of a model fibrillar adhesin, the Pseudomonas aeruginosa biofilm matrix protein called CdrA, and then explore the impact of these features on fibrillar adhesin function. The MIRA award will enable the PI (Reichhardt) to dedicate greater time and resources to addressing this knowledge gap as well as training and mentoring a diverse group of scientists at the interface between molecular biophysics and microbiology. Looking to the future, this proposed research will open the path to the structural and molecular biophysical studies of other fibrillar adhesins as well as other high molecular weight or repetitive proteins (e.g., eukaryotic extracellular matrix proteins).