Bacterial biofilm functionalization through Bap amyloid engineering

Abstract

Biofilm engineering has emerged as a controllable way to fabricate living structures with programmable functionalities. The amyloidogenic proteins comprising the biofilms can be engineered to create self-assembling extracellular functionalized surfaces. In this regard, facultative amyloids, which play a dual role in biofilm formation by acting as adhesins in their native conformation and as matrix scaffolds when they polymerize into amyloid-like fibrillar structures, are interesting candidates. Here, we report the use of the facultative amyloid-like Bap protein of Staphylococcus aureus as a tool to decorate the extracellular biofilm matrix or the bacterial cell surface with a battery of functional domains or proteins. We demonstrate that the localization of the functional tags can be change by simply modulating the pH of the medium. Using Bap features, we build a tool for trapping and covalent immobilizing molecules at bacterial cell surface or at the biofilm matrix based on the SpyTag/SpyCatcher system. Finally, we show that the cell wall of several Gram-positive bacteria could be functionalized through the external addition of the recombinant engineered Bap-amyloid domain. Overall, this work shows a simple and modulable system for biofilm functionalization based on the facultative protein Bap.

Hierarchical organization and assembly of the archaeal cell sheath from an amyloid-like protein

“…Our study shows that the outmost layer of M. hungatei cell is composed of a single small protein of just 40 kDa with an amyloid-like cross-β structure, thus forming a functional amyloid polymer. Other microbial functional amyloids are known, including the extracellular fibrillar structures described in both Gram-negative and positive bacteria with roles in biofilm formation and adhesion, for example, the curli, Bap and Esp orthologs 39 , P1 adhesions, Harpins and modulins (see review 40 ). Many structural and functional aspects of their biological roles remain to be fully elucidated.…”

Bacterial Patterning: A Promising Biofabrication Technique

“…98 Microfluidics creates controlled microenvironments for bacterial cells to grow and interact within microcavities of different shapes and sizes. 99 Additionally, it provides a platform for monitoring and measuring interspecies and biochemical interactions among bacteria with high precision. 100 However, microfluidics faces certain challenges in biopatterning, such as microchannel clogging, the risk of cross-contamination, and distortion of patterns.…”

“…Microfluidics, the science and technology of manipulating tiny volumes of fluid within devices with tailored microscale channels, has emerged as another promising class of biopatterning technologies for efficiently immobilizing bacteria into customized patterns. , Typically, microfluidic devices are made of elastomeric PDMS using multilayer soft lithography. , Common designs for microfluidic cultivating devices include microwells, microdroplets, and microchambers . Microfluidics creates controlled microenvironments for bacterial cells to grow and interact within microcavities of different shapes and sizes . Additionally, it provides a platform for monitoring and measuring interspecies and biochemical interactions among bacteria with high precision .…”

Rational design of functional amyloid fibrillar assemblies

“…123 Until now, several bacterial amyloid systems have been successfully engineered for such purposes, including E. coli curli CsgA, 124 Bacillus subtilis TasA 49 and Staphylococcus aureus Bap. 125 Indeed, these amyloid systems have been genetically modified with diverse functional domains ranging from short peptides (e.g., His tag (6 aa), FLAG tag (8 aa), mfp (48 aa)) to globular proteins (e.g., mCherry (237 aa), PETase (290 aa), organophosphate hydrolase (337 aa)) through the extracellular biofilm display technique. 126 The constructed living biofilms have demonstrated a wide range of applications in the environment, energy, and medicine.…”

“…123 Until now, several bacterial amyloid systems have been successfully engineered for such purposes, including E. coli curli CsgA, 124 Bacillus subtilis TasA 49 and Staphylococcus aureus Bap. 125 Indeed, these amyloid systems have been genetically modified with diverse functional domains ranging from short peptides ( e.g. , His tag (6 aa), FLAG tag (8 aa), mfp (48 aa)) to globular proteins ( e.g.…”