Inhibition of Staphylococcus aureus biofilm-forming functional amyloids by molecular tweezers

Bacterial biofilms are integrated communities of cells consisting of one or more species joined together in an extracellular polymeric matrix, enabling bacterial cells to efficiently adhere to surfaces and provide shielding from both host immune molecules and externally-provided antibiotics. In several bacterial species, amyloidogenic proteins, generally referred to as "functional amyloids" also participate in biofilm assembly. Functional amyloids function both as building blocks of the biofilm, mediate bacterial communication during biofilm assembly, and also constitute as cytotoxins. Development of substances that disrupt biofilm formation and/or affect biofilm constituents such as functional amyloids is a promising strategy for combating bacterial infections. This quest, however, is often hampered because of the exceptional resilience of biofilm networks and their pronounced hydrophobic properties, which limit the effectiveness of many water-soluble compounds. Staphylococcus aureus (S. aureus), a well-known Gram-positive pathogenic bacterium, generates extensive biofilm structures. S. aureus secretes functional amyloids called phenol-soluble modulins (PSMs). PSMs trigger inflammatory responses, lyse humane cells, contribute to biofilm structuring, and have been recently linked to the pronounced virulence of methicillin-resistant S. aureus (MRSA). PSMα1 and PSMα4 are known to structure the S. aureus biofilm by forming ultra-stable cross-β amyloid fibrils. A synthetic lysine-binding “molecular tweezer” called CLR01 is a powerful modulator of amyloid protein self-assembly and was shown to reduce amyloid proteins` toxicities. The mechanism of action of CLR01 is reversible binding to exposed lysine residues, which disrupts electrostatic and hydrophobic interactions among assembled peptides. CLR01 has been shown to be a generic inhibitor of amyloid formation in several protein families involving lysines and leads to remodeling of the assembly process into formation of nontoxic and nonamyloidogenic structures that can be effectively degraded by In light of the broad-based anti-fibrillar activity of CLR01, this work examines the impact of CLR01 derivatives upon S. aureus biofilms and self-assembly of the biofilm framework proteins PSMa1 and PSMa4.