• Energy Research

ABSTRACTPeptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here we developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we demonstrate this assay with PBP1B homologues fromEscherichia coli, Pseudomonas aeruginosaandAcinetobacter baumanniiin the presence or absence of their cognate lipoprotein activator. Our assay allows unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high throughput screening for new antimicrobials.

Biofilms are central to some of the most urgent global challenges across diverse fields of application, from medicine to industries to the environment, and exert considerable economic and social impact. A fundamental assumption in anti-biofilms has been that the coating on a substrate surface is solid. The invention of slippery liquid-infused porous surfaces─a continuously wet lubricating coating retained on a solid surface by capillary forces─has led to this being challenged. However, in situations where flow occurs, shear stress may deplete the lubricant and affect the anti-biofilm performance. Here, we report on the use of slippery omniphobic covalently attached liquid (SOCAL) surfaces, which provide a surface coating with short (ca. 4 nm) non-cross-linked polydimethylsiloxane (PDMS) chains retaining liquid-surface properties, as an antibiofilm strategy stable under shear stress from flow. This surface reduced biofilm formation of the key biofilm-forming pathogens Staphylococcus epidermidis and Pseudomonas aeruginosa by three-four orders of magnitude compared to the widely used medical implant material PDMS after 7 days under static and dynamic culture conditions. Throughout the entire dynamic culture period of P. aeruginosa, SOCAL significantly outperformed a typical antibiofilm slippery surface [i.e., swollen PDMS in silicone oil (S-PDMS)]. We have revealed that significant oil loss occurred after 2-7 day flow for S-PDMS, which correlated to increased contact angle hysteresis (CAH), indicating a degradation of the slippery surface properties, and biofilm formation, while SOCAL has stable CAH and sustainable antibiofilm performance after 7 day flow. The significance of this correlation is to provide a useful easy-to-measure physical parameter as an indicator for long-term antibiofilm performance. This biofilm-resistant liquid-like solid surface offers a new antibiofilm strategy for applications in medical devices and other areas where biofilm development is problematic.