Real-Time Observation of Antimicrobial Polycation Effects on Escherichia coli: Adapting the Carpet Model for Membrane Disruption to Quaternary Copolyoxetanes

• Published in: Langmuir Vol. 32; no. 12; pp. 2975 - 2984
• Main Authors: Wang, Congzhou, Zolotarskaya, Olga Y, Nair, Sithara S, Ehrhardt, Christopher J, Ohman, Dennis E, Wynne, Kenneth J, Yadavalli, Vamsi K
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 29-03-2016
• Subjects: Anti-Bacterial Agents - chemical synthesis; Anti-Bacterial Agents - pharmacology; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Escherichia coli - drug effects; Microscopy, Atomic Force; Polylysine - chemistry; Polyurethanes - chemistry; Polyurethanes - pharmacology; Surface-Active Agents - chemical synthesis; Surface-Active Agents - pharmacology
• ISSN: 0743-7463; 1520-5827
• DOI: 10.1021/acs.langmuir.5b04247

Real-time atomic force microscopy (AFM) was used for analyzing effects of the antimicrobial polycation copolyoxetane P­[(C12)-(ME2Ox)-50/50], C12–50 on the membrane of a model bacterium, Escherichia coli (ATCC# 35218). AFM imaging showed cell membrane changes with increasing C12–50 concentration and time including nanopore formation and bulges associated with outer bacterial membrane disruption. A macroscale bactericidal concentration study for C12–50 showed a 4 log kill at 15 μg/mL with conditions paralleling imaging (1 h, 1x PBS, physiological pH, 25 °C). The dramatic changes from the control image to 1 h after introducing 15 μg/mL C12–50 are therefore reasonably attributed to cell death. At the highest concentration (60 μg/mL) further cell membrane disruption results in leakage of cytoplasm driven by detergent-like action. The sequence of processes for initial membrane disruption by the synthetic polycation C12–50 follows the carpet model posited for antimicrobial peptides (AMPs). However, the nanoscale details are distinctly different as C12–50 is a synthetic, water-soluble copolycation that is best modeled as a random coil. In a complementary AFM study, chemical force microscopy shows that incubating cells with C12–50 decreased the hydrophobicity across the entire cell surface at an early stage. This finding provides additional evidence indicating that C12–50 polycations initially bind with the cell membrane in a carpet-like fashion. Taken together, real time AFM imaging elucidates the mechanism of antimicrobial action for copolyoxetane C12–50 at the single cell level. In future work this approach will provide important insights into structure–property relationships and improved antimicrobial effectiveness for synthetic amphiphilic polycations.