The function of peptide-mimetic anionic groups and salt bridges in the antimicrobial activity and conformation of cationic amphiphilic copolymers.

Herein we report the synthesis of ternary statistical methacrylate copolymers comprising cationic ammonium (amino-ethyl methacrylate: AEMA), carboxylic acid (propanoic acid methacrylate: PAMA) and hydrophobic (ethyl methacrylate: EMA) side chain monomers, to study the functional role of anionic groups on their antimicrobial and hemolytic activities as well as the conformation of polymer chains. The hydrophobic monomer EMA was maintained at 40 mol% in all the polymers, with different percentages of cationic ammonium (AEMA) and anionic carboxylate (PAMA) side chains, resulting in different total net charge for the polymers. The antimicrobial and hemolytic activities of the copolymer were determined by the net charge of +3 or larger, suggesting that there was no distinct effect of the anionic carboxylate groups on the antimicrobial and hemolytic activities of the copolymers. However, the pH titration and atomic molecular dynamics simulations suggest that anionic groups may play a strong role in controlling the polymer conformation. This was achieved via formation of salt bridges between cationic and anionic groups, transiently crosslinking the polymer chain allowing dynamic switching between compact and extended conformations. These results suggest that inclusion of functional groups in general, other than the canonical hydrophobic and cationic groups in antimicrobial agents, may have broader implications in acquiring functional structures required for adequate antimicrobial activity. In order to explain the implications, we propose a molecular model in which formation of intra-chain, transient salt bridges, due to the presence of both anionic and cationic groups along the polymer, may function as "adhesives" which facilitate compact packing of the polymer chain to enable functional group interaction but without rigidly locking down the overall polymer structure, which may adversely affect their functional roles.

Solution-Mediated Modulation of Pseudomonas aeruginosa Biofilm Formation by a Cationic Synthetic Polymer.

Bacterial biofilms and their associated infections are a continuing problem in the healthcare community. Previous approaches utilizing anti-biofilm coatings suffer from short lifetimes, and their applications are limited to surfaces. In this research, we explored a new approach to biofilm prevention based on the hypothesis that changing planktonic bacteria behavior to result in sub-optimal biofilm formation. The behavior of planktonic Pseudomonas aeruginosa exposed to a cationic polymer was characterized for changes in growth behavior and aggregation behavior, and linked to resulting P. aeruginosa biofilm formation, biomass, viability, and metabolic activity. The incubation of P. aeruginosa planktonic bacteria with a cationic polymer resulted in the aggregation of planktonic bacteria, and a reduction in biofilm development. We propose that cationic polymers may sequester planktonic bacteria away from surfaces, thereby preventing their attachment and suppressing biofilm formation.

Decoupling the Functional Roles of Cationic and Hydrophobic Groups in the Antimicrobial and Hemolytic Activities of Methacrylate Random Copolymers.

In this study, we report the antimicrobial and hemolytic activities of ternary statistical methacrylate copolymers consisting of cationic ammonium (amino-ethyl methacrylate: AEMA), hydrophobic alkyl (ethyl methacrylate: EMA), and neutral hydroxyl (hydroxyethyl methacrylate: HEMA) side chain monomers. The cationic and hydrophobic functionalities of copolymers mimic the cationic amphiphilicity of naturally occurring antimicrobial peptides (AMPs). The HEMA monomer units were used to separately modulate the compositions of cationic and hydrophobic monomers, and we investigated the effect of each component on the antimicrobial and hemolytic activities of copolymers. Our data indicated that increasing the number of cationic groups of the copolymers to be more than 30 mol % did not increase their antimicrobial activity against Escherichia coli. The number of cationic side chains in a polymer chain at this threshold is 5.5-7.7, which is comparable to those of natural antimicrobial peptides such as maginin (+6). The MIC values of copolymers with >30 mol % of AEMA depend on only the mol % of EMA, indicating that the hydrophobic interactions of the copolymers with E. coli cell membranes determine the antimicrobial activity of copolymers. These results suggest that the roles of cationic and hydrophobic groups can be controlled independently by design in the ternary copolymers studied here.

Monitoring Antimicrobial Mechanisms of Surface-Immobilized Peptides in Situ.

Antimicrobial peptides (AMPs) in free solution can kill bacteria by disrupting bacterial cell membranes. Their modes of action have been extensively studied, and various models ranging from pore formation to carpet-like mechanisms were proposed. Surface-immobilized AMPs have been used as coatings to kill bacteria and as sensors to capture bacteria, but the interaction mechanisms of surface-immobilized AMPs and bacteria are not fully understood. In this research, an analytical platform, sum frequency generation (SFG) microscope, which is composed of an SFG vibrational spectrometer and a fluorescence microscope, was used to probe molecular interactions between surface-immobilized AMPs and bacteria in situ in real time at the solid/liquid interface. SFG probed the molecular structure of surface-immobilized AMPs while interacting with bacteria, and fluorescence images of dead bacteria were monitored as a function of time during the peptide-bacteria interaction. It was believed that upon bacteria contact, the surface-immobilized peptides changed their orientation and killed bacteria. This research demonstrated that the SFG microscope platform can examine the structure and function (bacterial killing) at the same time in the same sample environment, providing in-depth understanding on the structure-activity relationships of surface-immobilized AMPs.