Role of lipopolysaccharides and lipoteichoic acids on C-Chrysophsin-1 interactions with model Gram-positive and Gram-negative bacterial membranes.

Antimicrobial peptides (AMPs) are attractive as biomaterial coatings because they have broad spectrum activity against different microbes, with a low likelihood of incurring antimicrobial resistance. Direct action against the bacterial membrane is the most common mechanism of action (MOA) of AMPs, with specific MOAs dependent on membrane composition, peptide concentration, and environmental factors that include temperature. Chrysophsin-1 (CHY1) is a broad spectrum salt-tolerant AMP that is derived from a marine fish. A cysteine modification was made to the peptide to facilitate attachment to a surface, such as a biomedical device. The authors used quartz crystal microbalance with dissipation monitoring to study how temperature (23 and 37 °C) and lipid composition influence the MOA of cysteine-modified peptide (C-CHY1) with model membranes comprised of supported lipid bilayers (SLBs). These two temperatures were used so that the authors could better understand the differences in behavior between typical lab temperatures and physiologic conditions. The authors created model membranes that mimicked properties of Gram-negative and Gram-positive bacteria in order to understand how the mechanisms might differ for different types of bacterial systems. SLB models of Gram-positive bacterial membranes were formed using combinations of phosphatidylcholine, phosphatidylglycerol (PG), and S. aureus-derived lipoteichoic acid (LTA). SLB models of Gram-negative bacterial membranes were formed using combinations of phosphatidylethanolamine (PE), PG, and E. coli-derived lipopolysaccharides (LPS). The molecules that distinguish Gram-positive and Gram-negative membranes (LTA and LPS) have the potential to alter the MOA of C-CHY1 with the SLBs. The authors' results showed that the MOA for the Gram-positive SLBs was not sensitive to temperature, but the LTA addition did have an effect. Specifically, similar trends in frequency and dissipation changes across all overtones were observed, and the same mechanistic trends were observed in the polar plots at 23 and 37 °C. However, when LTA was added, polar plots showed an association between C-CHY1 and LTA, leading to SLB saturation. This was demonstrated by significant changes in dissipation, while the frequency (mass) was not increasing after the saturation point. For the Gram-negative SLBs, the composition did not have a significant effect on MOA, but the authors saw more differences between the two temperatures studied. The authors believe this is due to the fact that the gel-liquid crystal transition temperature of PE is 25 °C, which means that the bilayer is more rigid at 23 °C, compared to temperatures above the transition point. At 23 °C, a significant energetic shift would be required to allow for additional AMP insertion. This could be seen in the polar plots, where there was a steep slope but there was very little mass addition. At 37 °C, the membrane is more fluid and there is less of an energetic requirement for insertion. Therefore, the authors observed greater mass addition and fewer changes in dissipation. A better understanding of C-CHY1 MOA using different SLB models will allow for the more rational design of future therapeutic solutions that make use of antimicrobial peptides, including those involving biomaterial coatings.

QCM-D characterization of time-dependence of bacterial adhesion.

Quartz crystal microbalance with dissipation monitoring (QCM-D) is becoming an increasingly popular technique that can be employed as part of experimental and modeling investigations of bacterial adhesion. The usefulness of QCM-D derives from this technique's ability to probe binding and interactions under dynamic conditions, in real time. Bacterial adhesion is an important first step in the formation of biofilms, the control of which is relevant to industries that include shipping, water purification, packaging, and biomedical devices. However, many questions remain unanswered in the bacterial adhesion process, despite extensive research in this area. With QCM-D, multiple variables affecting bacterial adhesion can be studied, including the roles of substrate composition, chemical modification, solution ionic strength, environmental temperature, shear conditions, and time. Recent studies demonstrate the utility of QCM-D in developing new bacterial adhesion models and studying different stages of biofilm formation. We provide a review of how QCM-D has been used to study bacterial adhesion at stages ranging from the first step of bacterial adhesion to mature biofilms, and how QCM-D studies are being used to promote the development of solutions to biofilm formation.

Proposed Mechanisms of Tethered Antimicrobial Peptide Chrysophsin-1 as a Function of Tether Length Using QCM-D.

Rising antibiotic resistance has led to a call for the development of alternative antibiotics. Antimicrobial peptides (AMPs) are promising, but their potential has not been fully explored because of toxicity and lack of stability in vivo. Multiple recent studies have focused on surface immobilization of AMPs to maximize antimicrobial activity and stability while mitigating toxicity. We covalently tethered cysteine-modified chrysophsin-1 (C-CHY1) via PEG of three molecular weights, 866, 2000, and 7500. Quartz crystal microbalance with dissipation (QCM-D) was used to characterize thickness and grafting density of tethered C-CHY1, which were related to its activity against Staphylococcus aureus and Escherichia coli and found to be important in determining mechanisms leading to activity. The PEG 866 tether promoted an antimicrobial mechanism that caused displacement of positive cations from bacterial membranes. The PEG 7500 tether maintained C-CHY1's ability to effectively form membrane pores, promoting the highest activity. When AMP was tethered with PEG 2000, antimicrobial activity was limited, apparently because neither mechanism of AMP activity was able to occur with this tether. Using QCM-D, we calculated thickness and density of PEG-tethered C-CHY1 and correlated it with antimicrobial effectiveness to determine the mechanisms by which tethered C-CHY1 acts against bacteria.