Determining structural changes in model membranes of Gram-negative bacteria during their interaction with antimicrobial peptides: spectroscopic and microscopic study under electrochemical control
Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
Final Report Abstract
Summarizing, this research project was realized successfully. The work plan of the project was well prepared and allowed in-time realization of individual research aims. One of the main achievements of this project is the elaboration of the fabrication protocols of stable and realistic models of the outer membranes of Gram-negative bacteria. This step of the project realization required a large number of testing and took longer than initially expected. Moreover, lipid bilayers with asymmetric composition and charge distribution displayed, distinct to commonly studied zwitterionic lipid bilayers, behaviour in electric fields. To understand potential-driven changes in the orientation and hydration of lipids molecules in the lipid bilayer, in situ polarization modulation infrared reflection-absorption spectroscopy (PM IRRAS) was employed. However, this technique is not sensitive for investigations of the flow of water into the bilayer. To understand the distinct spectroelectrochemical characteristic of the model lipid bilayers fabricated during the realization of this project, Dr. Brand established collaboration with Prof. Sek (University of Warsaw, Poland). In Warsaw in situ quartz crystal microbalance experiments with electrochemical control were done. In this way a holistic picture of molecular-scale changes in the supramolecular assembly of the lipid bilayer exposed to changing electric fields is available. Within realization of this project, preparation protocols of realistic model of the outer membrane of Gram-negative bacteria were successfully elaborated. Furthermore, the structural changes in the outer membrane exposed to changing, physiological electric fields were investigated. These results show that lipids in the bacterial cell membrane respond differently than zwitterionic phospholipids, to modulations of the membrane potential. This is a new result, which confirms first quantum chemical studies, published in the literature in the last three years. Clearly, results obtained within the realization of this project are important to understand structural changes occurring in the cell membrane envelope of bacteria. In the last part of the project realization, the interaction of the outer membrane (first contact surface) with an antimicrobial peptide (melittin) was studied. Of course, melittin is able to lyse the outer membrane. However, the interaction of melittin with lipopolysaccharides is different than with zwitterionic phospholipids. The binding of melittin affects the membrane stability in electric fields, suggesting that the potential drop across the membrane may significantly affect the antimicrobial peptide – bacterial cell membrane interaction. Direct electrostatic interactions (charge-charge and chargedipole) between amine groups in melittin and carboxylic and hydroxyl groups in lipopolysaccharide were detected. The electrostatic interactions anchor the peptide on the membrane surface. The anchored peptide undergoes pronounced conformational changes and penetrates the lipid bilayer forming channels and finally leads to membrane disruption. This work contributes to a general understanding of the mechanism of action of antimicrobial peptides on bacterial cell membranes. It also shows that a symmetric phospholipid bilayer is not a sufficient model of structurally complex bacterial membranes. It demonstrates the need of further investigations of the structure, dynamics, hydration and hydrogen bonds network in models of bacterial and viral cell membranes.
Publications
- The shape of lipid molecules affects potential-driven molecular-scale rearrangements in model cell membranes on electrodes. Bioelectrochemistry 2020, 132 (107443)
Khairalla, B.; Juhaniewicz-Debinska, J.; Sek, S.; Brand, I.
(See online at https://doi.org/10.1016/j.bioelechem.2019.107443)
