The action of antimicrobial peptides on supported lipid bilayers investigated by biophysical methods

2017-03-02T00:05:00Z (GMT) by Piantavigna, Stefania
The emergence of bacteria that have developed resistance towards “traditional” antibiotics is becoming a serious global health threat. Consequently, alternative approaches are needed to find new drugs that can act directly as antibiotics or to assist traditional drugs to improve efficacy. The emergence of antimicrobial peptides (AMPs) as a possible new class offers promise. AMPs represent a large and varied group of “natural antibiotics” present in virtually every organism. However, in order to develop new drugs derived from AMPs knowledge of the bioactivity of these is needed, such as concentration ranges and specific bacterial targets. Of great practical importance is to have a comprehensive understanding of the mechanism of action of AMPs, so that the risk of cross-reactivity and development of new bacterial resistance is minimised. All AMPs interact with the cell membrane, which is a complex and dynamic system, mostly containing phospholipids and proteins. Phospholipids are not simple “bricks” of the membrane, but they themselves are involved in various cellular processes. Therefore, biomimetic membranes, e.g. supported lipid bilayers (SLBs), represent a valid approach for investigating the interactions between lipids and AMPs. Creation of a supported membrane reduces the complexity of those studies to just one variable. Many variables influence the formation of SLBs and a protocol regarding the formation of SLBs assembled on gold-coated sensors is described in Paper 1. The membrane deposition and the peptide-membrane interactions were investigated using a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) (Paper 2). Thus, the action of various peptides were investigated with zwitterionic membranes, which contained negatively charged lipid (bacterial membranes), or cholesterol (mammalian membranes). The action of the two most widely studied AMPs, melittin and magainin 2, on SLBs, has been examined using QCM-D in Chapter 3. These peptides formed “toroidal pores”, which lead to membrane disruption. However, the action of these peptides has been found to be both concentration and composition dependent. Many AMPs are enriched in a particular amino acid residue. The influence of several of these peptide residues has been investigated using QCM-D in Chapters 4, 5 and 6. The action of proline-rich peptides apidaecins HbI and HbII, the variant Api88 and oncocin peptides on SLBs, are illustrated in Papers 3, 4 and 5, respectively. These peptides were found to insert into the membrane without any evidence of disruption. The influence of lipid composition on the activity of the arginine-rich peptide Tat has also been investigated with QCM together with scanning electrochemical microscopy (SECM) (Chapter 5). The cell-penetrating Tat peptide was shown to act as a lytic AMP in the presence of negatively charged membranes (Papers 6 and 7). The addition of tryptophan residues in the sequence of a short arginine-rich peptide, (RW)3, caused a dramatic switch from cell penetrating to lytic activity, while the inclusion of ruthenocene in the peptide RcCO-W(RW)2 did not affect the peptide activity (Chapter 6). Finally, in Chapter 7, Uperin 3.5, an amyloid-like AMP, demonstrated that the amyloid fibrils are not necessary for the membrane-disruption. However, the action of Uperin 3.5 towards zwitterionic membranes is switched to insertion if cholesterol is present in the membrane. Thus, QCM has been demonstrated to be an invaluable technique for characterising, in real time, the action of various peptides on SLBs of bacterial mimetic composition and mammalian. However, the combination of QCM with other techniques e.g. SECM, is always encouraged to reinforce this data and to gain a wide perspective of activity.