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Study of the action of antimicrobial peptides by spectroscopic methods : From model membrane to bacterial biofilm

Abstract : The emergence and multiplication of infections involving resistant and multi-resistant antibiotic-resistant bacteria is currently a major challenge in the field of health. Indeed, the resistance of microorganisms to antibiotic molecules has become an increasingly worrying phenomenon, particularly in hospitals, hence the need for new therapies and new antimicrobial agents that are more effective. Many conventional antibiotic agents have been developed in recent years, but many of them still present risks of more or less toxic side effects on eukaryotic cells, and despite their high effectiveness against multi-resistant microorganisms. Thus, antimicrobial peptides are considered good candidates in the fight against multi-resistant microorganisms, mainly because of their low toxicity to eukaryotic cells and their different modes of action compared to conventional antibiotics. Indeed, the latter are generally non-specific and are less likely to lead to the observed resistance phenomena for conventional antibiotics.The aim of the work carried out in this thesis was to study the modes of action of the two different antimicrobial agents; (I) colistin, a cyclic polypeptide already used to treat infections caused by multi-resistant bacteria; and (ii) bovine catestatin (CAT), a recently discovered linear peptide belonging to the Host Defense Peptides Ie produced by the endocrine and immune system of mammals. This study was carried out mainly using different physico-chemical characterization methods such as Atomic Force Microscopy (AFM) and Fourier Transform Infrared Spectroscopy (ATR-FTIR). The activity of colistin on pure and mixed phospholipid membranes (based on DPPC, DOPC and DPPE) was monitored in real time and in situ by these two techniques. The changes in the biochemical fingerprint of the membranes, in particular in the Amide II band and in the Amide II / C = O integrated intensity ratio allowed us to reinforce the hypothesis that the activity of the peptide was more intense On mixed membranes than on pure membranes. Similar changes in the biochemical footprint of these membranes were observed when they were exposed to catestatin. In addition, infrared spectroscopy has also demonstrated conformational changes in the structure of catestatin, in particular by the passage from a so-called random coil structure to an alpha-helix structure, and only in contact with the structure membrane. Such conformational changes could be implicated in the antimicrobial activity and mode of action of this peptide. In addition, we have also been interested in the action of the two peptides on more complex phospholipid membranes since they consist mainly of natural extracts of bacterial lipopolysaccharides (lipid A, LPS-s and LPS-re). Our results showed that the two antimicrobial agents were responsible for a reorganization of the structure of the membranes and in some cases the peptide was at the origin of the formation of the pores of different sizes. The influence of the elasticity of the membrane has also been studied using force spectroscopy (AFM). This study revealed a considerable impact of the peptides on the mechanical properties of the membranes and in particular on their elasticity. In order to approximate the actual conditions of antimicrobial treatment, we exhibited different bacterial E. coli biofilms from doses of two antimicrobial peptides. This latter study was carried out in real time and in situ using infrared spectroscopy and atomic force microscopy. Infrared spectroscopy allowed us to follow the modifications of the biochemical fingerprint of the biofilm on the course of the treatment. Also provided information on possible changes in bacterial metabolism. In parallel with these measurements, the AFM allowed us to observe the changes in the morphology and mechanical properties of the bacterial biofilm as a function of the antimicrobial treatment applied. [...]
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Submitted on : Tuesday, September 26, 2017 - 3:38:05 PM
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Oona Freudenthal. Study of the action of antimicrobial peptides by spectroscopic methods : From model membrane to bacterial biofilm. Medicinal Chemistry. Université de Lorraine, 2016. English. ⟨NNT : 2016LORR0197⟩. ⟨tel-01585915⟩



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