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Title: In vitro models of the bacterial outer membrane for antibiotic research
Authors: Paracini, Nicolo
Issue Date: 2019
Publisher: Newcastle University
Abstract: Antibiotic resistance is rapidly becoming a global health threat and the proportion of bacteria capable of evading the effects of antibiotics is progressively rising. In the case of Gramnegative bacteria this phenomenon is exacerbated by the outermost layer of their cellular envelope, the outer membrane (OM), an asymmetric lipid bilayer which represents an impenetrable barrier for many antibiotics. The defining characteristic of the OM is lipid asymmetry, with an inner leaflet of phospholipids and an outer layer of lipopolysaccharides (LPS), which confers on it its peculiar barrier properties. Due to its complexity, obtaining high-resolution structural information about the OM directly on the surface of bacteria is a challenging task. Thus, a reductionist approach to the problem consists of reconstituting OM components in vitro to create models that mimic the OM natural structure and composition, enabling its investigation under controlled conditions. Here, asymmetric OM models were used to investigate the effects of two antimicrobials, the last resort drug polymyxin B (PmB) and the bacterial toxin colicin N (ColN), providing insights into their mode of action and demonstrating the potential of these models as powerful research tools. The only structural technique capable of resolving the asymmetry of these 5 nm thin layers is neutron reflectometry, which was the key tool used throughout this work. The differential sensitivity of neutrons towards hydrogen (1H) and deuterium (2H), applied to the study of isotopically asymmetric OM model bilayers, enabled accurate determination of the lipid asymmetry of these systems which would not have been possible with any other biophysical technique. The studies on the effects of PmB clearly revealed the correlation between the antibiotic’s effect and the transition of the OM from a gel to a fluid phase confirming the previously disputed existence of a liquid crystalline phase in the OM. Furthermore, a comparison between different OM models highlighted the requirement for accurate systems to reproduce the behaviour displayed in vivo. The structural investigation of ColN binding to OM models led to the identification of two separate membrane-bound toxin conformations determined by the specificity of the interaction. Additionally, a disordered loop region of ColN responsible for both LPS binding and toxicity was identified. Finally, complex OM models integrating LPS from pathogenic bacteria were assembled and characterised, opening up new possibilities to study more realistic models of the Gram-negative bacterial outer membrane.
Description: PhD Thesis
Appears in Collections:Institute for Cell and Molecular Biosciences

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