Protein translocation across the gram negative bacterial membrane /

PhD ThesisGram-negative bacteria are becoming increasingly resistant to many antibiotics. Unlike other bacteria, gram-negative bacteria have an additional membrane, the so-called outer membrane, which protects them from harmful agents. This membrane is highly asymmetric and contains tightly-packed lipopolysaccharides (LPS) in the outer leaflet and phospholipids in the inner leaflet. This thesis describes a study of the outer membrane protein F (OmpF) which is the most abundant porin present in the outer membrane of Esherichia coli. OmpF plays a key role in the organization of outer membrane and is also a receptor and translocator for colicins, which are antibacterial toxins. The aim of this project is to better understand the defensive barrier of gram-negative bacteria and find routes to pass through it by studying the interaction of OmpF with colicin N (ColN) and LPS. The solution structure of ColN and the OmpFColN complex in neutral detergents, studied by AUC and small-angle scattering (SAS), indicated that translocation and receptor binding domain of ColN (ColNTR) became more compact when binding to outside of OmpF. Furthermore, OmpF in complex with the protein TolA, which forms part of the ColN translocon complex, was also studied by SAS. It showed that TolA bound to OmpF also became more compact though TolA remains a flexible structure. Furthermore, the interaction of OmpF with LPS was studied. Mutagenesis of positivelycharged residues on OmpF was used to disrupt its electrostatic interaction with LPS. The findings suggested that OmpF has two LPS-binding sites and that OmpF binds to LPS via the minimal Lipid A moiety. This was supported by SAS data. Dynamic light scattering experiments indicated that OmpF, LPS and divalent cations form larger-scale structures, which are reminiscent of the outer membrane of bacteria. Alternative approaches for studying OmpF in amphipol (APol) and nanodisc were also utilised. OmpF/APol assembled as filaments in the absence of free APol but these converted into 2D arrays in the presence of LPS and calcium ions. OmpF could be incorporated into Nanodiscs, significantly increasing their diameters compared to empty Nanodiscs.DPST Scholarship from the Royal Thai Government (RTG

An accurate in vitro model of the E. coli envelope

Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir-Blodgett and Langmuir-Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development

Colicin N Mediates Apoptosis and Suppresses Integrin-Modulated Survival in Human Lung Cancer Cells

The inherent limitations, including serious side-effects and drug resistance, of current chemotherapies necessitate the search for alternative treatments especially for lung cancer. Herein, the anticancer activity of colicin N, bacteria-produced antibiotic peptide, was investigated in various human lung cancer cells. After 24 h of treatment, colicin N at 5–15 µM selectively caused cytotoxicity detected by MTT assay in human lung cancer H460, H292 and H23 cells with no noticeable cell death in human dermal papilla DPCs cells. Flow cytometry analysis of annexin V-FITC/propidium iodide indicated that colicin N primarily induced apoptosis in human lung cancer cells. The activation of extrinsic apoptosis evidenced with the reduction of c-FLIP and caspase-8, as well as the modulation of intrinsic apoptosis signaling proteins including Bax and Mcl-1 were observed via Western blot analysis in lung cancer cells cultured with colicin N (10–15 µM) for 12 h. Moreover, 5–15 µM of colicin N down-regulated the expression of activated Akt (p-Akt) and its upstream survival molecules, integrin β1 and αV in human lung cancer cells. Taken together, colicin N exhibits selective anticancer activity associated with suppression of integrin-modulated survival which potentiate the development of a novel therapy with high safety profile for treatment of human lung cancer

An Accurate In Vitro Model of the E. coli Envelope

<p>Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir–Blodgett and Langmuir–Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development.</p