Secondary multidrug transporters use the energy stored in transmembrane ion gradients to bind and extrude a variety of weakly related chemical structures. These polyspecific antiporters challenge the notions of high-affinity conformation and strict ion-substrate coupling, inherent to the alternating-access model of transport. In order to investigate the mechanism of secondary multidrug transport at a molecular level, we study LmrP, a Major Facilitator Superfamily (MFS) multidrug transporter from Lactococcus lactis, which relies on the proton-motive force to achieve the transport of its diverse substrates. We carried out Double Electron Electron (DEER) distance measurements to elucidate the conformational dynamics underlying the transport cycle. We monitored the conformational response of a library of labeled double cysteine mutants to the presence of ligand(s) and proton(s). We investigated the role of the lipid environment by performing the measurements on mutants reconstituted in nanoscale soluble lipid bilayers (nanodiscs). During this work, we have demonstrated that the transporter oscillates between two main conformations, the outward-open and the inward-open. We have shown that the protonation of conserved acidic residues is the driving force of the conformational transition. The lipid bilayer modulates the equilibrium and allows the transition to occur at higher and more physiological pH values. By using specific lipid compositions, we observe that the lipid headgroup is crucial in the regulation of the conformational equilibrium. Based on our data, we propose a model of secondary multidrug transport wherein substrate binding initiates the transport cycle by catalysing proton entrance from the extracellular side. Subsequent protonation of membrane-embedded acidic residues triggers a cascade of conformational changes that results in substrate extrusion to the extracellular side and proton release in the cytosol. We suggest the opening and closing of the extracellular site is tightly regulated while the cytoplasmic side is more flexible. To our knowledge, this work provides the first direct structural evidence of the role of the lipids in the regulation of the conformational dynamics of a membrane transporter. / La surexpression de transporteurs capables d’expulser des molécules cytotoxiques est un mécanisme connu de résistance aux antibiotiques de la cellule bactérienne. Certains transporteurs ont développé la capacité de reconnaitre et d’expulser des substrats de structures diverses, donnant lieu à une résistance multidrogue de la part de leur hôte. Ces transporteurs multidrogues sont présents dans une variété de classes de protéines, distribués dans tous les règnes du vivant. Parmi celles - ci, la famille MFS (Major Facilitator Superfamily) comprend la majorité des transporteurs multidrogues activé par une source d’énergie secondaire, et jouent un rôle crucial dans la propagation de maladies nosocomiales d’origine bactérienne. Une meilleure compréhension des mécanismes fondamentaux du transport multidrogue secondaire est le prérequis indispensable à l’élaboration de thérapies adaptées. En particulier, une description détaillée des changements conformationnels impliqués dans le transport, et une identification des mécanismes moléculaires qui permettent de lier la source d’énergie au transport fait actuellement défaut. Afin de pallier ce manque, ce travail vise à étudier LmrP (Lactococcus lactis multidrug resistance Protein) un transporteur MFS qui confère à son hôte Lactococcus lactis la résistance à divers antibiotiques et agents cytotoxiques de structure et de charge variable. Cette extrusion active est alimentée par un cotransport énergétiquement favorable de protons. Nous avons étudié le mécanisme de transport de LmrP à l’échelle moléculaire en utilisant la technique spectroscopique Double Electron Electron Resonance (DEER), qui permet de mesurer des variations de distances à l’échelle nanométrique, idéale pour observer les mouvements intramoléculaires d’un transporteur MFS. Différents aspects moléculaires susceptibles de réguler le cycle de transport sont étudiés de façon indépendante et couplée :le rôle des protons, des différents substrats, et de l’environnement lipidique. Sur base de cette cartographie conformationnelle, un mécanisme de transport couplant tous les acteurs moléculaires est proposé :la liaison du proton à un motif d’acides aminés conservé constitue la base de la transition conformationnelle, les divers substrats ayant pour rôle de permettre aux protons d’accéder à ce motif. La compétition substrat-proton est la base du transport couplé. Notre travail a mis en évidence le rôle fondamental de l’environnement lipidique, qui module l’équilibre conformationnel du transporteur en interagissant avec un ou plusieurs motif(s) conservé(s). Par ailleurs, notre étude questionne le paradigme actuel de transport au sein de la famille MFS car elle démontre que les changements conformationnels globaux passent par des réarrangements locaux et coordonnés. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Introduction: Nosocomial infections remain a major cause of mortality and morbidity. The problem is most apparent in intensive care units (ICUs). Most ICU patients are compromised and vulnerable as a result of disease or severe trauma. One in ten people admitted to hospital is given an antibiotic for infection. The risk of acquiring a nosocomial infection in a European ICU is approximately 20%. It is vitally important that ways are found to prevent transmission between patients and personnel, and that local hygiene routines and antibiotic policies are developed. This thesis is a holistic work focused particularly on antimicrobial antibiotic resistance, antibiotic consumption and to some extent on hygiene in Swedish ICUs. Aims: The general aim of this thesis was to investigate bacterial resistance and antibiotic consumption in Swedish ICUs and to try to correlate ICU demographic data with antibiotic consumption and antibiotic resistance. Additional aims were to investigate on which clinical indications antibacterial drugs are prescribed in the ICU, and to investigate the emergence of resistance and transmission of Pseudomonas aeruginosa in the ICU using cluster analysis based on antibiograms and genotype data obtained by AFLP. Material and methods: In paper 1-3, antibiotic consumption data together with bacterial antibiotic resistance data and specific ICU-demographic data were collected from an increasing number of ICUs over the years 1997-2001. Data from ICUs covering up to six million out of Sweden’s nine million inhabitants were included. In paper 4, the indications for antibiotic prescribing were studied during two weeks in 2000. Paper 5 investigated Pseudomonas aeruginosa isolates in order to detect cross-transmission with genotype obtained by AFLP, and antibiogram-based cluster analysis was also performed in order to see if this could be a quicker and easier substitute for AFLP. Results: This thesis has produced three important findings. Firstly, antibiotic consumption in participating ICUs was relatively high during the study period, and every patient received on average more than one antimicrobial drug per day (I-IV). Secondly, levels of antimicrobial drug resistance seen in S. aureus, E. coli and Klebsiella spp remained low when data were pooled from all ICUs throughout the study period, despite relatively high antibiotic consumption (I-V). Thirdly, the prevalence of antibiotic resistance in CoNS and E. faecium, cefotaxime resistance in Enterobacter, and ciprofloxacin and imipenem resistance in P. aeruginosa was high enough to cause concern. Conclusion: For the period studied, multidrug resistance in Swedish ICUs was not a major problem. Signs of cross-transmission with non-multiresistant bacteria were observed, indicating a hygiene problem and identifying simple improvements that could be made in patient care guidelines and barrier precautions. A need for better follow up of prescribed antibiotics was evident. With further surveillance studies and monitoring of antibiotics and bacterial resistance patterns in the local setting as well as on a national and international level, some of the strategic goals in the prevention and control of the emergence of antimicrobial-resistant microbes may be achievable.