Etude du rôle joué par les porines dans la persistance des infections par Providencia stuartii

Present in the outer membrane of bacteria, porins are the main gateway for soluble molecules, such as nutrients and ions, into the bacteria. They are also the way taken by hydrophilic antibiotics to reach their targets and kill the cell. Under the strong selective pressure caused by antibiotic overuse, bacteria have evolved modified porins that are less permeable to antibiotics. Although not the only strategy developed by bacteria to survive drug treatment, it is an important factor in the spreading phenomenon of multidrug resistant infections.In order to gain further insights into the molecular determinants of antibiotic translocation, the first part of my thesis work aimed at resolving the crystallographic structures of Omp-Pst1 and Omp-Pst2, two non-specific porins encoded in the genome of Providencia stuartii. This bacterial species is not very invasive and, therefore, causes endemic rather than epidemic infections. However, these infections are often fatal given the intrinsically stringent MDR phenotype of this species. It has been shown that Omp-Pst1 is the main entrance for β-lactam antibiotics. To provide structural and functional insights into the contribution of P. stuartii porins to antibiotic resistance phenotypes, structural analysis was undertaken, not only from the wild type strain but also from two clinical mutant strains i.e. Omp-Pst1-99645 and Omp-Pst1-Nea16. Mutations result in more pronounced anion selectivity due to an increased number of positively charged amino acids lining the pore and mostly in the extracellular loops in both mutants compared to the wild type. To further determine whether these mutations contributed to a decrease in antibiotic uptake, we undertook the characterization of β-lactam antibiotics transport kinetics using electrophysiology studies at the single protein level. For the zwitterionic β-lactam tested, single-molecule conductance measurements evidenced a decrease in the association rate constant, in both mutants compared to the wild type. However, we observed instead an increase in these values for the negatively charged β-lactam, which is in good agreement with our structure-based analysis. All together, our results point towards porins playing a major role in the antibiotic resistance mechanism by reducing drug uptake.In the second part of my thesis work, we discovered that porins could self-associate to form adhesive junctions between two cells and could provide the initial scaffold for the establishment of biofilms at early stages of their developpement. The self-matching interaction is mediated by a steric zipper interaction and involves their extracellular loops. In order to confirm the adhesive proprieties of porins, we exploited a large panel of biophysical and imaging methods both in vitro and in vivo. Furthermore, we studied their diffusive proprieties in reconstituted liposomes, to explore whether these self-matching interactions between porins could play a role in cell-to-cell communication. Our results point at a major role of P. stuartii porins, Omp-Pst1 and Omp-Pst2, in cell-to-cell adhesion and make them promising targets to disrupt bacterial biofilm infections.Les porines sont des protéines « canal » qui assurent la diffusion non-spécifique des ions et nutriments au sein des bactéries à Gram-négatif. Elles sont également la voie d'entrée des antibiotiques hydrophiles, en particulier les β-lactames. Des mutations au sein des porines, ou leur sous-expression, ont été rapportées dans de nombreux cas d'infections multi-résistantes au cours de la dernière décade, soulignant le rôle de ces protéines dans la résistance aux antibiotiques.La première partie de ma thèse a porté sur l'étude des relations structure fonction au sein des deux porines non spécifiques de Providencia stuartii, Omp-Pst1 et Omp-Pst2. Il a été montré qu'Omp-Pst1 est majoritairement responsable de l'entrée des antibiotiques. Afin de comprendre comment évolue cette porine in situ, nous avons réalisé une étude comparative sur les variantes d'Omp-Pst1 issues de la souche sauvage et de deux isolats cliniques. Globalement, ces structures pointent vers un consensus dans l'adaptation des porines in situ, lequel repose sur l'accumulation de résidus chargés positivement dans les boucles extracellulaires et dans le canal. Cette observation est en accord avec les mesures de translocation effectuées à l'échelle de la porine unique, lesquelles montrent une diffusion ralentie des antibiotiques chargés négativement au travers des porines issues des isolats cliniques. Mis ensemble, nos résultats démontrent le rôle critique joué par les porines dans la résistance aux antibiotiques, lequel vise à diminuer l'influx de ces derniers tout en conservant l'habilité pour la bactérie de se nourrir.La deuxième partie de ma thèse s'est focalisée sur une fonction inédite des porines, à savoir leur rôle dans l'association intercellulaire et la genèse de biofilms bactériens. Les porines sont généralement exprimées sous la forme de trimères fonctionnels enchâssés dans la membrane externe des bactéries à Gram-négatif. Le mécanisme d'adhésion mis en évidence par mes travaux de thèse repose sur la formation de dimères de trimères de porines, associées face à face par leurs boucles externes, grâce à une interaction de type steric zipper. En exploitant un large panel de méthodologies biophysiques et d'imageries, nous avons caractérisé les propriétés adhésives d'Omp-Pst1 et Omp-Pst2, à la fois in vitro et in vivo. Nous avons également investigué le transport de petites molécules fluorescentes au travers de ces dimères de porines, afin de vérifier leur putative implication dans la communication intercellulaire. Nos résultats démontrent la capacité des porines Omp-Pst1 et Omp-Pst2 à former des jonctions intercellulaires et les suggèrent donc comme des cibles thérapeutiques prometteuses dans la lutte contre les infections bactériennes

Etude du rôle joué par les porines dans la persistance des infections par Providencia stuartii

Present in the outer membrane of bacteria, porins are the main gateway for soluble molecules, such as nutrients and ions, into the bacteria. They are also the way taken by hydrophilic antibiotics to reach their targets and kill the cell. Under the strong selective pressure caused by antibiotic overuse, bacteria have evolved modified porins that are less permeable to antibiotics. Although not the only strategy developed by bacteria to survive drug treatment, it is an important factor in the spreading phenomenon of multidrug resistant infections.In order to gain further insights into the molecular determinants of antibiotic translocation, the first part of my thesis work aimed at resolving the crystallographic structures of Omp-Pst1 and Omp-Pst2, two non-specific porins encoded in the genome of Providencia stuartii. This bacterial species is not very invasive and, therefore, causes endemic rather than epidemic infections. However, these infections are often fatal given the intrinsically stringent MDR phenotype of this species. It has been shown that Omp-Pst1 is the main entrance for β-lactam antibiotics. To provide structural and functional insights into the contribution of P. stuartii porins to antibiotic resistance phenotypes, structural analysis was undertaken, not only from the wild type strain but also from two clinical mutant strains i.e. Omp-Pst1-99645 and Omp-Pst1-Nea16. Mutations result in more pronounced anion selectivity due to an increased number of positively charged amino acids lining the pore and mostly in the extracellular loops in both mutants compared to the wild type. To further determine whether these mutations contributed to a decrease in antibiotic uptake, we undertook the characterization of β-lactam antibiotics transport kinetics using electrophysiology studies at the single protein level. For the zwitterionic β-lactam tested, single-molecule conductance measurements evidenced a decrease in the association rate constant, in both mutants compared to the wild type. However, we observed instead an increase in these values for the negatively charged β-lactam, which is in good agreement with our structure-based analysis. All together, our results point towards porins playing a major role in the antibiotic resistance mechanism by reducing drug uptake.In the second part of my thesis work, we discovered that porins could self-associate to form adhesive junctions between two cells and could provide the initial scaffold for the establishment of biofilms at early stages of their developpement. The self-matching interaction is mediated by a steric zipper interaction and involves their extracellular loops. In order to confirm the adhesive proprieties of porins, we exploited a large panel of biophysical and imaging methods both in vitro and in vivo. Furthermore, we studied their diffusive proprieties in reconstituted liposomes, to explore whether these self-matching interactions between porins could play a role in cell-to-cell communication. Our results point at a major role of P. stuartii porins, Omp-Pst1 and Omp-Pst2, in cell-to-cell adhesion and make them promising targets to disrupt bacterial biofilm infections.Les porines sont des protéines « canal » qui assurent la diffusion non-spécifique des ions et nutriments au sein des bactéries à Gram-négatif. Elles sont également la voie d'entrée des antibiotiques hydrophiles, en particulier les β-lactames. Des mutations au sein des porines, ou leur sous-expression, ont été rapportées dans de nombreux cas d'infections multi-résistantes au cours de la dernière décade, soulignant le rôle de ces protéines dans la résistance aux antibiotiques.La première partie de ma thèse a porté sur l'étude des relations structure fonction au sein des deux porines non spécifiques de Providencia stuartii, Omp-Pst1 et Omp-Pst2. Il a été montré qu'Omp-Pst1 est majoritairement responsable de l'entrée des antibiotiques. Afin de comprendre comment évolue cette porine in situ, nous avons réalisé une étude comparative sur les variantes d'Omp-Pst1 issues de la souche sauvage et de deux isolats cliniques. Globalement, ces structures pointent vers un consensus dans l'adaptation des porines in situ, lequel repose sur l'accumulation de résidus chargés positivement dans les boucles extracellulaires et dans le canal. Cette observation est en accord avec les mesures de translocation effectuées à l'échelle de la porine unique, lesquelles montrent une diffusion ralentie des antibiotiques chargés négativement au travers des porines issues des isolats cliniques. Mis ensemble, nos résultats démontrent le rôle critique joué par les porines dans la résistance aux antibiotiques, lequel vise à diminuer l'influx de ces derniers tout en conservant l'habilité pour la bactérie de se nourrir.La deuxième partie de ma thèse s'est focalisée sur une fonction inédite des porines, à savoir leur rôle dans l'association intercellulaire et la genèse de biofilms bactériens. Les porines sont généralement exprimées sous la forme de trimères fonctionnels enchâssés dans la membrane externe des bactéries à Gram-négatif. Le mécanisme d'adhésion mis en évidence par mes travaux de thèse repose sur la formation de dimères de trimères de porines, associées face à face par leurs boucles externes, grâce à une interaction de type steric zipper. En exploitant un large panel de méthodologies biophysiques et d'imageries, nous avons caractérisé les propriétés adhésives d'Omp-Pst1 et Omp-Pst2, à la fois in vitro et in vivo. Nous avons également investigué le transport de petites molécules fluorescentes au travers de ces dimères de porines, afin de vérifier leur putative implication dans la communication intercellulaire. Nos résultats démontrent la capacité des porines Omp-Pst1 et Omp-Pst2 à former des jonctions intercellulaires et les suggèrent donc comme des cibles thérapeutiques prometteuses dans la lutte contre les infections bactériennes

Enhanced sampling methods and their application in the study of molecular permeation in gram-negative bacteria

Antimicrobial resistance is inhibiting our ability to fight against pathogens. By selectively changing the composition and expression of influx water-filled proteins filling their outer membrane, gram- negative bacteria are able to reduce the rates at which specific polar compounds are able to permeate. A clear comprehension of the mechanism determining substrates diffusion through these pores is still missing. In this thesis, we show how biased computer simulations may offer a unique perspective in the study of molecular permeation through porins, overcoming the intrinsic limitations of both experimental techniques and standard molecular dynamics. The first test-case is Acinetobacter baumannii’s CarO. The use of substrates with varying charge and molecular weight, as well as the creation of a loop-less mutant missing the extracellular domain of the protein, allowed to determine the charge selectivity and the transition rates of polar molecules. We obtained good agreement with the results of liposome swelling assays experiments. Further, we compared the passage of two carbapenem antibiotics in a series of mutated proteins extracted from a patient undergoing long term hospital infection. We connected the mutation of few key residues to a drastic change in the internal electric field of the proteins, showing that the antibiotics follow the choreography of water molecules inside the channels. In the last section, we present a kinetic model that allows to determine for a molecule the relative probability of different conformations and the time required for the translocation through a pore. This approach allowed to connect the results of enhanced sampling MD methods with current blockages in single channel experiments.All these results together show that multiscale MD techniques can offer an exhaustive view on the mechanism of molecular diffusion through pores, helping to understand the most important charac- teristics that determine the rates of translocation of different com- pounds in gram-negative bacteria. We can use these data to com- plement experimental results and to design the next generation of antibiotics

Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current

One major determinant of the efficacy of antibiotics on Gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within the pore, thus partially blocking the ionic current. The modulation of one affinity site by Mg2+ reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg2+-free media are converted to flickery sojourns at the highest apparent current level (all three pores flickering). This indicates weaker binding in the presence of Mg2+. Analysis of the resulting amplitude histograms with beta distributions revealed the rate constants of blocking (k(OB)) and unblocking (k(BO)) in the range of 1,000 to 120,000 s(-1). As expected for a bimolecular reaction, k(OB) was proportional to blocker concentration and k(BO) independent of it. k(OB) was approximately three times lower for enrofloxacin coming from the cis side than from the trans side. The block was not complete, leading to a residual conductivity of the blocked state being similar to 25% of that of the open state. Interpretation of the results has led to the following model: fast flickering as caused by interaction of Mg2+ and enrofloxacin is related to the binding site at the trans side, whereas the cis site mediates slow blocking events which are also found without Mg2+. The difference in the accessibility of the binding sites also explains the dependency of k(OB) on the side of enrofloxacin addition and yields a means of determining the most plausible orientation of OmpF in the bilayer. The voltage dependence suggests that the dipole of the antibiotic has to be adequately oriented to facilitate binding

Characterisation of the major porins OmpU and OmpT of Vibrio cholerae

PhD ThesisThe asymmetric outer membrane (OM) of a Gram-negative bacterium has many proteins embedded as β-barrel structures in it called outer membrane proteins (OMPs). The majority of these OMPs (porins) form non-selective channels across the OM to allow passive uptake of substrates. The treatment for infections caused by such bacteria mostly involves the administration of drugs/antibiotics, for which these porins play a very crucial role by providing an efficient (although not yet fully understood) route through their channel. The goal of this study is to study small-molecule permeation through the major porins, OmpU and OmpT, of Vibrio cholerae (the causative agent of cholera) for potential use of these proteins as the target for designing antibiotics or vaccines. Towards this project, we have succeeded in solving the 3D X-ray crystal structures of OmpU and OmpT as well as the structures of the major porins from Klebsiella pneumoniae (OmpK36) and Enterobacter cloacae (OmpE36, OmpE35). The proteins (OmpU/T, OmpE35/E36 and OmpK36) show the typical arrangement of porins with three β-barrel monomers arranged into a trimer. Each monomer displays 16 antiparallel β-strands forming the hollow β-barrel formed by 8 long extracellular loops and 8 short periplasmic turns. The latching loop L2 stabilises the trimer while loop L3 departs from the β-barrel fold and constricts the pore half-way through the channel. An unusual feature is observed in the channels of OmpU and OmpT that distinguishes them from other typical porins. In OmpU, the first 10 residues of N-terminus insert into the barrel and constrict the pore. In contrast, the structure of OmpT reveals that the extracellular loop L8 folds inwards to constrict the lumen of the channel. Such constriction elements not only reduce the pore sizes of OmpU and OmpT but may also dramatically affect the internal electrostatics of these channels, which is very important for small-molecule permeation. In addition, we also performed single channel electrophysiology experiments with OmpU and OmpT which revealed interesting features with the addition of carbapenems.European Union’s Seventh Framework Programme (FP7/2007–2013) and European Federation of Pharmaceutical Industries and Associations companies in kind contribution. Therefore, a very special gratitude goes out to all down to EU Marie Curie network (ITN) for funding my PhD

Understanding Voltage Gating of Providencia stuartii Porins at Atomic Level.

International audienceBacterial porins are water-filled β-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis

Novel ruthenium metal-based complexes as antimicrobial agents

Antimicrobial resistance (AMR) is becoming increasingly prevalent amongst clinically significant bacteria. The World Health Organization (WHO) has declared AMR as one of the greatest public health threats facing humanity. There has been a sharp decline in the number of new clinically approved antibiotics, with most new antibiotics being based on pre-existing antibiotic scaffolds. As a result, there is an urgent need for new novel ways to treat infections caused by AMR bacteria. There has been an increased focus on Ruthenium (Ru) complexes acting as antimicrobial agents. This is in part due to their biological compatibility, multiple oxidation states and bonding configuration allowing for specific geometries that are ideally suited for biological applications. This study evaluated the antimicrobial activity of 12 repurposed Ru complexes. Preliminary screening against a diverse selection of clinically significant bacteria identified Ru complexes 1 (C22H23Cl3N2SRu) and 7 ([Ru(NH3)6]Cl3) as potential lead candidates with the Ru complexes acted as bactericidal agents against S. aureus USA300 JE2 and P. aeruginosa PAO1 respectively. Eukaryotic cytotoxicity testing against HeLa and HEK 293T cell lines demonstrated Ru complex 7 exhibited no significant cytotoxic effects against both cell lines (p>0.05), whilst Ru complex 1 was significantly cytotoxic (p<0.05). S. aureus USA300 JE2 and E. coli EC958 were able to tolerate an 11-fold increase in MIC after long term incrementally increasing concentrations of Ru complex 1. Comparative genome analysis of S. aureus USA300 JE2 showed long term exposure to Ru complex 1 increased the rate of mutagenesis and led to 17 de novo mutations being identified within eight genes. Furthermore, significant gene expression changes in clpP, katA and norA were reported in S. aureus USA300 JE2 after exposure to Ru complex 1, indicating Ru complex 1 affected a wide array of cellular functions. The mechanisms of action for antimicrobials Ru complex 1 and 7 were investigated. Ru complex 1 displayed no significant outer membrane or inner membrane permeabilising effects, whilst Ru complex 7 caused no significant outer membrane permeabilising but did stimulate elevated depolarisation of the inner membrane in a number of bacterial species. Scanning electron microscopy confirm that both complexes appeared not to be directly targeting the outer membrane as no cellular morphological changes were observed. Cellular metal uptake studies using Ru complexes 1 and 7 showed elevated intracellular concentrations in S. aureus USA300 JE2 and P. aeruginosa PAO1 respectively compared to the exposure concentrations. Electrophoretic mobility shift assays (EMSA) and competitive binding assays showed that intracellular concentrations of Ru complexes 1 and 7 had a significant impact on DNA mobility and displacement of SYTO 9 from the SYTO 9/DNA complex. Exposure to Ru complexes 1 and 7 caused elevated but not significant levels of reactive oxygen species generation (ROS) in S. aureus USA300 JE2, P. aeruginosa PAO1 and E. coli EC958 The results of the thesis demonstrate the potential to use mononuclear Ru complexes as antimicrobial agents. Notably, the potent antimicrobial activity of Ru complex 7 against P. aeruginosa PAO1, coupled with low levels of cytotoxicity make this an ideal candidate for further in vivo investigation

THE ROLE OF CHAPERONES AND BAMA IN THE OUTER MEMBRANE PROTEIN BIOGENESIS PATHWAY OF ESCHERICHIA COLI

Outer membrane protein (OMP) biogenesis in Gram-negative bacteria is a multi-step, complex process that spans several cellular compartments. OMPs are post-translationally secreted across the bacterial inner membrane and subsequently encounter the aqueous periplasm. In this milieu, nascent OMPs interact with chaperone proteins that prevent the formation of off-folding-pathway species and deleterious aggregates. Here we investigate how two Escherichia coli periplasmic chaperones, FkpA and SurA, bind to unfolded OMP clients to facilitate trafficking. We find that FkpA populates both monomeric and dimeric species and these oligomers have differential affinities for unfolded OMP clients. We present the first structural model for a SurA-OMP complex, in which the unfolded OMP is expanded and makes delocalized contacts with the SurA chaperone. Upon reaching the outer membrane, OMPs are assembled via interactions with the E. coli β-Barrel Assembly Machinery (BAM) multi-protein complex. We consider the functional mechanism for the OMP component of the BAM complex, BamA, and determine that this enzyme works via a catalytic cycle to facilitate the folding of OMPs with an activity similar to the entire BAM complex. Lastly, we interrogate this pathway in a holistic manner by constructing a computational model to simulate OMP flux through this pathway. Our modeling suggests that together the concentrations of periplasmic chaperones and the kinetic and thermodynamic parameters for chaperone-OMP binding are poised for this system to act as a reservoir for OMP flux towards the OM. Our studies highlight the importance of both chaperone-OMP and BAM-OMP interactions in the accurate and efficient process of OMP biogenesis