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

Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria

Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria

Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria

Structural and functional insights into the contribution of Providencia stuartii's porins to the persistence of 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.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

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

Direct coupling of detergent purified human mGlu5 receptor to the heterotrimeric G proteins Gq and Gs

The metabotropic glutamate (mGlu) receptors are class C G protein-coupled receptors (GPCRs) that modulate synaptic activity and plasticity throughout the mammalian brain. Signal transduction is initiated by glutamate binding to the venus flytrap domains (VFT), which initiates a conformational change that is transmitted to the conserved heptahelical domains (7TM) and results ultimately in the activation of intracellular G proteins. While both mGlu1 and mGlu5 activate Gαq G-proteins, they also increase intracellular cAMP concentration through an unknown mechanism. To study directly the G protein coupling properties of the human mGlu5 receptor homodimer, we purified the full-length receptor, which required careful optimisation of the expression, N-glycosylation and purification. We successfully purified functional mGlu5 that activated the heterotrimeric G protein Gq. The high-affinity agonist-PAM VU0424465 also activated the purified receptor in the absence of an orthosteric agonist. In addition, it was found that purified mGlu5 was capable of activating the G protein Gs either upon stimulation with VU0424465 or glutamate, although the later induced a much weaker response. Our findings provide important mechanistic insights into mGlu5 G protein-dependent activity and selectivity. © 2018 The Author(s).The authors would like to acknowledge Laurent Prézeau, Julie Kniazeff, Philippe Rondard and Cyril Goudet for helpful discussion and Chris Tate for a critical reading of the manuscript. We are grateful to Eric Trinquet (Cisbio, France) for providing us the SNAP tag full-length mGlu5 expression plasmid, as well as to Arpege and FFP platforms at the IGF. Karine Rottier was supported by FRM “ingenieur de Recherche” program and Chady Nasrallah is supported by ATIP grant (2014–2016) and the University of Montpellier, Postodocotral scientist program (2016–2018). Joan Font and Amadeu Llebaria acknowledge MINECO (PCIN-2013–017 C03-01 and CTQ2014-57020-R), the Catalan Government (2014SGR109 and 2014CTP0002) and to ERANET Neuron project ‘LIGHTPAIN’for support. Guillaume Lebon acknowledges Program ATIP (2013–2016), the CNRS, INSERM and the University of Montpellier for their support.Peer reviewe

Providencia stuartii form biofilms and floating communities of cells that display high resistance to environmental insults.

Biofilms are organized communities of bacterial cells that are responsible for the majority of human chronic bacterial infections. Providencia stuartii is a Gram-negative biofilm-forming bacterium involved in high incidence of urinary tract infections in catheterized patients. Yet, the structuration of these biofilms, and their resistance to environmental insults remain poorly understood. Here, we report on planktonic cell growth and biofilm formation by P. stuartii, in conditions that mimic its most common pathophysiological habitat in humans, i.e. the urinary tract. We observed that, in the planktonic state, P. stuartii forms floating communities of cells, prior to attachment to a surface and subsequent adoption of the biofilm phenotype. P. stuartii planktonic and biofilm cells are remarkably resistant to calcium, magnesium and to high concentrations of urea, and show the ability to grow over a wide range of pHs. Experiments conducted on a P. stuartii strain knocked-out for the Omp-Pst2 porin sheds light on the role it plays in the early stages of growth, as well as in the adaptation to high concentration of urea and to varying pH

The calculated current, conductance and ion selectivity from MD simulations.

<p>(A) The currents are calculated every 50 ns monomer-wise and the total channel currents are shown in in-set panels in comparison to the experimentally determined values (dash lines). (B) The conductances of the porins’ channel are obtained from the slope of their I-V curve. The raw channel conductances are shown. (C) The ion selectivity is determined as the ratio of K⁺ to Cl^- current. The value shown at each voltage is the averaged ratio from three monomers and the error bar corresponds to the standard deviation among the three.</p