Activity Monitoring of Functional OprM Using a Biomimetic Microfluidic Device

International audienceThis paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for 1) fluidic and electrical access to both sides of the BLM, 2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (Gi), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity during more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishing of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance

PpiA, a Surface PPIase of the Cyclophilin Family in Lactococcus lactis

Background: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. Results: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H2O2) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H2O2. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. Conclusions: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displaye

Etudes structurales et fonctionnelles de la pompe d'efflux MexAB-OprM impliquée dans la résistance aux antibiotiques chez Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. This bacteria has developed various strategies to resist antibiotics treatments, one of them being the activation of membrane efflux pumps. These tripartite systems consist of an OMF (Outer Membrane Factor) family porin, localized in the outer membrane, an active transporter in the inner membrane, belonging to the RND (Resistance Nodulation Division) family and a periplasmic adaptator protein, member of the MFP (Membrane Fusion Protein) family which consolidates the whole complex. Results obtained during this thesis contribute to a better understanding of efflux pumps’ assembly and opening thanks to the development of new research tools borrowed from physic, biochemistry and microbiology. The first study describes the binding stoechiometry of MexA with its cognate partner OprM by Blue Native Polyacrylamide gel Electrophoresis (Ferrandez, Monlezun et al. 2012). Secondly, a study, in collaboration with B. Le Pioufle’s team (ENS Cachan), was dedicated to the electrophysiologic caracterization of OprM opening using a microfluidic device incorporated with a miniaturized artificial bilayer membrane (Wang, Monlezun et al. 2012). Then, to complete this analysis in vivo, in the third part of this thesis, complementation experiments were initiated in a Pseudomonas aeruginosa strain deleted of its chromosomal oprM gene. Finally, in collaboration with P. Plésiat’s team (Laboratoire de Bactériologie, Besançon), modelling of MexZ, the MexXY/OprM pump’s regulator and modelling of the carbapenems’ porin OprD were made in order to link structural modifications to mutations observed in clinical strains.Pseudomonas aeruginosa est un pathogène opportuniste impliqué dans les infections nosocomiales. Sa multi résistance aux antibiotiques s’exerce notamment grâce à l’activation de pompes d’efflux membranaires. Il s’agit de systèmes tripartites composés d’une porine de la famille OMF (Outer Membrane Factor) ancrée dans la membrane externe, d’un transporteur de la famille des RND (Resistance Nodulation Division) localisé dans la membrane interne et d’un adaptateur périplasmique de la famille des MFP (Membrane Fusion Protein) qui consolide l’ensemble. Le travail réalisé au cours de cette thèse apporte une contribution à la compréhension des mécanismes d’assemblage et d’ouverture des pompes d’efflux ainsi qu’à leur régulation grâce au développement de nouveaux outils empruntés à la physique, à la biochimie et à la microbiologie. Une première étude a permis de déterminer la stoechiométrie d’interaction entre MexA et OprM par gel bleu natif (Ferrandez, Monlezun et al. 2012). Une deuxième étude a été consacrée, dans le cadre d’une collaboration avec l’équipe de B. Le Pioufle (ENS Cachan), à la caractérisation par électrophysiologie de l'ouverture de la porine OprM, insérée dans une membrane artificielle reconstituée sur une biopuce (Wang, Monlezun et al. 2012). Puis, afin d’étudier cette fois ci, le mécanisme d’ouverture de la porine OprM in vivo, une étude fonctionnelle par complémentation chez Pseudomonas aeruginosa a été initiée. Enfin, dans le cadre d'une collaboration avec l’équipe de P. Plésiat (Laboratoire de Bactériologie, Besançon), deux analyses de mutants cliniques par modélisation ont été réalisées sur le régulateur MexZ de la pompe MexXY/OprM et de la porine d’influx des carbapénèmes OprD

Structural and functional studies of MexAB-OprM efflux pump involved in Pseudomonas aeruginosa antibiotics resistance

Pseudomonas aeruginosa est un pathogène opportuniste impliqué dans les infections nosocomiales. Sa multi résistance aux antibiotiques s’exerce notamment grâce à l’activation de pompes d’efflux membranaires. Il s’agit de systèmes tripartites composés d’une porine de la famille OMF (Outer Membrane Factor) ancrée dans la membrane externe, d’un transporteur de la famille des RND (Resistance Nodulation Division) localisé dans la membrane interne et d’un adaptateur périplasmique de la famille des MFP (Membrane Fusion Protein) qui consolide l’ensemble. Le travail réalisé au cours de cette thèse apporte une contribution à la compréhension des mécanismes d’assemblage et d’ouverture des pompes d’efflux ainsi qu’à leur régulation grâce au développement de nouveaux outils empruntés à la physique, à la biochimie et à la microbiologie. Une première étude a permis de déterminer la stoechiométrie d’interaction entre MexA et OprM par gel bleu natif (Ferrandez, Monlezun et al. 2012). Une deuxième étude a été consacrée, dans le cadre d’une collaboration avec l’équipe de B. Le Pioufle (ENS Cachan), à la caractérisation par électrophysiologie de l'ouverture de la porine OprM, insérée dans une membrane artificielle reconstituée sur une biopuce (Wang, Monlezun et al. 2012). Puis, afin d’étudier cette fois ci, le mécanisme d’ouverture de la porine OprM in vivo, une étude fonctionnelle par complémentation chez Pseudomonas aeruginosa a été initiée. Enfin, dans le cadre d'une collaboration avec l’équipe de P. Plésiat (Laboratoire de Bactériologie, Besançon), deux analyses de mutants cliniques par modélisation ont été réalisées sur le régulateur MexZ de la pompe MexXY/OprM et de la porine d’influx des carbapénèmes OprD.Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. This bacteria has developed various strategies to resist antibiotics treatments, one of them being the activation of membrane efflux pumps. These tripartite systems consist of an OMF (Outer Membrane Factor) family porin, localized in the outer membrane, an active transporter in the inner membrane, belonging to the RND (Resistance Nodulation Division) family and a periplasmic adaptator protein, member of the MFP (Membrane Fusion Protein) family which consolidates the whole complex. Results obtained during this thesis contribute to a better understanding of efflux pumps’ assembly and opening thanks to the development of new research tools borrowed from physic, biochemistry and microbiology. The first study describes the binding stoechiometry of MexA with its cognate partner OprM by Blue Native Polyacrylamide gel Electrophoresis (Ferrandez, Monlezun et al. 2012). Secondly, a study, in collaboration with B. Le Pioufle’s team (ENS Cachan), was dedicated to the electrophysiologic caracterization of OprM opening using a microfluidic device incorporated with a miniaturized artificial bilayer membrane (Wang, Monlezun et al. 2012). Then, to complete this analysis in vivo, in the third part of this thesis, complementation experiments were initiated in a Pseudomonas aeruginosa strain deleted of its chromosomal oprM gene. Finally, in collaboration with P. Plésiat’s team (Laboratoire de Bactériologie, Besançon), modelling of MexZ, the MexXY/OprM pump’s regulator and modelling of the carbapenems’ porin OprD were made in order to link structural modifications to mutations observed in clinical strains

Aim, Load, Fire:The Type VI Secretion System, a Bacterial Nanoweapon

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Les mécanismes d’efflux et la résistance chez Pseudomonas aeruginosa

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Carbapenem resistance in cystic fibrosis strains of Pseudomonas aeruginosa as a result of amino acid substitutions in porin OprD

International audienceThe aim of this work was to investigate the impact of single amino acid substitutions occurring in specific porin OprD on carbapenem resistance of cystic fibrosis (CF) strains of Pseudomonas aeruginosa. A PAO1ΔoprD mutant was complemented with the oprD genes from five carbapenem-resistant CF strains exhibiting very low amounts of mutated OprD porins in their outer membrane despite wild-type levels of oprD transcripts. Compared with wild-type porin from strain PAO1, single amino acid substitutions S403P (in periplasmic loop 8), Y242H, S278P and L345P (in β-sheets 10, 12 and 14, respectively) were found to result in reduced amounts of OprD in the outer membrane, increased carbapenem resistance, and slower growth in minimal medium containing gluconate, an OprD substrate, as the sole source of carbon and energy. This indicates that in CF strains of P. aeruginosa, loss of porin OprD may not only result from mutations downregulating the expression of or disrupting the oprD gene, but also from mutations generating deleterious amino acid substitutions in the porin structure

Multiple mutations lead to MexXY-OprM-dependent aminoglycoside resistance in clinical strains of Pseudomonas aeruginosa.

International audienceConstitutive overproduction of the pump MexXY-OprM is recognized as a major cause of resistance to aminoglycosides, fluoroquinolones, and zwitterionic cephalosporins in Pseudomonas aeruginosa. In this study, 57 clonally unrelated strains recovered from non-cystic fibrosis patients were analyzed to characterize the mutations resulting in upregulation of the mexXY operon. Forty-four (77.2%) of the strains, classified as agrZ mutants were found to harbor mutations inactivating the local repressor gene (mexZ) of the mexXY operon (n = 33; 57.9%) or introducing amino acid substitutions in its product, MexZ (n = 11; 19.3%). These sequence variations, which mapped in the dimerization domain, the DNA binding domain, or the rest of the MexZ structure, mostly affected amino acid positions conserved in TetR-like regulators. The 13 remaining MexXY-OprM strains (22.8%) contained intact mexZ genes encoding wild-type MexZ proteins. Eight (14.0%) of these isolates, classified as agrW1 mutants, overexpressed the gene PA5471, which codes for the MexZ antirepressor ArmZ [corrected], with 5 strains exhibiting growth defects at 37°C and 44°C, consistent with mutations impairing ribosome activity. Interestingly, one agrW1 mutant appeared to harbor a 7-bp deletion in the coding sequence of the leader peptide, PA5471.1, involved in ribosome-dependent, translational attenuation of PA5471 expression. Finally, DNA sequencing and complementation experiments revealed that 5 (8.8%) strains, classified as agrW2 mutants, harbored single amino acid variations in the sensor histidine kinase of ParRS, a two-component system known to positively control mexXY expression. Collectively, these results demonstrate that clinical strains of P. aeruginosa exploit different regulatory circuitries to mutationally overproduce the MexXY-OprM pump and become multidrug resistant, which accounts for the high prevalence of MexXY-OprM mutants in the clinical setting