Anne Delcour

Publications (43) View all

• Article: Effects of conjugated and unconjugated bile acids on the activity of the Vibrio cholerae porin OmpT.

  Melissa Pagel, Anne H Delcour
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  ABSTRACT: During infection, the enteric pathogen Vibrio cholerae encounters a bile-containing environment. Previous studies have shown that bile and/or bile acids exert several effects on the virulence and physiology of the bacterial cells. These observations have led to the suggestion that bile acids may play a signaling role in infection. We have previously reported that the bile component deoxycholic acid blocks the general diffusion porin OmpT in a dose-dependent manner, presumably as it transits through the pore. V. cholerae colonizes the distal jejunum and ileum, where a mixture of various conjugated and unconjugated bile acids are found. In this work, we have used patch clamp electrophysiology to investigate the effects of six bile acids on OmpT. Two bile acids (deoxycholic and chenodeoxycholic acids) were found to block OmpT at physiological concentrations below 1 mM, while glycodeoxycholic acid was mildly effective and cholic, lithocholic and taurodeoxycholic acids were ineffective in this range. The block was also voltage-dependent. These observations suggest the presence of a specific binding site inside the OmpT pore. Since deconjugation is due to the activity of the endogenous flora, the preferential uptake of some unconjugated bile acids by OmpT may signal the presence of a hospitable environment. The results are also discussed in terms of the possible molecular interactions between the penetrating bile acid molecule and the channel wall.
  Molecular Membrane Biology 11/2010; 28(1):69-78. · 2.86 Impact Factor
• Article: Disulfide bond tethering of extracellular loops does not affect the closure of OmpF porin at acidic pH.

  Beau Wager, Arnaud Baslé, Anne H Delcour
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  ABSTRACT: The permeability of the outer membrane of gram-negative bacteria is essentially controlled by pore-forming proteins of the porin family. The trimeric E. coli porin OmpF is assembled as a triple β-barrel, where each monomer contains a central pore and extracellular loops. Electrophysiological analysis of the behavior of OmpF at acidic pH reveals that the protein undergoes a conformational change leading to the sequential step-wise closure of the three monomers. A previous atomic force microscopy study suggested that the conformational change might be due to a bending of extracellular loops over the pore opening, and loop deletion experiments suggested that loops L1, L7, and L8 are involved. In order to test the hypothesis for loop movement, we engineered a series of double cysteine mutants in loops L1, L6, L7 and L8 in order to create disulfide bonds linking two loops to each other, or the two branches of a loop, or a loop to the β-barrel. Five out of the six mutants showed the formation of the disulfide bond. However, none of these had an altered response to acidic pH relative to the wildtype channel. Although we cannot dismiss the possibility that the mobility restriction introduced by each disulfide bond was too localized to impact a more global conformational change of the three loops, the fact that all of the different types of disulfide bond tethering were similarly ineffective suggests that the extracellular loops L1, L7, and L8 may not undergo a major acidic-pH induced conformational change leading to channel closure.
  Proteins Structure Function and Bioinformatics 11/2010; 78(14):2886-94. · 3.39 Impact Factor
• Article: Size and dynamics of the Vibrio cholerae porins OmpU and OmpT probed by polymer exclusion.

  Guillaume Duret, Anne H Delcour
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  ABSTRACT: The trimeric OmpU and OmpT porins form large, triple-barrel hydrophilic channels in the outer membrane of the pathogen Vibrio cholerae. They have distinct pore properties, such as conductance, block by deoxycholic acid, and sensitivity to acidic pH. Their three-dimensional structures are unknown, but they share significant sequence homologies. To gain insight into the molecular basis for the distinct functional properties of these two similar porins, we carried out polymer exclusion experiments using planar lipid bilayer and patch-clamp electrophysiology. By studying the partitioning of polyethylene glycols (PEGs) of different molecular weights into each porin, we determined an effective radius of 0.55 nm and 0.43 nm for OmpU and OmpT respectively, and found an increased OmpU effective radius at acidic pH. PEGs or high buffer ionic strength promotes the appearance of single step closures in OmpU similar to the acidic-pH induced closures we documented previously. In addition, these closing events can be triggered by nonpenetrating PEGs applied asymmetrically. We believe our results support a model whereby acidic pH, high ionic strength, or exposure to PEGs stabilizes a less conductive state that corresponds to the appearance of an additional resistive element on one side of the OmpU protein and common to the three monomers.
  Biophysical Journal 05/2010; 98(9):1820-9. · 3.65 Impact Factor
• Source

  Available from: PubMed Central

  Article: Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli

  Muriel Masi, Guillaume Duret, Anne H Delcour, Rajeev Misra
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  ABSTRACT: TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique α/β-barrel structure. Previous studies have shown that unlike the biogenesis of β-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of β-barrel OMPs, is dependent on BamA and BamD, two essential components of the β-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCΔ2–22). A significant amount of TolCΔ2–22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCΔ2–22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCΔ2–22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCΔ2–22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCΔ2–22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble α-helical domain.
  Microbiology 05/2009; · 3.06 Impact Factor
• Article: Altered pore properties and kinetic changes in mutants of the Vibrio cholerae porin OmpU.

  Brian Lauman, Melissa Pagel, Anne H Delcour
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  ABSTRACT: The electrophysiological technique of patch-clamp was used to characterize the pore properties of site-directed mutants in the Vibrio cholerae general diffusion porin OmpU. Changes in conductance and selectivity were observed, thus confirming the predicted pore location of these residues, based on homology with the Escherichia coli porins OmpF and OmpC. Some mutants acquire a weak selectivity for cations, which mirrors the properties of the homologous, deoxycholic acid sensitive, OmpT porin of V. cholerae. However, the mutants remain insensitive to deoxycholic acid, like wildtype OmpU. This result suggests that channel selectivity is not an important determinant in the sensitivity to this drug, and is in agreement with our finding that the neutral deoxycholic acid, and not deoxycholate, is the actual active form in channel block. Modifications in the kinetics of spontaneous closures were also noted, and are similar to those found for the E. coli channels. In addition, mutants at the D116 residue on the L3 loop display marked transitions to sub-conductance states. The results reported here are compared to a phenotypical characterization of the mutants in terms of permeability to maltodextrins and beta-lactam antibiotic sensitivity. No strict correlations are observed, suggesting that distinct, but somewhat overlapping, molecular determinants control electrophysiological properties and substrate permeability.
  Molecular Membrane Biology 11/2008; 25(6-7):498-505. · 2.86 Impact Factor