title: Structure-function relationships of CarO, the carbapenem resistance-associated outer membrane protein of Acinetobacter baumannii.
abstract: OBJECTIVE
  In the context of the increasing worldwide occurrence of imipenem-resistant Acinetobacter baumannii strains, we investigated a possible porin-mediated mechanism relating to the carbapenem resistance-associated outer membrane protein, CarO. The aim of this study was to determine whether this porin may be a diffusion pathway for carbapenems in A. baumannii.
  
  
  METHODS
  By analysing and comparing the sequences of CarO with protein databanks, we identified two major groups of sequences that we named CarOa and CarOb. We overproduced in Escherichia coli, extracted, purified by affinity chromatography and refolded in Triton X-100 rCarO from both groups. Their functional properties were investigated and compared by reconstitution in planar lipid bilayers.
  
  
  RESULTS
  This functional study showed that rCarOa and rCarOb exhibit identical single-channel conductances (i.e. 20 pS in 1 M KCl) and similar poor cationic selectivity. Both channels were not specific towards meropenem and glutamic acid and poorly specific towards arginine, but they presented a marked specificity towards imipenem. From the calculated binding constants, we highlight that the CarOb channel was twice as specific as the CarOa channel for this antibiotic. Moreover, the CarOa channel could facilitate ornithine diffusion when the CarOb channel would not.
  
  
  CONCLUSIONS
  We provide here the first evidence that CarO channels possess an imipenem (but not meropenem) binding site, and that their specificities depend on their primary structure. Any decrease in CarO expression would thus reduce the susceptibility of A. baumannii to this antibiotic.
authors.affiliation: University of Rouen, Laboratoire Polymères Biopolymères Surfaces, UMR 6270 & FR 3038 CNRS, IFRMP23, F76821 Mont Saint Aignan, France.
authors.forename: Manuella
authors.initials: M
authors.lastname: Catel-Ferreira
authors.forename: Gael
authors.initials: G
authors.lastname: Coadou
authors.forename: Virginie
authors.initials: V
authors.lastname: Molle
authors.forename: Pauline
authors.initials: P
authors.lastname: Mugnier
authors.forename: Patrice
authors.initials: P
authors.lastname: Nordmann
authors.forename: Axel
authors.initials: A
authors.lastname: Siroy
authors.forename: Thierry
authors.initials: T
authors.lastname: Jouenne
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Dé
mesh_terms: D040981:Acinetobacter baumannii / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D000465:Algorithms; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D015780:Carbapenems / Q000494:pharmacology*; D003001:Cloning, Molecular; D030541:Databases, Genetic; D024881:Drug Resistance, Bacterial / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D015378:Imipenem / Q000494:pharmacology; D007476:Ionophores / Q000737:chemistry; D008051:Lipid Bilayers; D018272:Porins / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; C561703:CarO protein, Acinetobacter baumannii; D015780:Carbapenems; D007476:Ionophores; D008051:Lipid Bilayers; D018272:Porins; D011994:Recombinant Proteins; D015378:Imipenem