The Acinetobacter Outer Membrane Contains Multiple Specific Channels for Carbapenem β-Lactams as Revealed by Kinetic Characterization Analyses of Imipenem Permeation into Acinetobacter baylyi Cells

• Published in: Antimicrobial agents and chemotherapy 2017-03, Vol.61 (3)
• Main Authors: Morán-Barrio, Jorgelina, Cameranesi, María M, Relling, Verónica, Limansky, Adriana S, Brambilla, Luciano, Viale, Alejandro M
• Format: Article
• Language: English
• Subjects: Acinetobacter; Acinetobacter - genetics; Acinetobacter - metabolism; Acinetobacter baumannii - genetics; Acinetobacter baumannii - metabolism; Amino Acids, Basic; Amino Acids, Basic - metabolism; Anti-Bacterial Agents; Anti-Bacterial Agents - metabolism; Bacterial Outer Membrane Proteins; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; beta-Lactams - metabolism; Biological Transport; Cell Membrane - chemistry; Cell Membrane - metabolism; Evolution, Molecular; Gene Deletion; Gene Expression; Imipenem; Imipenem - metabolism; Kinetics; Mechanisms of Resistance; Porins; Porins - deficiency; Porins - genetics; Thienamycins - metabolism
• ISSN: 0066-4804; 1098-6596
• DOI: 10.1128/AAC.01737-16

The number and type of outer membrane (OM) channels responsible for carbapenem uptake in are still not well defined. Here, we addressed these questions by using as a model species and a combination of methodologies aimed to characterize OM channels in their original membrane environment. Kinetic and competition analyses of imipenem (IPM) uptake by whole cells allowed us to identify different carbapenem-specific OM uptake sites. Comparative analyses of IPM uptake by wild-type (WT) cells and Δ mutants lacking CarO indicated that this OM protein provided a carbapenem uptake site displaying saturable kinetics and common binding sites for basic amino acids compatible with a specific channel. The kinetic analysis uncovered another carbapenem-specific channel displaying a somewhat lower affinity for IPM than that of CarO and, in addition, common binding sites for basic amino acids as determined by competition studies. The use of gene deletion mutants lacking OM proteins proposed to function in carbapenem uptake in indicated that CarO and OprD/OccAB1 mutants displayed low but consistent reductions in susceptibility to different carbapenems, including IPM, meropenem, and ertapenem. These two mutants also showed impaired growth on l-Arg but not on other carbon sources, further supporting a role of CarO and OprD/OccAB1 in basic amino acid and carbapenem uptake. A multiple-carbapenem-channel scenario may provide clues to our understanding of the contribution of OM channel loss or mutation to the carbapenem-resistant phenotype evolved by pathogenic members of the genus.