Functional characterisation of the Acinetobacter baylyi outer membrane protein CarO homolog as a channel for basic amino acids and imipenem in intact bacterial cells

RELLING, V.; LIMANSKY, A.S.; JORGELINA MORÁN BARRIO; MUSSI, M.A.; BRAMBILLA, L.; VIALE, A.M.

Colonia

Simposio; 9th International Symposium on the Biology of Acinetobacter.; 2013

Objectives: We previously described a new family of outer membrane (OM) proteins, CarO, present only in members of the Moraxellaceae family of the Gammaproteobacteria and whose members in clinical strains of Acinetobacter baumannii function as OM channels for carbapenem b-lactams and basic amino acids, with a marked preference for ornithine among the latter. Here, we describe the functional characterisation of the CarO homolog present in A. baylyi ADP1 cells concerning its imipenem (IPM) and basic amino acids channel roles. Methods: We constructed a carO deletion mutant (Delta-carO) of the A. baylyi ADP1 strain. Both the wild-type (wt) and the Delta-carO mutant were transformed with plasmid pVIM encoding blaVIM-11 directing production of a metallo-b-lactamase displaying imipenemase activity and secreted to the periplasmic space. The IPM and basic amino acids channel properties of A. baylyi CarO were characterized in both wt and Delta-carO bacteria by analysing IPM degradation kinetics by intact cells in the absence and presence of different competitors including series of different amino acids and polyamines, following procedures applied for whole cells of other bacterial species. Results: Determinations of IPM uptake kinetics at various concentration of this carbapenem in A. baylyi wt whole cells indicated the presence of a saturable specific channel in the OM through which permeation occurs mainly at low IPM concentrations. Moreover, similar procedures indicated significantly decreased IPM permeability and the loss of this channel in Delta-carO mutants of A. baylyi cells. A number of basic amino acids including arginine, ornithine, lysine, and histidine, were effective inhibitors of IPM uptake by A. baylyi wt cells, while other amino acids including glutamate, glutamine, as well as basic polyamines such as putrescine, failed to inhibit IPM uptake. Conclusion: Acinetobacter CarO family members function physiologically as specific OM channels for basic amino acids also allowing the permeation of structurally related carbapenems such as IPM, a feature that acquires clinical relevance when pathogenic members of the genus are concerned and selective pressure by carbapenem therapy is applied to treat infections.