Acinetobacter baumannii CarO outer membrane proteins are involved in basic amino acids uptake

VIALE , ALEJANDRO MIGUEL; SÁNCHEZ ROCÍO INÉS; MORAN-BARRIO, JORGELINA

Paraná

Congreso; SAIB :Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2018

Acinetobacter baumannii CarO outer membrane proteins are involved in basic amino acids uptake Sanchez RI, Morán-Barrio J, Viale AMIBR-CONICET ? FCByF, UNR, Rosario.The family of outer membrane (OM) proteins CarO is restricted to the family Moraxellaceae, where Acinetobacter baumannii belongs. We previously described four polymorphic carO variants with the corresponding CarO proteins exhibiting distinctive properties such as differential migrations in gels, antigenicity, formation of oligomeric structures, and L-ornithine permeation abilities. To further characterize the different A. baumannii CarO variants we used A. baylyi ADP1 as model. For this purpose we constructed ADP1 recombinant cells harboring genetic replacements for each of the A. baumannii carO variants. We evaluated the physiology of the mutants concerning growth in rich and minimal medium, susceptibility to external aggressors, and CarO production. All of the replacement mutants showed similar growth rates comparable to that of the wild-type ADP1, comparable susceptibility to ionic detergents and NaCl stress, and same levels of CarO accumulation in the OM. Motility assays indicated that restitution of CarO to the OM restored the motility of the recombinants in semisolid medium. Nutrient utilization assays conducted in minimal media supplemented with different compounds, including amino acids, sugars, and different phenolic compounds, indicated differential growth of the mutants only on the basic amino acid arginine. Mutants carrying CarO variants III and IV were impeded to grow in arginine, while cells carrying variants I and II could grow similarly to wild-type cells. The overall results indicate that CarO participates in the selective uptake of basic amino acids and suggest roles in the interaction with abiotic and biotic surfaces.  revealing roles for CarO in the uptake of this nutrient. were observed among the mutants to this family. CarO function as OM channel for carbapenem β-lactams and basic amino acids with a marked preference for ornithine (1). We describe here the functional characterization of the CarO homolog present in A. baylyi ADP1 cells concerning its imipenem (IPM) and basic amino acids channel roles. A carO deletion mutant (DcarO) of A. baylyi ADP1 and the corresponding wild-type strain were transformed with plasmid pVIM directing production of the VIM-11 metallo-b-lactamase displaying imipenemase activity, which is secreted to the periplasmic space. The IPM and basic amino acids channel properties of A. baylyi CarO were characterized in both wt and DcarO bacteria by analyzing IPM degradation kinetics in intact cells. The possible competitor role of different compounds with IPM for influx through CarO, including a series of different amino acids and polyamines, was assayed following procedures applied for whole cells of other bacterial species (2). Determinations of IPM uptake kinetics in A. baylyi wt whole cells at various concentration of this carbapenem indicated the presence of a saturable specific channel in the OM through which permeation occurs mainly at low IPM concentrations. Similar procedures indicated significantly decreased IPM permeability and therefore loss of this channel in DcarO 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. The above results lead us to hypothesize that CarO family members function physiologically as specific OM channels for basic amino acids also allowing the permeation of structurally related carbapenems such as IPM in species Acinetobacter, a feature that acquires relevance when pathogenic members of the genus are concerned and selective carbapenem therapy pressure is applied.1. Mussi et al. (2011) J Bacteriol. 193:4736-4748.2. Trias et al. (1989) Antimicrob Agents Chemother. 33:1202-1206.