Expression and biogenesis of Acinetobacter baumannii OXA-58 in Escherichia coli

FABBRI C; CAMERANESI MM; LIMANSKY AS; VIALE AM; MORAN BARRIO J

Mar del Plata

Congreso; LI Reunión Anual SAIB; 2015

We studied the biogenesis of the carbapenem hydrolyzing class D b-lactamase (CHDL), OXA-58 from Acinetobacter baumannii in E. coli as a model system. The mature blaOXA-58 sequence was fused to a pelB signal sequence in a pBAD-derived plasmid and expressed in this host. OXA-58 biogenesis was studied following b-lactam resistance, level of protein production, and subcellular localization. High levels of a 29 kDa OXA-58 band were observed in whole A. baumannii and E. coli cell extracts which correlated with resistance to imipenem only in A. baumannii, although in both cases resistance to ampicilin was obtained. Subcellular fractionation showed that OXA-58 accumulated in both species mostly in the periplasmic fraction, although a significant percentage was also associated to membranes. In vitro enzymatic assays revealed imipenemase activity in the soluble periplasmic fraction of A. baumannii cells but much lower activity in those of E. coli. The presence of molecular crowders such as PEG, ficoll or PVP did not affect substantially the imipenemase activity of the extracts, ruling out in principle an effect of macromolecular crowding in the regulation of this activity in the periplasm. Overall these results suggest that different post-traductional events occur in A. baumannii and E. coli which may differentially affect substrate specificity towards b-lactam substrates.