IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
BIOGENESIS OF OXA-58 FROM Acinetobacter baumannii CLINICAL ISOLATE IN GRAM-NEGATIVE BACTERIA
Autor/es:
FABBRI C; CAMERANESI MM; LIMANSKY AS; VIALE AM; MORAN BARRIO J
Lugar:
Córdoba
Reunión:
Congreso; XI Congreso Argentino de Microbiología General - SAMIGE 2015; 2015
Resumen:
Acinetobacter baumannii is an important Gram-negative opportunistic pathogen responsible for a variety of nosocomial infections. It can rapidly evolve multi-drug resistance (MDR) when confronted with antibiotic therapy, and the emerging resistance to carbapenems among nosocomial strains represents a major concern worldwide. One of the mechanisms proposed to play a significant role in carbapenem resistance in A. baumannii is the overexpression of carbapenem hydrolyzing class D, OXA-type, b-lactamases (CHDL). Many studies were aimed to uncover the genetic location and context of the blaOXA genes, and to kinetically characterize the encoded enzyme. However, less attention has been given to OXA lactamase biogenesis, a process that includes translocation of the newly synthesized precursors from the cytoplasm across the inner membrane, and folding in the actual subcellular location. Most b-lactamases belonging to classes A, B and C are periplasmic soluble enzymes in the periplasm of Gram-negative bacteria, but still no information exists about CHDL. We analyze here OXA-58 biogenesis regarding to the level of protein production and carbapenem resistance conferred, using A. baumanni ATCC 17978 as well as E. coli K12 as model hosts of Gram-negative bacteria. Plasmid pWH1266-blaOXA-58 was used for OXA-58 production in A. baumannii, and pBAD-derived plasmids were constructed for E. coli. We also developed different protocols and optimized procedures for proper subcellular localization analysis. Periplasmic contents were obtained by osmotic shock procedures. Membrane fractions were obtained by Sarcosynate treatment of sonic extracts and ultracentrifugation, thus allowing partition of inner and outer membranes. SDS-PAGE analysis indicated that OXA-58 was associated to the inner membrane fraction in A. baumannii when produced in low quantities, and also in the periplasmic space as a soluble protein when over-produced. In these cases the levels of OXA-58 showed correlation with the MIC values obtained towards IPM. In E. coli however, though different conditions inducing blaOXA-58 expression were used, no carbapenem resistance was observed when the complete blaOXA-58 gene was expressed. Substituting the original signal sequence with that of PelB resulted in OXA-58 production as judged by SDS-PAGE analysis and E. coli ampicillin resistance. Altogether, these results indicate the association of OXA-58 to the A. baumannii inner-cell membrane probably as a mechanism of enzyme stabilization.