DacD PENICILLIN BINDING PROTEIN PARTICIPATES IN PEPTIDOGLYCAN REMODELING AND METALLO-B-LACTAMASE BIOGENESIS IN ENTERIC BACTERIA

BRAMBILA, L; MORAN BARRIO, J; VIALE, A. M.

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General; 2012

The expression of b-lactam-degrading enzymes (b-lactamases) is the most prevalent mechanism of antibiotic resistance in bacteria. Metallo-b-lactamases (MBLs) are particularly worrisome in the clinical setting, in that they can hydrolyze a broad spectrum of b-lactam substrates including the latest-generation carbapenems and they are resistant to all clinically employed inhibitors. In fact, the design of an efficient pan-MBL inhibitor has been limited by a large diversity of active-site structures, catalytic properties, and Zn(II) requirements for activity among different members of this family of enzymes. Some aspects that have received less attention are the cellular processes involved in biogenesis of MBLs that ultimately result in resistance, a possible target for antimicrobial design. We have previously characterized GOB-18 MBL from the Gram-negative pathogen Elizabethkingia meningoseptica. GOB-18 precursor is synthesized in the bacterial cytoplasm and secreted to the periplasmic space by the Sec machinery, aided by the DnaK chaperone system, where the polypeptide folds and binds the Zn(II) ion. Subtle differences observed between the folding process in vitro and in vivo suggest the involvement of extrinsic factors (i.e. periplasmic proteins) during in vivo biogenesis process. In an effort to identify periplasmic assisting proteins, we generated an insertional mutant library using MudJ transposon in Salmonella enterica expressing GOB-18 from a plasmid. Screening of MudJ insertional mutants for reduced cefotaxime resistance resulted in the identification of a dacD gene disruptional mutant, coding for PBP6b DD-carboxipeptidase. S. enterica mutants lacking dacD contained decreased amounts of GOB-18 in the periplasmic space and reduced cefotaxime MIC values, suggesting a role of DacD in GOB biogenesis and/or stability. Our additional phenotypic analysis of dacD mutants showed that DacD is involved in peptidoglican (PG) structure/remodeling, as judge by Congo Red binding. Based on the above results, we propose that DacD has a role in PG remodeling and that the PG architecture provides a scaffold for GOB stability, in addition to its barrier function.