Low-Molecular-Mass Penicillin Binding Protein 6b (DacD) Is Required for Efficient GOB-18 Metallo-β-Lactamase Biogenesis in Salmonella enterica and Escherichia coli

Abstract

Metallo-␤-lactamases (MBLs) are Zn 2؉-containing secretory enzymes of clinical relevance, whose final folding and metal ion assembly steps in Gram-negative bacteria occur after secretion of the apo form to the periplasmic space. In the search of periplasmic factors assisting MBL biogenesis, we found that dacD null (⌬dacD) mutants of Salmonella enterica and Escherichia coli expressing the pre-GOB-18 MBL gene from plasmids showed significantly reduced resistance to cefotaxime and concomitant lower accumulation of GOB-18 in the periplasm. This reduced accumulation of GOB-18 resulted from increased accessibility to proteolytic attack in the periplasm, suggesting that the lack of DacD negatively affects the stability of secreted apo MBL forms. Moreover, ⌬dacD mutants of S. enterica and E. coli showed an altered ability to develop biofilm growth. DacD is a widely distributed low-molecular-mass (LMM) penicillin binding protein (PBP6b) endowed with low DD-carboxypeptidase activity whose functions are still obscure. Our results indicate roles for DacD in assisting biogenesis of particular secretory macromolecules in Gram-negative bacteria and represent to our knowledge the first reported phenotypes for bacterial mutants lacking this LMM PBP.␤-Lactamase production represents a common mechanism of bacterial resistance and cause of failure in the treatment of infections (1). Among the different groups of ␤-lactamases defined on the basis of structural similarity and catalytic mechanisms, the metallo-␤-lactamases (MBLs) are especially worrisome (2, 3). This group is constituted entirely of metalloenzymes employing Zn 2ϩ for catalysis; members of this group can hydrolyze a broad spectrum of substrates, including the latest generations of clinically relevant ␤-lactams, and can be rapidly spread by horizontal gene transfer (1-3). Among the different strategies for controlling MBL dissemination, efforts have so far been focused on the design of a general MBL inhibitor, a goal so far hindered by the diversity of active-site structures among these metalloenzymes (4).A less known aspect of MBL research, but one that may provide potential targets for antimicrobial drug design, is the MBL biogenesis pathway (5). We previously reported that the productive biogenesis of the GOB-18 MBL in Escherichia coli requires an "expanded" DnaK chaperone system to assist the cytoplasmic transit of the preapoprotein to the secretion system (5). Moreover, we also found that secretion of the apo GOB-18 form is driven by the Sec (SecA-SecYEG) machinery, implying that final folding and Zn 2ϩ ion assembly to the native MBL conformation occur in the periplasm (5). Unfolded proteins emerging from the Sec channel are greeted by a highly complex macromolecule-crowded environment containing an array of modifying enzymes, some of them endowed with the ability to act as folding assistants (6-8). This complex scenario can certainly affect individual folding landscapes (6), but whether the final steps of MBL folding and Zn 2ϩ assembly are assisted by periplasmic ...

Loss of membrane‐bound lytic transglycosylases increases outer membrane permeability and β‐lactam sensitivity in Pseudomonas aeruginosa

“…; Brambilla et al. ), altering adhesion to surfaces or the ability to form the protective matrix (Monteiro et al. ; Liu et al.…”

“…; Brambilla et al. ). Only the mltD mutant in this study was defective for biofilm formation, while four strains lacking mLTs and Family 1 LTs produced significantly more biofilm.…”

“…Cells in a biofilm are protected from exposure to many antibiotics by the surrounding extracellular polymeric substance matrix and are heterogeneous with respect to growth rates, with reduced sensitivity in slow-growing or persister subpopulations (Kostakioti et al 2013). Factors affecting cell wall metabolism can modulate bacterial surface properties (Reid et al 2004;Liu et al 2012) and biofilm formation (Monteiro et al 2011;Payne et al 2013;Brambilla et al 2014), altering adhesion to surfaces or the ability to form the protective matrix (Monteiro et al 2011;Liu et al 2014). Therefore, we asked whether the ability of LT mutants to form biofilms was altered.…”

Expression of theEscherichia coliompWcolicin S4 receptor gene is regulated by temperature and modulated by the H-NS and StpA nucleoid-associated proteins

“…(b) SDS ‐ PAGE analysis of OM fractions derived from the above cells grown at 37 °C. The final positions of O mp W as well as the O mp C / O mp F and O mp A porins (Brambilla et al ., ) are shown on the left margin. The final positions of the molecular mass markers are also indicated.…”

“…Polyclonal antibodies were elicited in rabbits following conventional procedures (Sambrook et al, 1989). Immunoblot analysis was performed by transferring the proteins from the SDS-PAGE gels to polyvinylidene fluoride (PVDF) membranes (Amersham Pharmacia) followed by OmpW detection with rabbit polyclonal antibodies as first antibody and alkaline phosphatase-conjugated anti-rabbit IgG as second antibody as described (Brambilla et al, 2014).…”