β-Lactam Resistance Response Triggered by Inactivation of a Nonessential Penicillin-Binding Protein

et al. (2009) b-Lactam Resistance Response Triggered by Inactivation of a Nonessential Penicillin- Binding Protein. PLoS Pathog 5(3): e1000353. doi:10.1371/journal.ppat.1000353 b-Lactam Resistance Response Triggered by Inactivation of a Nonessential Penicillin-Binding Protein Bartolome Moya 0 Andreas Do tsch 0 Carlos Juan 0 Jesu s Bla zquez 0 Laura Zamorano 0 Susanne 0 Haussler 0 Antonio Oliver 0 Frederick M. Ausubel, Massachusetts General Hospital, United States of America 0 1 Servicio de Microbiolog a and Unidad de Investigacio n, Hospital Son Dureta, Instituto Universitario de Investigacio n en Ciencias de la Salud (IUNICS) Palma de Mallorca, Spain, 2 Helmholtz Centre for Infection Research , Braunschweig, Germany , 3 Centro Nacional de Biotecnolog a, Consejo Superior de Investigaciones Cient ficas (CSIC), Campus UAM , Madrid , Spain It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for b-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance. Among Gramnegative rods (GNR), however, this mechanism has been considered unusual, and restricted to clinically irrelevant laboratory mutants for most species. Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing lifethreatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in b-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including wholegenome analysis approaches, we demonstrate that high-level (clinical) b-lactam resistance in vitro, in vivo, and in the clinical setting is driven by the inactivation of the dacB-encoded nonessential PBP4, which behaves as a trap target for b-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex b-lactam resistance response, triggering overproduction of the chromosomal b-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance. These findings are a major step forward in our understanding of blactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases. - Funding: This work was supported by the Ministerio de Educacion y Ciencia of Spain (SAF2006-08154), the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III through the Spanish Network for the Research in Infectious Diseases (REIPI C03/14 and RD06/0008), and the Govern de les Illes Balears (PROGECIB-9A). AD is a recipient of a predoctoral stipend provided by the DFG-sponsored European Graduate School program Pseudomonas: Pathogenicity and Biotechnology. Financial support from the Helmholtz Gemeinschaft is also gratefully acknowledged. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Decades after their discovery, b-lactams remain key components of our antimicrobial armamentarium for the treatment of infectious diseases. Bacterial resistance to them is generally driven either by the production of enzymes that inactivate them (b-lactamases), or by the modification of their targets in the cell wall (penicillin-binding Proteins, PBPs), sometimes in conjunction with mechanisms leading to diminished permeability or active efflux [1]. While the acquisition of modified PBPs showing low affinity for b-lactams is well known to be a major resistance mechanism in Gram-positive cocci, such as penicillin-resistant Streptococcus pneumoniae or the much-feared methicillin-resistant Staphylococcus aureus, this mechanism has not been thought to be important for most species of Gram-negative rods (GNR) [2]. The production of intrinsic or horizontally acquired b-lactamases is undoubtedly the predominant resistance mechanism in the latter organisms [3]. Among GNRs, the most widely distributed b-lactamases are chromosomally-encoded AmpC variants, produced by most Enterobacteriaceae and Pseudomonas aeruginosa, high up the list of pathogens causing life-threatening infections in hospitalized patients world-wide [4]. Although AmpC is produced at very low basal levels in wildtype strains, its expression is highly inducible in the presence of certain b-lactams (b-lactamase inducers) such as cefoxitin or imipenem [3]. In fact, the efficacy of the widely-used broad spectrum penicillins (such as piperacillin) and cephalosporins (such as ceftazidime) relies on the fact that they are very weak AmpC inducers, even though they are efficiently hydrolyzed by this enzyme [3]. Unfortunately, mutants showing constitutive high level AmpC production (AmpC derepressed mutants) are frequently selected during treatment with these b-lactams, leading to the failure of antimicrobial therapy [5,6]. In some natural strains of Enterobacteriaceae and P. aeruginosa [69], the inactivation of AmpD (cytosolic N-acetyl-anhydromuramyl-L-alanine amidase involved in peptidoglycan recycling [1012]), and point mutations in AmpR (LysR-type transcriptional regulator required for ampC induction [1315]) have been found to lead to AmpC overexpression, and thus to b-lactam resistance. In this paper we show that, in contrast to the current expectations, the mutations triggering b-lactam resistance in P. aeruginosa, whether in vitro, in vivo, or in the clinical setting, frequently arise within a PBP gene. Inactivation of the E. coli dacB ortholog, encoding the nonessential low molecular mass PBP4 Decades after their discovery, b-lactams remain key components of our antimicrobial armamentarium for the treatment of infectious diseases. Nevertheless, resistance to these antibiotics is increasing alarmingly. There are two major bacterial strategies to develop resistance to blactam antibiotics: the production of enzymes that inactivate them (b-lactamases), or the modification of their targets in the cell wall (the essential penicillin-binding proteins, PBPs). Using the pathogen Pseudomonas aeruginosa as a model microorganism, we show that high-level (clinical) b-lactam resistance in vitro and in vivo frequently occurs through a previously unrecognized, totally distinct resistance pathway, driven by the mutational inactivation of a nonessential PBP (PBP4) that behaves as a trap target for b-lactams. We show that mutation of this PBP determines a highly efficient and complex b-lactam resistance response, triggering overproduction of the chromosomal b-lactamase AmpC and the specific activation of a two-component regulator, which in turn plays a key role in resistance. These findings are a major step forward in our understanding of b-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious di (...truncated)