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

Abstract

It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for β-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance. Among Gram-negative 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 life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in β-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including whole-genome analysis approaches, we demonstrate that high-level (clinical) β-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 β-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex β-lactam resistance response, triggering overproduction of the chromosomal β-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 β-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases.

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…aeruginosa has three LMM PBPs (PBP4, PBP5 and PBP7) whose role in b-lactam resistance has been investigated [125,216]. Clinical mutants of PBP4 (PA3047) are associated with increased b-lactam resistance [216].…”

“…Clinical mutants of PBP4 (PA3047) are associated with increased b-lactam resistance [216]. In fact, a mutation in PBP4 leads to a one-step upregulation of ampC resulting in clinical b-lactam resistance [216]. Similar to PBP6b in E. coli, the role of PBP5 (PA3999) is not evident, unless in the PBP4 background where the double mutant further increases b-lactam resistance (Table 3).…”

“…This may be reflective of the different mechanism of resistance, as E. coli has a non-inducible ampC [217]. One speculation is that E. coli PBP5 could act as a 'trap' for b-lactams thereby preventing access to their targets, the HMM PBPs whereas in P. aeruginosa loss of PBP4 results in increased production of the AmpR effector which escalates ampC b-lactamase expression and thereby the MIC [124,215,216,218] Indirect regulation of b-lactam resistance through AmpR Although P. aeruginosa AmpR acts as the nexus for AmpC b-lactamase induction and cell-wall recycling processes, it also modulates the activity of other proteins that regulate blactam resistance. One of these is the second chromosomally encoded b-lactamase, PoxB (PA5514) in P. aeruginosa [219].…”

“…Another mechanism of b-lactamase induction is mediated through the two-component system CreBC (PA0463-PA0464) and its effector, an inner membrane protein CreD (PA0465) [216]. In E. coli, CreB has been identified as a transcriptional factor that differentially regulates up to eight genes under diverse nutritional conditions [224].…”

“…The increased expression of ampC is accompanied by elevated MIC of b-lactams, which reverts to wild-type levels upon deletion of creBC [226]. The association of creBC and b-lactam resistance was seen only in dacB mutants and not with other mutations that confer high resistance, namely ampD, ampDh2 and ampDh3 [216]. Moreover, these resistance phenotypes regulated through creBC were lost in the absence of NagZ and AmpG [207,216,226] (Table 3).…”