Targeting of PBP1 by β-lactams Determines recA/SOS Response Activation in Heterogeneous MRSA Clinical Strains

Abstract

The SOS response, a conserved regulatory network in bacteria that is induced in response to DNA damage, has been shown to be associated with the emergence of resistance to antibiotics. Previously, we demonstrated that heterogeneous (HeR) MRSA strains, when exposed to sub-inhibitory concentrations of oxacillin, were able to express a homogeneous high level of resistance (HoR). Moreover, we showed that oxacillin appeared to be the triggering factor of a β-lactam-mediated SOS response through lexA/recA regulators, responsible for an increased mutation rate and selection of a HoR derivative. In this work, we demonstrated, by selectively exposing to β-lactam and non-β-lactam cell wall inhibitors, that PBP1 plays a critical role in SOS-mediated recA activation and HeR-HoR selection. Functional analysis of PBP1 using an inducible PBP1-specific antisense construct showed that PBP1 depletion abolished both β-lactam-induced recA expression/activation and increased mutation rates during HeR/HoR selection. Furthermore, based on the observation that HeR/HoR selection is accompanied by compensatory increases in the expression of PBP1,-2, -2a, and -4, our study provides evidence that a combination of agents simultaneously targeting PBP1 and either PBP2 or PBP2a showed both in-vitro and in-vivo efficacy, thereby representing a therapeutic option for the treatment of highly resistant HoR-MRSA strains. The information gathered from these studies contributes to our understanding of β-lactam-mediated HeR/HoR selection and provides new insights, based on β-lactam synergistic combinations, that mitigate drug resistance for the treatment of MRSA infections.

Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for β-Lactam–Daptomycin Synergy

“…Previous work has suggested that the degree to which different ␤-lactams potentiate the anti-MRSA activity of DAP may be associated with the relative affinity for PBP1 (13). This specific differential effect echoes other recent studies that observed increased toxin expression (12) and induction of DNA repair systems (14) upon inhibition of PBP1 but not upon inhibition of other PBPs (12,(15)(16)(17).…”

Ceftaroline Is Active against Heteroresistant Methicillin-Resistant Staphylococcus aureus Clinical Strains despite Associated Mutational Mechanisms and Intermediate Levels of Resistance

“…RNA extractions for real-time reverse transcription-PCR (RT-PCR) analysis were performed as previously described (17). Total RNA was extracted using an RNeasy isolation kit (Qiagen); all RNA samples were analyzed by A 260 /A 280 spectrophotometry to assess concentration and integrity and cleaned of potential DNA contamination by treating them with DNase per manufacturer recommendations (Ambion, Inc., Life Technologies/Thermo Fisher, Austin, TX).…”

“…We used a wax worm model to investigate whether CPT therapy regimens have enhanced in vivo efficacy against clinical HeR-and HoR-MRSA compared with the efficacies of other ␤-lactam regimens, including IPM, NAF, and FOX. Larvae of the greater wax moth (Galleria mellonella) have recently been used as an alternative to vertebrates as a model host for studying pathogenic microbes, virulence, and therapeutic regimens (21)(22)(23), including the assessment of the efficacy of antistaphylococcal agents (17,20). G. mellonella larvae possess an immune system relatively homologous to the innate immune system of vertebrates, a digestive tract, a loosely organized muscular system, a biosynthetic fat body, and hemolymph that, analogous to blood, transports nutrients, hemocytes, and immune molecules.…”

Antimicrobial Heteroresistance: an Emerging Field in Need of Clarity

“…Increased expression of the agr (accessory gene regulator) system during HeR-HoR selection was required to tightly modulate SOS-mediated mutation rates, which then led to full expression of homogeneous oxacillin resistance in very heterogeneous clinical MRSA strains (130). The PBP1 protein played a role in SOS-mediated RecA activation and HeR-HoR selection (131). Conversely, a mutation in the less resistant cells of a heterogeneous population seemed to be responsible for their increased susceptibility.…”