903. Resensitization to β-Lactams in Enterococci Depends on Penicillin-Binding Protein (PBP) Mislocalization and Is Mediated by a Single Protein That Modulates Cell Membrane (CM) Adaptation to Daptomycin (DAP)

Abstract

Background

DAP disrupts bacterial CM by binding to septal anionic phospholipids (APLs). LiaX, an effector of the LiaFSR stress system, modulates DAP-R by diverting APLs away from the septum. Enterococci are intrinsically resistant to β-lactams due to the presence of PBPs (e.g., PBP5) with low affinity to these drugs. However, emergence of DAP-R leads to increased susceptibility to β-lactams, a phenomenon designated as the see-saw effect. Here, we dissect the molecular mechanism of this phenomenon.

Methods

We studied a clinical strain pair of DAP-S (S613) and DAP-R (R712) E. faecalis strains recovered from a patient before and after DAP therapy. We generated deletions of liaX and PBPs ( ponA and pbp5) in DAP-susceptible (DAP-S) E. faecalis OG1RF and JH2-2. APLs and membrane structures were visualized with NAO and/or FM4-64. PBPs and LiaX localization were evaluated with bocillin-FL or immunofluorescence. PBP transcripts and PBP5 protein levels were measured by qRT-PCR or immunoblotting, respectively. β-Lactam binding affinity of PBPs was assessed by SDS-PAGE of bocillin-FL stained membranes and a LiaX–PBP5 interaction was evaluated by the bacterial two-hybrid (BACTH) system. MICs were determined via E-test.

Results

Deletion of liaX led to DAP-R and redistribution of APL microdomains (nonseptal foci with CM aberrations; Figure 1A) in all strains, with a marked decrease in ceftriaxone (CRO) MICs. Only PBP5 was essential for β-lactam resistance but not for DAP-R. DAP-R was associated with mislocalization of PBPs to the sites of CM aberrations (Figure 2). Notably, LiaX and PBP5 were localized to the septum in DAP-S strains but redistributed away from septal areas upon development of DAP-R (Figure 3). An interaction of LiaX and PBP5 was confirmed by the BACTH system. Mislocalized PBPs, most notably PonA and PBP5, had increased affinity for β-lactams in all DAP-R strains. The increased affinity of PBPs to β-lactams was not associated with increased transcripts or PBP5 levels.

Conclusion

LiaX regulates CM adaptation and cell wall synthesis via membrane remodeling and direct interactions with key PBPs. Changes in LiaX that cause DAP-R results in mislocalization of PBPs to nonseptal areas and likely increases access of β-lactam to the active site, explaining the see-saw effect.

Disclosures

All Authors: No reported Disclosures.