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)

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. [Image: see text] [Image: see text] [Image: see text] DISCLOSURES: All Authors: No reported Disclosures.