Oral Abstract Submission
William R. Miller, MD
Center for Antimicrobial Resistance and Microbial Genomics, UTHealth
Disclosure: Entasis: Grant/Research Support
Merck: Grant/Research Support
Shionogi: Advisory Board
Cesar A. Arias, MD, MSc, PhD
University of Texas Health Science Center
Disclosure: American Society for Microbiology: Fees for Editor of Antimicrobial Agents and Chemotherapy. Money paid to University, Other Financial or Material Support
Entasis Pharmaceuticals: Grant/Research Support, Research Grant
MeMed Diagnostics: Grant/Research Support, Research Grant
Merck: Grant/Research Support, Research Grant
UptoDate: Other Financial or Material Support, Royalties from a Chapter
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 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.
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 was 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.
Deletion of liaX led to DAP-R and redistribution of APL microdomains (non-septal foci with CM aberrations; Fig 1A) in all strains, with a marked decrease in ceftriaxone (CRO) MICs. Only PBP5was essential for β-lactam resistance but not for DAP-R. DAP-R was associated with mislocalization of PBPs to the sites of CM aberrations (Fig. 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 (Fig 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.
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 non-septal areas and likely increases access of β-lactam to the active site, explaining the seesaw effect.