An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State

Abstract

The cyclic antimicrobial lipopeptide daptomycin triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, nonsusceptibility to daptomycin by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaRW73C). We have determined the structure of the activated Enterococcus faecium LiaR protein at 3.2 Å resolution and, in combination with solution studies, show that activation of LiaR induces formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaRW73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to daptomycin by upregulation of the LiaFSR regulon. A comparison of the Enterococcus faecalis LiaR, Enterococcus faecium LiaR and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with daptomycin resistance suggest that physicochemical properties such as oligomerization state and DNA specificity, though tuned to the biology of each organism, share some features that could be targeted for new antimicrobials.

Enterococcus faecalis Adapts to Antimicrobial Conjugated Oligoelectrolytes by Lipid Rearrangement and Differential Expression of Membrane Stress Response Genes

“…While there seems to be an obvious role of membrane lipids in COE resistance, we have been unable to fully explain this role at a mechanistic and regulatory level. To better understand how the change of an alanine for a valine might impact the function of LiaR, we compared the amino acid sequence of the LiaR from OG1RF with the that of E. faecium SD3B-2 (or E. faecium R494) since the crystal structure of the entire LiaR sequence of the latter has been published (Davlieva et al, 2016). At the amino acid level, alanine is conserved at position 98 in the two proteins.…”

The dimerization interface in VraR is essential for induction of the cell wall stress response in Staphylococcus aureus: a potential druggable target

“…VraR belongs to the NarL/FixJ family of RR, which use the helix-turn-helix motif to bind to DNA. The dimerization interface found in VraR is seen in a subset of this family of proteins, for example in Escherichia coli UhpA [19], a response regulator that regulates sugar uptake, Mycobacterium tuberculosis NarL [20], S. aureus LuxR (PDB ID 3B2N), a response regulator implicated in regulation of cell-density, Bacillus subtillis DesR [21], and Enterococcus faecium LiaR, a response regulator involved in daptomycin induced cell wall stress response [22]. The response regulator proteins that belong to the OmpR/PhoB family of proteins, which use a winged helix-turn-helix to target DNA, implicate the α4-β5-α5 interface for dimerization [23].…”

Antibiotic Production and Antibiotic Resistance: The Two Sides of AbrB1/B2, a Two-Component System of Streptomyces coelicolor

“…Relevant structural data have been published on VraR and LiaR ( Donaldson, 2008 ; Leonard et al, 2013 ; Davlieva et al, 2015 , 2016 ). All the relevant residues identified in both regulators were conserved in AbrB1, both at the amino acid sequence level and at the level of the three-dimensional structure [homology modeling of AbrB1 was performed in SWISS-MODEL using the structure of VraR ( 4GVP.1.A ) as a template] ( Figure 5 ):…”