Deletion of liaR Reverses Daptomycin Resistance in Enterococcus faecium Independent of the Genetic Background

Abstract

fWe have shown previously that changes in LiaFSR, a three-component regulatory system predicted to orchestrate the cell membrane stress response, are important mediators of daptomycin (DAP) resistance in enterococci. Indeed, deletion of the gene encoding the response regulator LiaR in a clinical strain of Enterococcus faecalis reversed DAP resistance (DAP-R) and produced a strain hypersusceptible to antimicrobial peptides. Since LiaFSR is conserved in Enterococcus faecium, we investigated the role of LiaR in a variety of clinical E. faecium strains representing the most common DAP-R genetic backgrounds. Deletion of liaR in DAP-R E. faecium R446F (DAP MIC of 16 g/ml) and R497F (MIC of 24 g/ml; harboring changes in LiaRS) strains fully reversed resistance (DAP MICs decreasing to 0.25 and 0.094 g/ml, respectively). Moreover, DAP at concentrations of 13 g/ml (achieved with human doses of 12 mg/kg body weight) retained bactericidal activity against the mutants. Furthermore, the liaR deletion derivatives of these two DAP-R strains exhibited increased binding of boron-dipyrromethene difluoride (BODIPY)-daptomycin, suggesting that high-level DAP-R mediated by LiaR in E. faecium involves repulsion of the calcium-DAP complex from the cell surface. In DAP-tolerant strains HOU503F and HOU515F (DAP MICs within the susceptible range but bacteria not killed by DAP concentrations of 5؋ the MIC), deletion of liaR not only markedly decreased the DAP MICs (0.064 and 0.047 g/ ml, respectively) but also restored the bactericidal activity of DAP at concentrations as low as 4 g/ml (achieved with human doses of 4 mg/kg). Our results suggest that LiaR plays a relevant role in the enterococcal cell membrane adaptive response to antimicrobial peptides independent of the genetic background and emerges as an attractive target to restore the activity of DAP against multidrug-resistant strains.

A Novel Phosphodiesterase of the GdpP Family Modulates Cyclic di-AMP Levels in Response to Cell Membrane Stress in Daptomycin-Resistant Enterococci

“…To determine whether c-di-AMP signaling is present in other enterococcal strains, daptomycin-tolerant and vancomycin-resistant Enterococcus faecium clinical isolate HOU503 and its fusidic acid-resistant derivative E. faecium HOU503F were tested (Table 1) (27). The c-di-AMP levels of E. faecium HOU503 and E. faecium HOU503F were 766 Ϯ 318 and 542 Ϯ 133 pM/OD 600 at late exponential phase (OD 600 , ϳ1.2) ( Table 1 and Fig.…”

“…To demonstrate that changes in LiaFSR signaling lead to changes in c-di-AMP more broadly, we measured the c-di-AMP levels in nonpolar liaR deletion mutants of E. faecium strains HOU503F and R497F (E. faecium HOU503F_ΔliaR and R497F_ΔliaR, Table 1) which are tolerant of and resistant to daptomycin (27). As observed with E. faecalis, deletion of liaR in E. faecium HOU503F_ΔliaR and R497F_ΔliaR showed modest increases in c-di-AMP levels compared to E. faecium HOU503F and R497F (622 Ϯ 104 pM/OD 600 versus 542 Ϯ 133 pM/OD 600 and 736 Ϯ 186 pM/OD 600 versus 549 Ϯ 150 pM/OD 600 , Fig.…”

Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci

“…It is also significant that genes protective against daptomycin (liaXYZ) are highly upregulated in response to chlorhexidine. It was recently demonstrated that deletion of liaR, encoding an activator of liaXYZ expression (45), restores daptomycin susceptibility to daptomycin-nonsusceptible enterococci (76). Further studies will be required to determine whether gene expression "priming" by chlorhexidine impacts treatment outcomes with daptomycin, or if frequent exposure to subinhibitory chlorhexidine is selective for strains that constitutively activate liaXYZ.…”

The action mechanism of daptomycin

“…116 In such strains, the activation of LiaRS greatly reduces the efficiency of daptomycin to bacterial cells. 117 When activated, LiaRS induces the expression of LiaI and LiaH, which colocalize within segregated domains in the cytoplasmic membrane. 118 The phenotypic effects of these effector molecules, and accordingly their role in daptomycin resistance, are not yet understood.…”