A liaR deletion restores susceptibility to daptomycin and antimicrobial peptides in multidrug-resistant Enterococcus faecalis

Abstract

Daptomycin is a lipopeptide antibiotic that is used clinically against many gram-positive bacterial pathogens and is considered a key frontline bactericidal antibiotic to treat multidrug-resistant enterococci. Emergence of daptomycin resistance during therapy of serious enterococcal infections is a major clinical issue. In this work, we show that deletion of the gene encoding the response regulator, LiaR (a member of the LiaFSR system that controls cell envelope homeostasis), from daptomycin-resistant Enterococcus faecalis not only reversed resistance to 2 clinically available cell membrane-targeting antimicrobials (daptomycin and telavancin), but also resulted in hypersusceptibility to these antibiotics and to a variety of antimicrobial peptides of diverse origin and with different mechanisms of action. The changes in susceptibility to these antibiotics and antimicrobial peptides correlated with in vivo attenuation in a Caenorhabditis elegans model. Mechanistically, deletion of liaR altered the localization of cardiolipin microdomains in the cell membrane. Our findings suggest that LiaR is a master regulator of the enterococcal cell membrane response to diverse antimicrobial agents and peptides; as such, LiaR represents a novel target to restore the activity of clinically useful antimicrobials against these organisms and, potentially, increase susceptibility to endogenous antimicrobial peptides.

Keywords: E. faecalis; LiaFSR; antimicrobial peptides; daptomycin.

Figure 1.

Minimum inhibitory concentrations (MICs) and susceptibility to antimicrobial peptides of Enterococcus faecalis derivatives. A, Change of daptomycin (DAP) and telavancin (TLV) MICs determined by the Etest (Biomerieux) on Mueller-Hinton agar. MIC determination was performed in triplicate for all strains, with the same result in each experiment. Of note, breakpoints for TLV are not available. B, Survival after exposure to antimicrobial peptides (AMPs). Results are from 3 independent experiments. *P < .001, compared with S613FΔ_{liaF177gdpD170cls61}. Abbreviations: FRU, friulimicin, naturally occurring lipopeptide; GAL, gallidermin, type A lantibiotic; HBD3, human β-defensin 3; LL-37, human cathelicidin; MER, mersacidin, type B lantibiotic; nisin, type A lantibiotic; RP-1, synthetic antimicrobial peptide.

Figure 2.

Killing of Caenorhabditis elegans by Enterococcus faecalis derivatives. Exposure of C. elegans to E. faecalis S613FΔ_{liaF177gdpD170cls61}ΔliaR results in significant attenuation, compared with the parental strain (S613FΔ_{liaF177gdpD170cls61}). The in vivo phenotype was restored by introducing liaR back into its native chromosomal location (S613FΔ_{liaF177gdpD170cls61}ΔliaR::liaR). The experiment was repeated 3 times with similar results (n = 60–90 worms). ***P < .0001.

Figure 3.

Staining of representative cells of Enterococcus faecalis S613F and derivatives with 10-N-nonyl acridine orange (500 nM). A–D and I–K, Fluorescence microscopy images of bacterial cells. E–H, Phase-contrast images of the same cells in panels A–D. A, S613F displays cardiolipin microdomains in septum and poles of the cell (white arrows). B and I, S613FΔ_{liaF177gdpD170cls61} exhibits cardiolipin microdomains located away from the main septum and distributed throughout the cells. C and J, Deletion of liaR redistributes the cardiolipin microarrays back to the septum and poles, as shown in DAP-susceptible E. faecalis S613F. D and K, Reintroducing liaR in its native chromosomal location restored the remodeling of cardiolipin microdomains in the cell membrane. White arrows indicate the cardiolipin domains in the cell membrane, and bars denote lengths of 1 µm.