LiaS-dependent activation of the MadR regulon enables cross-talk between Enterococcus faecalis cell envelope defense systems

Enterococci are gastrointestinal commensals that must defend their cell envelope against antimicrobial peptides derived from the host and other members of the microbiota. The signaling systems LiaFSR and MadRS are pivotal for survival in the presence of antimicrobial peptides and antimicrobial peptide-like antibiotics such as daptomycin. Both systems possess a signaling histidine kinase (LiaS, MadS) and cognate response regulator (LiaR, MadR) that activate transcription of distinct sets of effector genes. Using isogenic deletion strains, we noted differences in daptomycin minimum inhibitory concentration (MIC) between the laboratory strain E. faecalis OG1RF (1.5 µg/mL), OG1RFΔ madR (0.38 µg/mL), and OG1RFΔ madS (4 µg/mL). Transcriptional analysis of the MadR regulon showed a daptomycin-dependent increase in madG, madL , and dltA gene expression in the OG1RFΔ madS background, suggesting activation of the LiaFSR system may provide a cross-regulatory role. Deletion of the liaS gene in OG117Δ madS was associated with a significant decrease in daptomycin MIC and loss of madG expression, one of the most differentially expressed genes on activation of the MadR regulon. Using microscale thermophoresis, LiaS showed a similar binding affinity to both LiaR (K _d 2.42 µM) and MadR (K _d 5.02 µM), while MadS showed higher affinity for its cognate regulator MadR (K _d 8.08 µM) than for LiaR (K _d 25.7 µM). Taken together, our findings indicate that the MadR regulon can be expressed independent of MadS-induced signaling, likely through cross-talk between LiaS and MadR. Thus, enterococci have evolved an interconnected network of cell envelope signaling that permits bacterial survival in the presence of antibiotics and antimicrobial peptides.