LiaS-dependent activation of the MadR regulon enables cross-talk between 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 OG1RF (1.5 μg/mL), OG1RFΔ (0.38 μg/mL), and OG1RFΔ (4 μg/mL). Transcriptional analysis of the MadR regulon showed a daptomycin-dependent increase in and gene expression in the OG1RFΔ background, suggesting activation of the LiaFSR system may provide a cross-regulatory role. Deletion of the gene in OG117Δ was associated with a significant decrease in daptomycin MIC and loss of 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 2.42 μM) and MadR (K 5.02 μM), while MadS showed higher affinity for its cognate regulator MadR (K 8.08 μM) than for LiaR (K 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.