27. The Membrane Antimicrobial Peptide Defense (MadRS) System Orchestrates Resistance Against Antibiotics and Host Innate Immune Peptides in enterococcus Faecalis

Abstract

BackgroundEnterococci have evolved a network of two component-signaling systems (TCS) to evade killing by cationic antimicrobial peptides (AP) of the host innate immune response. Activation of these pathways can also lead to cross-resistance to AP-like antibiotics such as daptomycin (DAP), complicating treatment of severe enterococcal infections. Our previous work implicated the MadRS (formerly YxdJK) TCS in resistance to DAP. In this work, we show that the MadRS system regulates a network of membrane defense proteins that provide targeted resistance to killing by LL-37.MethodsWe performed a global transcriptional analysis comparing wild type (WT) E. faecalis OG1RF and an OG1RF derivative possessing a mutated allele of the madS gene that activates the MadRS system (OG1RFmadSA202E). Selected genes identified in this analysis were knocked out using a CRISPR-cas9 gene editing system and confirmed by sequencing. Antibiotic minimum inhibitory concentrations (MIC) were determined using gradient diffusion strips. LL-37 killing assays were performed in triplicate in RPMI+5% LB broth. Caenorhabditis elegans assays were performed in triplicate, and representative results are shown.ResultsAnalysis of the MadRS regulon revealed a significant upregulation of a network of gene targets, including madLM (formerly yxdLM, involved in bacitracin resistance), madEFG, the dlt operon, salA, and EF2211 (Fig 1). The madSA202E allele was associated with increased bacterial survival on exposure to LL-37, while the madEFG deletion mutant was more susceptible to killing (Fig 2A). Deletion of madLM decreased bacitracin MICs, but did not alter survival on exposure to LL-37. In C. elegans, inactivation of the MadRS system by deletion of the madR response regulator resulted in attenuation as compared to WT OG1RF. The effect was reversed by complementing with wild-type madR on a plasmid (Fig 2B). The attenuation effect of the mutant was abolished in pmk-1 knockout worms, which lack the ability to produce innate immune peptides (Fig 2C).Figure 1 Figure 2ConclusionThe MadRS system plays an important role in mediating resistance to AP both in vitro and in vivo. The novel ABC-transporter MadEFG is likely to play a major role in preventing enterococcal killing by the human cathelicidin LL-37. Disclosures William R. Miller, MD, Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support)Shionogi (Advisor or Review Panel member) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)