Enterococcus faecalis is an opportunistic pathogen and a major cause of severe nosocomial infections. Treatment options against enterococcal infections are declining due to the resistance of enterococci to numerous antibiotics. A key risk factor for developing enterococcal infections is treatment with cephalosporin antibiotics, to which enterococci are intrinsically resistant. For susceptible organisms, cephalosporins inhibit bacterial growth by acylating the active site of penicillin-binding proteins (PBPs), key enzymes that catalyze peptidoglycan cross-linking. Two specific PBPs of enterococci, Pbp4(5) and PbpA(2b), exhibit low reactivity toward cephalosporins, allowing these PBPs to cross-link peptidoglycan in the presence of cephalosporins to drive resistance in enterococci, but the mechanisms by which these PBPs are regulated are poorly understood. The CroS/R two-component signal transduction system (TCS) is also required for cephalosporin resistance. Activation of CroS/R by cephalosporins leads to CroR-dependent changes in gene expression. However, the specific genes regulated by CroS/R that are responsible for cephalosporin resistance remain largely unknown. In this study, we characterized CroR-dependent transcriptome remodeling by RNA-seq, identifying pbp4(5) as a CroR regulon member in multiple, diverse lineages of E. faecalis. Through genetic analysis of the pbp4(5) and croR promoters, we uncovered a CroR-dependent regulatory motif. Mutations in this motif to disrupt CroR-dependent upregulation of pbp4(5) in the presence of cell wall stress resulted in a reduction of resistance to cephalosporins in E. faecalis, demonstrating that enhanced production of Pbp4(5) and likely other proteins involved in peptidoglycan biogenesis by the CroS/R system drives enterococcal cephalosporin resistance. IMPORTANCE Investigation into molecular mechanisms used by enterococci to subvert cephalosporin antibiotics is imperative for preventing and treating life-threatening infections. In this study, we used genetic means to investigate the functional output of the CroS/R TCS required for enterococcal resistance to cephalosporins. We found that enhanced production of the penicillin-binding protein Pbp4(5) upon exposure to cell wall stress was mediated by CroS/R and was critical for intrinsic cephalosporin resistance of E. faecalis.

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