The role of the CroR response regulator in resistance of Enterococcus faecalis to D-cycloserine is defined using an inducible receiver domain.

Abstract

Enterococcus faecalis is an opportunistic multidrug-resistant human pathogen causing severe nosocomial infections. Previous investigations revealed that the CroRS two-component regulatory pathway likely displays a pleiotropic role in E. faecalis, involved in virulence, macrophage survival, oxidative stress response as well as antibiotic resistance. Therefore, CroRS represents an attractive potential new target for antibiotherapy. In this report, we further explored CroRS cellular functions by characterizing the CroR regulon: the 'domain swapping' method was applied and a CroR chimera protein was generated by fusing the receiver domain from NisR to the output domain from CroR. After demonstrating that the chimera CroR complements a croR gene deletion in E. faecalis (stress response, virulence), we conducted a global gene expression analysis using RNA-Seq and identified 50 potential CroR targets involved in multiple cellular functions such as cell envelope homeostasis, substrate transport, cell metabolism, gene expression regulation, stress response, virulence and antibiotic resistance. For validation, CroR direct binding to several candidate targets was demonstrated by EMSA. Further, this work identified alr, the gene encoding the alanine racemase enzyme involved in E. faecalis resistance to D-cycloserine, a promising antimicrobial drug to treat enterococcal infections, as a member of the CroR regulon.