Functional Dissection of the CroRS Two-Component System Required for Resistance to Cell Wall Stressors in Enterococcus faecalis

Abstract

Bacteria use two-component signal transduction systems (TCSs) to sense and respond to environmental changes via a conserved phosphorelay between a sensor histidine kinase and its cognate response regulator. The opportunistic pathogen Enterococcus faecalis utilizes a TCS comprised of the histidine kinase CroS and the response regulator CroR to mediate resistance to cell wall stresses such as cephalosporin antibiotics, but the molecular details by which CroRS promotes cephalosporin resistance have not been elucidated. Here, we analyzed mutants of E. faecalis carrying substitutions in CroR and CroS to demonstrate that phosphorylated CroR drives resistance to cephalosporins, and that CroS exhibits kinase and phosphatase activities to control the level of CroR phosphorylation in vivo. Deletion of croS in various lineages of E. faecalis revealed a CroS-independent mechanism for CroR phosphorylation and led to the identification of a noncognate histidine kinase capable of influencing CroR (encoded by OG1RF_12162; here called cisS). Further analysis of this TCS network revealed that both systems respond to cell wall stress. IMPORTANCETCSs allow bacteria to sense and respond to many different environmental conditions. The opportunistic pathogen Enterococcus faecalis utilizes the CroRS TCS to mediate resistance to cell wall stresses, including clinically relevant antibiotics such as cephalosporins and glycopeptides. In this study, we use genetic and biochemical means to investigate the relationship between CroRS signaling and cephalosporin resistance in E. faecalis cells. Through this, we uncovered a signaling network formed between the CroRS TCS and a previously uncharacterized TCS that also responds to cell wall stress. This study provides mechanistic insights into CroRS signaling and cephalosporin resistance in E. faecalis. Although Enterococcus faecalis is a normal commensal of the gut microbiome, it poses a serious risk to humans as an opportunistic pathogen. A major risk factor for the pathogenic transition of enterococci is therapy with broad-spectrum cephalosporin antibiotics. Cephalosporins are commonly used to treat bacterial infections; however, nearly all enterococcal isolates are resistant to these compounds. This intrinsic resistance allows for the expansion of resident enterococcal populations during cephalosporin treatment, which is thought to promote dissemination from the gastrointestinal tract, enabling enterococci to establish infections (1, 2). With the emergence of multidrug-resistant isolates of E. faecalis, few options for treating infections are available. Therefore, understanding the mechanisms that support cephalosporin resistance in E. faecalis is crucial for designing new therapies that limit growth during cephalosporin treatment or that can be used as adjuvants with cephalosporins to treat E. faecalis infections. An essential determinant of cephalosporin resistance in E. faecalis is the CroRS two-component system (TCS), consisting of CroS (a transmembrane sensor-histidine kinase) and ...

Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium

“…As noted above, previous studies indicate that CroRS Efs modulates gene expression in response to stress from cell wall-targeting antibiotics (12,26), and the functional conservation identified here suggests this is also the case with CroRS Efm . Hence, we hypothesize that CroRS Efm enhances expression of genes that are important for resistance upon sensing cell wall stress.…”

“…We previously identified a second TCS (CisRS), found in a subset of E. faecalis strains, that is capable of influencing the activity of CroRS Efs under certain conditions due to mutual overlap in the identity of so-called "specificity" residues of the CroS and CisS kinases that dictate response regulator specificity for TCS kinases (26)(27)(28). However, extensive studies indicated that "cross talk" between CisS and CroR Efs only occurs in the absence of CroS Efs and is not physiologically relevant (26).…”

“…However, extensive studies indicated that "cross talk" between CisS and CroR Efs only occurs in the absence of CroS Efs and is not physiologically relevant (26). Nevertheless, we searched for homologs of CisRS in sequenced E. faecium genomes, but none were found.…”

“…Upon sensing their signal, histidine kinases autophosphorylate and subsequently transfer the phosphoryl group to their cognate response regulators. We monitored this process using Phos-Tag SDS-PAGE and immunoblotting for CroR (26). In E. faecalis, a slower-mobility isoform of CroR Efs is observed with Phos-Tag SDS-PAGE after treatment of cells with bacitracin and vancomycin (Fig.…”

“…The MICs of antibiotics were determined as described previously (26). Briefly, bacteria from stationary-phase cultures in MHB (plus 10 g/ml chloramphenicol for plasmid carrying strains) were inoculated at a cell density of ϳ10 5 CFU/ml into microplate wells containing 2-fold serial dilutions of antibiotic.…”