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

MECHANISMS OF RESISTANCE crossm Requirement of the CroRS TwoComponent System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium Stephanie L. Kellogg, Jaime L. Little, Jessica S. Hoff, Christopher J. Kristich Department of Microbiology and Immunology, Center for Infectious Disease Research, Medical College of Wisconsin, Milwaukee, Wisconsin, USA Enterococci are serious opportunistic pathogens that are resistant to many cell wall-targeting antibiotics. The CroRS two-component signaling system responds to antibiotic-mediated cell wall stress and is critical for resistance to cell wall-targeting antibiotics in Enterococcus faecalis. Here, we identify and characterize an orthologous two-component system found in Enterococcus faecium that is functionally equivalent to the CroRS system of E. faecalis. Deletion of croRS in E. faecium resulted in marked susceptibility to cell wall-targeting agents including cephalosporins and bacitracin, as well as moderate susceptibility to ampicillin and vancomycin. As in E. faecalis, exposure to bacitracin and vancomycin stimulates signaling through the CroRS system in E. faecium. Moreover, the CroRS system is critical in E. faecium for enhanced beta-lactam resistance mediated by overexpression of Pbp5. Expression of a Pbp5 variant that confers enhanced beta-lactam resistance cannot overcome the requirement for CroRS function. Thus, the CroRS system is a conserved signaling system that responds to cell wall stress to promote intrinsic resistance to important cell wall-targeting antibiotics in clinically relevant enterococci. ABSTRACT Received 17 November 2016 Returned for modification 6 December 2016 Accepted 13 February 2017 Accepted manuscript posted online 21 February 2017 Citation Kellogg SL, Little JL, Hoff JS, Kristich CJ. 2017. Requirement of the CroRS two-component system for resistance to cell wall-targeting antimicrobials in Enterococcus faecium. Antimicrob Agents Chemother 61:e02461-16. https://doi.org/10.1128/AAC.02461-16. Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to Christopher J. Kristich, . KEYWORDS enterococcus, antibiotic resistance, two-component regulatory systems E nterococcus faecalis and Enterococcus faecium represent serious opportunistic pathogens that are responsible for many nosocomial infections. Treatment of enterococcal infections is particularly challenging due to intrinsic and acquired resistance toward many clinically relevant antibiotics, including beta-lactams, aminoglycosides, glycopeptides, and trimethoprim (1). Because all clinical isolates of E. faecalis and E. faecium are intrinsically resistant to cephalosporins (a subset of beta-lactam antibiotics), disabling cephalosporin resistance with small molecule therapeutics may be a viable strategy to overcome antibiotic-resistant enterococcal infections. Both species use transpeptidase activity of a low-affinity penicillin-binding protein (Pbp5) in cooperation with the glycosyltransferase activity of the penicillin-binding proteins (PBPs) PonA or PbpF to continue transpeptidation and transglycosylation reactions required for cell wall assembly during cephalosporin exposure (2–5). However, additional determinants contributing to cephalosporin resistance have also been explored in E. faecalis and E. faecium. In E. faecalis, two enzymes involved in cell wall synthesis (the UDP-N-acetylglucosamine 1-carboxyvinyl transferase MurAA [6] and the alanine transferase BppA2 [7]) are known to be required for normal cephalosporin resistance. In addition, two signal transduction pathways mediate intrinsic resistance to cephalosporins and other cell wall-targeting antibiotics. One pathway includes a eukaryotic-like Ser/Thr kinase, IreK, and its cognate phosphatase, IreP, which act antagonistically to regulate a pathway leading to cephalosporin resistance (8, 9). An ortholog of IreK in E. faecium has May 2017 Volume 61 Issue 5 e02461-16 Antimicrobial Agents and Chemotherapy aac.asm.org 1 Kellogg et al. Antimicrobial Agents and Chemotherapy also been implicated in cephalosporin resistance of that species (10). In E. faecalis, a substrate for phosphorylation by IreK has been described, designated IreB, which acts as a negative regulator of the pathway (11). However, the specific output of the pathway that drives cephalosporin resistance remains unknown. In addition to the IreK/IreP signaling pathway, the two-component signal transduction system (TCS) consisting of the CroS sensor kinase and its cognate response regulator CroR has a role in resistance to cell wall-targeting antibiotics. Disruption of the CroRS TCS in E. faecalis renders strains more sensitive to diverse cell wall-targeting agents such as cephalosporins, ampicillin, bacitracin, and vancomycin (12, 13). Consistent with a role for the CroRS TCS in responding to antibiotic-mediated cell wall stress, these agents can also stimulate CroR-dependent transcription (12). However, only three genes regulated by CroR have been identified (12, 14, 15), with croR itself the only of those that possesses a clear role in antimicrobial resistance. Thus, the downstream effectors in the CroR regulon that drive resistance remain to be identified. Although E. faecium is resistant to cephalosporins, most studies have analyzed ampicillin resistance in clinical isolates. High levels of ampicillin resistance have been associated with mutations in Pbp5. However, specific variants do not always correlate with MIC values in different E. faecium lineages (16–18), implying that additional factors modulate ampicillin resistance. A genome-wide study identified several determinants required for ampicillin resistance in E. faecium, including the L,D-transpeptidase Ldtfm, the D-alanyl–D-alanine carboxypeptidase DdcP, and the glycosyltransferase Pgt (19). The Ldtfm pathway was also identified as providing high-level ampicillin resistance after successive in vitro selections for ampicillin resistance (20–23). Collectively, these studies indicate that factors involved in enterococcal cell wall remodeling, distinct from the traditional biosynthetic PBPs, modulate resistance to ampicillin in E. faecium. However, the extent to which they also influence resistance to other beta-lactams such as cephalosporins remains largely unknown. Moreover, these factors are poorly conserved in E. faecalis, which tends to be considerably less prone to development of enhanced ampicillin resistance compared to E. faecium. Ideally, any target for new therapeutics designed to disable enterococcal resistance to cephalosporins will be conserved in both E. faecalis and E. faecium. To explore whether mechanisms mediating cephalosporin resistance in E. faecalis are conserved in E. faecium, we identified and functionally characterized a TCS encoded in the E. faecium genome that is homologous to the CroRS TCS of E. faecalis. We report that deletion of the E. faecium croRS orthologs render E. faec (...truncated)