Antibiotic-resistant enterococci are major causes of hospital-acquired infections and therefore represent a serious public health problem. One well-known risk factor for the acquisition of hospital-acquired enterococcal infections is prior therapy with broad-spectrum cephalosporin antibiotics. Enterococci can proliferate in patients undergoing cephalosporin therapy due to intrinsic cephalosporin resistance, a characteristic of the genus Enterococcus. However, the molecular basis for cephalosporin resistance in E. faecalis has yet to be adequately elucidated. Previously we determined that a putative Ser/Thr kinase, IreK (formerly PrkC), is required for intrinsic cephalosporin resistance in E. faecalis. Here we show that kinase activity is required for cephalosporin resistance and, further, that resistance in E. faecalis is reciprocally regulated by IreK and IreP, a PP2C-type protein phosphatase encoded immediately upstream of IreK. Mutants of two divergent lineages of E. faecalis lacking IreP exhibit remarkable hyperresistance to cephalosporins but not to antibiotics targeting other cellular processes. Further genetic analyses indicate that hyperresistance of the IreP mutant is mediated by the IreK kinase. Additionally, competition experiments reveal that hyperresistant ΔireP mutants exhibit a substantial fitness defect in the absence of antibiotics, providing an evolutionary rationale for the use of a complex signaling system to control intrinsic cephalosporin resistance. These results support a model in which IreK and IreP act antagonistically via protein phosphorylation and dephosphorylation as part of a signal transduction circuit to regulate cellular adaptation to cephalosporin-induced stress.

Enterococci are dangerous opportunistic pathogens with the potential to cause life-threatening infections due in part to their intrinsic resistance to cephalosporin antibiotics. Elucidating the molecular mechanisms that provide this resistance is critical for the development of strategies to both prevent and treat enterococcal infections.

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.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

Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired resistance to commonly used antibiotics.

Antibiotic-resistant enterococci are major causes of hospital-acquired infections. All enterococci are intrinsically resistant to most cephalosporins, antibiotics in the beta-lactam family that impair peptidoglycan synthesis by inactivating the transpeptidases responsible for cross-linking. In addition, clinical isolates of enterococci often possess acquired resistance to vancomycin, a glycopeptide antibiotic that impairs peptidoglycan biosynthesis by a mechanism distinct from that of the beta-lactams, namely, by binding to the D-Ala-D-Ala termini found in peptidoglycan precursors to prevent their utilization by biosynthetic transglycosylases. Antimicrobial synergism between vancomycin and beta-lactams against vancomycin-resistant enterococci was originally described decades ago, but the genetic basis for synergy has remained unknown. Because a complete understanding of the mechanism underlying synergy between vancomycin and beta-lactams might suggest new targets or strategies for therapeutic intervention against antibiotic-resistant enterococci, we explored the genetic basis for synergy between vancomycin and cephalosporins in Enterococcus faecalis. To do so, we developed a counterselection strategy based on a dominant-negative mutant of thymidylate synthase and implemented this approach to create a panel of mutants in vancomycin-resistant E. faecalis. Our results confirm that vancomycin promotes synergy by inducing expression of the van resistance genes, as a mutant in which the van genes are expressed in the absence of vancomycin exhibits susceptibility to cephalosporins. Further, we show that peptidoglycan precursors substituted with D-Ala-D-Lac are not required for vancomycin-enhanced cephalosporin sensitivity. Instead, production of the D,D-carboxypeptidase VanY B is both necessary and sufficient to dramatically sensitize E. faecalis to cephalosporins.