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 ...

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

Two common signal transduction mechanisms used by bacteria to sense and respond to changing environments are two-component systems (TCSs) and eukaryotic-like Ser/Thr kinases and phosphatases (eSTK/Ps). is a Gram-positive bacterium and serious opportunistic pathogen that relies on both a TCS and an eSTK/P pathway for intrinsic resistance to cell wall-targeting antibiotics. The TCS consists of a histidine kinase (CroS) and response regulator (CroR) that become activated upon exposure of cells to cell wall-targeting antibiotics, leading to modulation of gene expression. The eSTK/P pathway consists of a transmembrane kinase (IreK) and its cognate phosphatase (IreP), which act antagonistically to mediate antibiotic resistance through an unknown mechanism. Because both CroS/R and IreK/P contribute to enterococcal resistance towards cell wall-targeting antibiotics, we hypothesized these signaling systems are intertwined. To test this hypothesis, we analyzed CroR phosphorylation and CroS/R-dependent gene expression to probe the influence of IreK and IreP on CroS/R signaling. In addition, we analyzed the phosphorylation state of CroS which revealed IreK-dependent phosphorylation of a Thr residue important for CroS function. Our results are consistent with a model in which IreK positively influences CroR-dependent gene expression through phosphorylation of CroS to promote antimicrobial resistance in Two-component signaling systems (TCSs) and eukaryotic-like Ser/Thr kinases (eSTKs) are used by bacteria to sense and adapt to changing environments. Understanding how these pathways are regulated to promote bacterial survival is critical for a more complete understanding of bacterial stress responses and physiology. The opportunistic pathogen relies on both a TCS (CroS/R) and an eSTK (IreK) for intrinsic resistance to cell wall-targeting antibiotics. We probed the relationship between CroS/R and IreK, revealing convergence of IreK and the sensor kinase CroS to enhance signaling through CroS/R and increase antimicrobial resistance in This newly described example of eSTK/TCS convergence adds to our understanding of the signaling networks mediating antimicrobial resistance in .

Transmembrane Ser/Thr kinases containing extracellular PASTA (penicillin-binding protein [PBP] and Ser/Thr-associated) domains are ubiquitous among Actinobacteria and Firmicutes species. Such PASTA kinases regulate critical bacterial processes, including antibiotic resistance, cell division, cell envelope homeostasis, and virulence, and are sometimes essential for viability. Previous studies of purified PASTA kinase fragments revealed they are capable of autophosphorylation in vitro, typically at multiple sites on the kinase domain. Autophosphorylation of a specific structural element of the kinase known as the activation loop is thought to enhance kinase activity in response to stimuli. However, the role of kinase phosphorylation at other sites is largely unknown. Moreover, the mechanisms by which PASTA kinases are deactivated once their stimulus has diminished are poorly understood. Enterococcus faecalis is a Gram-positive intestinal bacterium and a major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, and such antimicrobials trigger enhanced phosphorylation of IreK in vivo. Here we identify multiple sites of phosphorylation on IreK and evaluate their function in vivo and in vitro. While phosphorylation of the IreK activation loop is required for kinase activity, we found that phosphorylation at a site distinct from the activation loop reciprocally modulates IreK activity in vivo, leading to diminished activity (and diminished antimicrobial resistance). Moreover, this site is important for deactivation of IreK in vivo upon removal of an activating stimulus. Our results are consistent with a model in which phosphorylation of IreK at distinct sites reciprocally regulates IreK activity in vivo to promote adaptation to cell wall stresses. IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes species and regulate critical processes, including antibiotic resistance, cell division, and cell envelope homeostasis. Previous studies of PASTA kinase fragments revealed autophosphorylation at multiple sites. However, the functional role of autophosphorylation and the relative impacts of phosphorylation at distinct sites are poorly understood. The PASTA kinase of Enterococcus faecalis, IreK, regulates intrinsic resistance to antimicrobials. Here we identify multiple sites of phosphorylation on IreK and show that modification of IreK at distinct sites reciprocally regulates IreK activity and antimicrobial resistance in vivo. Thus, these results provide new insights into the mechanisms by which PASTA kinases can regulate critical physiological processes in a wide variety of bacterial species.

Enterococci are ubiquitous inhabitants of the gastrointestinal (GI) tract. However, antibiotic-resistant enterococci are also major causes of hospital-acquired infections. Enterococci are intrinsically resistant to cephalosporins, enabling growth to abnormally high densities in the GI tract in patients during cephalosporin therapy, thereby promoting dissemination to other sites where they cause infection. Despite its importance, many questions about the underlying basis for cephalosporin resistance remain. A specific two-component signaling system, composed of the CroS sensor kinase and its cognate response regulator (CroR), is required for cephalosporin resistance in Enterococcus faecalis, but little is known about the factors that control this signaling system to modulate resistance. To explore the signaling network in which CroR participates to influence cephalosporin resistance, we employed a protein fragment complementation assay to detect protein-protein interactions in E. faecalis cells, revealing a previously unknown association of CroR with the HPr protein of the phosphotransferase system (PTS) responsible for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses indicate that association with HPr restricts the ability of CroR to promote cephalosporin resistance and gene expression in a nutrient-dependent manner. Mutational analysis suggests that the interface used by HPr to associate with CroR is distinct from the interface used to associate with other cellular partners. Our results define a physical and functional connection between a critical nutrient-responsive signaling system (the PTS) and a two-component signaling system that drives antibiotic resistance in E. faecalis, and they suggest a general strategy by which bacteria can integrate their nutritional status with diverse environmental stimuli. Enterococci are ubiquitous Gram-positive bacteria usually found as commensals of the gastrointestinal (GI) tract in humans, animals, and insects. In the competitive environment of the GI tract, the ability to utilize a diverse repertoire of nutrients for growth may represent an important selective advantage. Enterococci can utilize a wide variety of carbohydrates for growth (1-3), a trait that is reflected in the abundance of carbohydrate uptake and utilization pathways identified in the Enterococcus faecalis genome (4). The enterococcal genome is especially replete with genes encoding components of the phosphoenol pyruvate (PEP)-dependent phosphotransferase system (PTS) that is widely used by bacteria both to transport carbohydrates into the cell and to control gene expression in response to nutrient availability (see references 5 and 6 for thorough reviews). PTSs mediate transport and phosphorylation of substrate carbohydrates through the coupled action of carbohydrate-specific transporters (called "EIIs") and the general PTS components EI and HPr. EI and HPr participate in sequential phosphotransfer reactions in which phosphoryl groups derived from PEP are s...