Reciprocal regulation of enterococcal cephalosporin resistance by products of the autoregulated yvcJ-glmR-yvcL operon enhances fitness during cephalosporin exposure.

Enterococci are commensal members of the gastrointestinal tract and also major nosocomial pathogens. They possess both intrinsic and acquired resistance to many antibiotics, including intrinsic resistance to cephalosporins that target bacterial cell wall synthesis. These antimicrobial resistance traits make enterococcal infections challenging to treat. Moreover, prior therapy with antibiotics, including broad-spectrum cephalosporins, promotes enterococcal proliferation in the gut, resulting in dissemination to other sites of the body and subsequent infection. As a result, a better understanding of mechanisms of cephalosporin resistance is needed to enable development of new therapies to treat or prevent enterococcal infections. We previously reported that flow of metabolites through the peptidoglycan biosynthesis pathway is one determinant of enterococcal cephalosporin resistance. One factor that has been implicated in regulating flow of metabolites into cell wall biosynthesis pathways of other Gram-positive bacteria is GlmR. In enterococci, GlmR is encoded as the middle gene of a predicted 3-gene operon along with YvcJ and YvcL, whose functions are poorly understood. Here we use genetics and biochemistry to investigate the function of the enterococcal yvcJ-glmR-yvcL gene cluster. Our results reveal that YvcL is a DNA-binding protein that regulates expression of the yvcJ-glmR-yvcL operon in response to cell wall stress. YvcJ and GlmR bind UDP-GlcNAc and reciprocally regulate cephalosporin resistance in E. faecalis, and binding of UDP-GlcNAc by YvcJ appears essential for its activity. Reciprocal regulation by YvcJ/GlmR is essential for fitness during exposure to cephalosporin stress. Additionally, our results indicate that enterococcal GlmR likely acts by a different mechanism than the previously studied GlmR of Bacillus subtilis, suggesting that the YvcJ/GlmR regulatory module has evolved unique targets in different species of bacteria.

CCR2-dependent CX3CR1+ colonic macrophages promote Enterococcus faecalis dissemination.

Enterococci are common commensal bacteria that colonize the gastrointestinal tracts of most mammals, including humans. Importantly, these bacteria are one of the leading causes of nosocomial infections. This study examined the role of colonic macrophages in facilitating Enterococcus faecalis infections in mice. We determined that depletion of colonic phagocytes resulted in the reduction of E. faecalis dissemination to the gut-draining mesenteric lymph nodes. Furthermore, we established that trafficking of monocyte-derived CX3CR1-expressing macrophages contributed to E. faecalis dissemination in a manner that was not reliant on CCR7, the conventional receptor involved in lymphatic migration. Finally, we showed that E. faecalis mutants with impaired intracellular survival exhibited reduced dissemination, suggesting that E. faecalis can exploit host immune cell migration to disseminate systemically and cause disease. Our findings indicate that modulation of macrophage trafficking in the context of antibiotic therapy could serve as a novel approach for preventing or treating opportunistic infections by disseminating enteric pathobionts like E. faecalis.

PASTA-kinase-mediated signaling drives accumulation of the peptidoglycan synthesis protein MurAA to promote cephalosporin resistance in Enterococcus faecalis.

The bacterial PASTA kinase, IreK, is required for intrinsic cephalosporin resistance in the Gram-positive opportunistic pathogen, Enterococcus faecalis. IreK activity is enhanced in response to cell wall stress, such as cephalosporin exposure. The downstream consequences of IreK activation are not well understood in E. faecalis, but recent work in other low-GC Gram-positive bacteria demonstrated PASTA kinase-dependent regulation of MurAA, an enzyme that performs the first committed step in the peptidoglycan synthesis pathway. Here, we used genetic suppressor selections to identify MurAA as a downstream target of IreK signaling in E. faecalis. Using complementary genetic and biochemical approaches, we demonstrated that MurAA abundance is regulated by IreK signaling in response to physiologically relevant cell wall stress to modulate substrate flux through the peptidoglycan synthesis pathway. Specifically, the IreK substrate, IreB, promotes proteolysis of MurAA through a direct physical interaction in a manner responsive to phosphorylation by IreK. MurAB, a homolog of MurAA, also promotes MurAA proteolysis and interacts directly with IreB. Our results therefore establish a connection between the cell wall stress sensor IreK and one critical physiological output to modulate peptidoglycan synthesis and drive cephalosporin resistance.

Pbp4 provides transpeptidase activity to the FtsW-PbpB peptidoglycan synthase to drive cephalosporin resistance in Enterococcus faecalis.

Enterococci exhibit intrinsic resistance to cephalosporins, mediated in part by the class B penicillin-binding protein (bPBP) Pbp4 that exhibits low reactivity toward cephalosporins and thus can continue crosslinking peptidoglycan despite exposure to cephalosporins. bPBPs partner with cognate SEDS (shape, elongation, division, and sporulation) glycosyltransferases to form the core catalytic complex of peptidoglycan synthases that synthesize peptidoglycan at discrete cellular locations, although the SEDS partner for Pbp4 is unknown. SEDS-bPBP peptidoglycan synthases of enterococci have not been studied, but some SEDS-bPBP pairs can be predicted based on sequence similarity. For example, FtsW (SEDS)-PbpB (bPBP) is predicted to form the catalytic core of the peptidoglycan synthase that functions at the division septum (the divisome). However, PbpB is readily inactivated by cephalosporins, raising the question-how could the FtsW-PbpB synthase continue functioning to enable growth in the presence of cephalosporins? In this work, we report that the FtsW-PbpB peptidoglycan synthase is required for cephalosporin resistance of Enterococcus faecalis, despite the fact that PbpB is inactivated by cephalosporins. Moreover, Pbp4 associates with the FtsW-PbpB synthase and the TPase activity of Pbp4 is required to enable growth in the presence of cephalosporins in an FtsW-PbpB-synthase-dependent manner. Overall, our results implicate a model in which Pbp4 directly interacts with the FtsW-PbpB peptidoglycan synthase to provide TPase activity during cephalosporin treatment, thereby maintaining the divisome SEDS-bPBP peptidoglycan synthase in a functional state competent to synthesize crosslinked peptidoglycan. These results suggest that two bPBPs coordinate within the FtsW-PbpB peptidoglycan synthase to drive cephalosporin resistance in E. faecalis.

Use of an Interspecies Chimeric Receptor for Inducible Gene Expression Reveals that Metabolic Flux through the Peptidoglycan Biosynthesis Pathway is an Important Driver of Cephalosporin Resistance in Enterococcus faecalis.

Cephalosporins are commonly prescribed antibiotics that impair cross-linking of the bacterial cell wall. The Gram-positive opportunistic pathogen, Enterococcus faecalis, is intrinsically resistant to these antibiotics and proliferates substantially during cephalosporin therapy. As a result, the usage of cephalosporins has the potential to lead to life-threatening enterococcal infections. Yet, the molecular mechanisms that drive cephalosporin resistance (CR) are incompletely understood. Previously, we demonstrated that MurAA, an enzyme that catalyzes the first committed step in peptidoglycan (PG) synthesis, is required for CR. However, the mechanism by which MurAA contributes to CR remained unknown. Here, we tested the hypothesis that MurAA drives CR by controlling metabolic flux through the PG synthesis pathway. To do so, we developed and exploited an inducible gene expression system for E. faecalis based on an interspecies chimeric receptor that responds to exogenous nitrate for control of expression from a NisR-regulated promoter (PnisA). We used this tool to demonstrate synthetic lethality of MurAA with its homolog MurAB, to titrate expression of MurAA, and to conditionally deplete multiple PG synthesis enzymes downstream of MurAA that are predicted to be essential. These genetic manipulations, in addition to pharmacological inhibition of the PG synthesis pathway, all led to reductions in PG synthesis that correlated with reductions in CR. Our findings are consistent with a model in which control of metabolic flux through the PG synthesis pathway is a major driver of CR. IMPORTANCE 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. Here, we report that the cell wall synthesis enzyme, MurAA, drives cephalosporin resistance at least in part by controlling metabolic flux through the peptidoglycan synthesis pathway. To demonstrate this, we designed and validated an inducible gene expression system based on a chimeric receptor that is functional in multiple lineages of E. faecalis. In doing so, we provided a new tool for inducible gene expression with broad applications beyond our studies, including studies of essential genes.

GpsB Promotes PASTA Kinase Signaling and Cephalosporin Resistance in Enterococcus faecalis.

Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired mechanisms of resistance to commonly used antibiotics, including cephalosporins. The transmembrane serine/threonine PASTA kinase, IreK, is an important determinant of enterococcal cephalosporin resistance. Upon exposure to cephalosporins, IreK becomes autophosphorylated, which stimulates its kinase activity to phosphorylate downstream substrates and drive cephalosporin resistance. However, the molecular mechanisms that modulate IreK autophosphorylation in response to cell wall stress, such as that induced by cephalosporins, remain unknown. A cytoplasmic protein, GpsB, promotes signaling by PASTA kinase homologs in other bacterial species, but the function of enterococcal GpsB has not been previously investigated. We used in vitro and in vivo approaches to test the hypothesis that enterococcal GpsB promotes IreK signaling in response to cephalosporins to drive cephalosporin resistance. We found that GpsB promotes IreK activity both in vivo and in vitro. This effect is required for cephalosporins to trigger IreK autophosphorylation and activation of an IreK-dependent signaling pathway, and thereby is also required for enterococcal intrinsic cephalosporin resistance. Moreover, analyses of GpsB mutants and a ΔireK gpsB double mutant suggest that GpsB has an additional function, beyond regulation of IreK activity, which is required for optimal growth and full cephalosporin resistance. Collectively, our data provide new insights into the mechanism of signal transduction by the PASTA kinase IreK and the mechanism of enterococcal intrinsic cephalosporin resistance. IMPORTANCE 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. In particular, enterococci are intrinsically resistant to cephalosporin antibiotics, a trait that requires the activity of a transmembrane serine/threonine kinase, IreK, which belongs to the bacterial PASTA kinase family. The mechanisms by which PASTA kinases are regulated in cells are poorly understood. Here, we report that the cytoplasmic protein GpsB directly promotes IreK signaling in enterococci to drive cephalosporin resistance. Thus, we provide new insights into PASTA kinase regulation and control of enterococcal cephalosporin resistance, and suggest that GpsB could be a promising target for new therapeutics to disable cephalosporin resistance.

IreK-Mediated, Cell Wall-Protective Phosphorylation in Enterococcus faecalis.

Enterococcus faecalis is a Gram-positive bacterium that is a major cause of hospital-acquired infections due, in part, to its intrinsic resistance to cell wall-active antimicrobials. One critical determinant of this resistance is the transmembrane kinase IreK, which belongs to the penicillin-binding protein and serine/threonine kinase-associated kinase family of bacterial signaling proteins involved with the regulation of cell wall homeostasis. The activity of IreK is enhanced in response to cell wall stress, but direct substrates of IreK phosphorylation, leading to antimicrobial resistance, are largely unknown. To better understand stress-modulated phosphorylation events contributing to antimicrobial resistance, wild type E. faecalis cells treated with cell wall-active antimicrobials, chlorhexidine or ceftriaxone, were examined via phosphoproteomics. Among the most prominent changes was increased phosphorylation of divisome components after both treatments, suggesting that E. faecalis modulates cell division in response to cell wall stress. Phosphorylation mediated by IreK was then determined via a similar analysis with a E. faecalis ΔireK mutant strain, revealing potential IreK substrates involved with the regulation of peptidoglycan biosynthesis and within the E. faecalis CroS/R two-component system, another signal transduction pathway that promotes antimicrobial resistance. These results reveal critical insights into the biological functions of IreK.

Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract.

Enterococcus faecalis is both a common commensal of the human gastrointestinal tract and a leading cause of hospital-acquired infections. Systemic infections with multidrug-resistant enterococci occur subsequent to gastrointestinal colonization. Preventing colonization by multidrug-resistant E. faecalis could therefore be a valuable approach towards limiting infection. However, little is known about the mechanisms E. faecalis uses to colonize and compete for stable gastrointestinal niches. Pheromone-responsive conjugative plasmids encoding bacteriocins are common among enterococcal strains and could modulate niche competition among enterococci or between enterococci and the intestinal microbiota. We developed a model of colonization of the mouse gut with E. faecalis, without disrupting the microbiota, to evaluate the role of the conjugative plasmid pPD1 expressing bacteriocin 21 (ref. 4) in enterococcal colonization. Here we show that E. faecalis harbouring pPD1 replaces indigenous enterococci and outcompetes E. faecalis lacking pPD1. Furthermore, in the intestine, pPD1 is transferred to other E. faecalis strains by conjugation, enhancing their survival. Colonization with an E. faecalis strain carrying a conjugation-defective pPD1 mutant subsequently resulted in clearance of vancomycin-resistant enterococci, without plasmid transfer. Therefore, bacteriocin expression by commensal bacteria can influence niche competition in the gastrointestinal tract, and bacteriocins, delivered by commensals that occupy a precise intestinal bacterial niche, may be an effective therapeutic approach to specifically eliminate intestinal colonization by multidrug-resistant bacteria, without profound disruption of the indigenous microbiota.

Multiple Low-Reactivity Class B Penicillin-Binding Proteins Are Required for Cephalosporin Resistance in Enterococci.

Enterococcus faecalis and Enterococcus faecium are commensals of the gastrointestinal tract of most terrestrial organisms, including humans, and are major causes of health care-associated infections. Such infections are difficult or impossible to treat, as the enterococcal strains responsible are often resistant to multiple antibiotics. One intrinsic resistance trait that is conserved among E. faecalis and E. faecium is cephalosporin resistance, and prior exposure to cephalosporins is one of the most well-known risk factors for acquisition of an enterococcal infection. Cephalosporins inhibit peptidoglycan biosynthesis by acylating the active-site serine of penicillin-binding proteins (PBPs) to prevent the PBPs from catalyzing cross-linking during peptidoglycan synthesis. For decades, a specific PBP (known as Pbp4 or Pbp5) that exhibits low reactivity toward cephalosporins has been thought to be the primary PBP required for cephalosporin resistance. We analyzed other PBPs and report that in both E. faecalis and E. faecium, a second PBP, PbpA(2b), is also required for resistance; notably, the cephalosporin ceftriaxone exhibits a lethal effect on the ΔpbpA mutant. Strikingly, PbpA(2b) exhibits low intrinsic reactivity with cephalosporins in vivo and in vitro Unlike the Δpbp5 mutant, the ΔpbpA mutant exhibits a variety of phenotypic defects in growth kinetics, cell wall integrity, and cellular morphology, indicating that PbpA(2b) and Pbp5(4) are not functionally redundant and that PbpA(2b) plays a more central role in peptidoglycan synthesis. Collectively, our results shift the current understanding of enterococcal cephalosporin resistance and suggest a model in which PbpA(2b) and Pbp5(4) cooperate to coordinately mediate peptidoglycan cross-linking in the presence of cephalosporins.

Reciprocal Regulation of PASTA Kinase Signaling by Differential Modification.

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.

Sortase-Dependent Proteins Promote Gastrointestinal Colonization by Enterococci.

The human gastrointestinal tract (GIT) is inhabited by a dense microbial community of symbionts. Enterococci are among the earliest members of this community and remain core members of the GIT microbiota throughout life. Enterococci have also recently emerged as opportunistic pathogens and major causes of nosocomial infections. Although recognized as a prerequisite for infection, colonization of the GIT by enterococci remains poorly understood. One way that bacteria adapt to dynamic ecosystems like the GIT is through the use of their surface proteins to sense and interact with components of their immediate environment. In Gram-positive bacteria, a subset of surface proteins relies on an enzyme called sortase for covalent attachment to the cell wall. Here, we show that the housekeeping sortase A (SrtA) enzyme promotes intestinal colonization by enterococci. Furthermore, we show that the enzymatic activity of SrtA is key to the ability of Enterococcus faecalis to bind mucin (a major component of the GIT mucus). We also report the GIT colonization phenotypes of E. faecalis mutants lacking selected sortase-dependent proteins (SDPs). Further examination of the mucin binding ability of these mutants suggests that adhesion to mucin contributes to intestinal colonization by E. faecalis.

Convergence of PASTA Kinase and Two-Component Signaling in Response to Cell Wall Stress in Enterococcus faecalis.

Two common signal transduction mechanisms used by bacteria to sense and respond to changing environments are two-component systems (TCSs) and eukaryote-like Ser/Thr kinases and phosphatases (eSTK/Ps). Enterococcus faecalis is a Gram-positive bacterium and a 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 a response regulator (CroR) that become activated upon exposure of cells to cell wall-targeting antibiotics, leading to a 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 toward cell wall-targeting antibiotics, we hypothesized that 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 the 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 the phosphorylation of CroS to promote antimicrobial resistance in E. faecalisIMPORTANCE Two-component signaling systems (TCSs) and eukaryote-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 Enterococcus faecalis 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 the convergence of IreK and the sensor kinase CroS to enhance signaling through CroS/R and increase antimicrobial resistance in E. faecalis This newly described example of eSTK/TCS convergence adds to our understanding of the signaling networks mediating antimicrobial resistance in E. faecalis.

Modulators of Enterococcus faecalis Cell Envelope Integrity and Antimicrobial Resistance Influence Stable Colonization of the Mammalian Gastrointestinal Tract.

The Gram-positive bacterium Enterococcus faecalis is both a colonizer of the gastrointestinal tract (GIT) and an agent of serious nosocomial infections. Although it is typically required for pathogenesis, GIT colonization by E. faecalis is poorly understood. E. faecalis tolerates high concentrations of GIT antimicrobials, like cholate and lysozyme, leading us to hypothesize that resistance to intestinal antimicrobials is essential for long-term GIT colonization. Analyses of E. faecalis mutants exhibiting defects in antimicrobial resistance revealed that IreK, a determinant of envelope integrity and antimicrobial resistance, is required for long-term GIT colonization. IreK is a member of the PASTA kinase protein family, bacterial transmembrane signaling proteins implicated in the regulation of cell wall homeostasis. Among several determinants of cholate and lysozyme resistance in E. faecalis, IreK was the only one found to be required for intestinal colonization, emphasizing the importance of this protein to enterococcal adaptation to the GIT. By studying ΔireK suppressor mutants that recovered the ability to colonize the GIT, we identified two conserved enterococcal proteins (OG1RF_11271 and OG1RF_11272) that function antagonistically to IreK and interfere with cell envelope integrity, antimicrobial resistance, and GIT colonization. Our data suggest that IreK, through its kinase activity, inhibits the actions of these proteins. IreK, OG1RF_11271, and OG1RF_11272 are found in all enterococci, suggesting that their effect on GIT colonization is universal across enterococci. Thus, we have defined conserved genes in the enterococcal core genome that influence GIT colonization through their effect on enterococcal envelope integrity and antimicrobial resistance.

Ceftriaxone Administration Disrupts Intestinal Homeostasis, Mediating Noninflammatory Proliferation and Dissemination of Commensal Enterococci.

Enterococci are Gram-positive commensals of the mammalian intestinal tract and harbor intrinsic resistance to broad-spectrum cephalosporins. Disruption of colonization resistance in humans by antibiotics allows enterococci to proliferate in the gut and cause disseminated infections. In this study, we used Enterococcus faecalis (EF)-colonized mice to study the dynamics of enterococci, commensal microbiota, and the host in response to systemic ceftriaxone administration. We found that the mouse model recapitulates intestinal proliferation and dissemination of enterococci seen in humans. Employing a ceftriaxone-sensitive strain of enterococci (E. faecalis JL308), we showed that increased intestinal abundance is critical for the systemic dissemination of enterococci. Investigation of the impact of ceftriaxone on the mucosal barrier defenses and integrity suggested that translocation of enterococci across the intestinal mucosa was not associated with intestinal pathology or increased permeability. Ceftriaxone-induced alteration of intestinal microbial composition was associated with transient increase in the abundance of multiple bacterial operational taxonomic units (OTUs) in addition to enterococci, for example, lactobacilli, which also disseminated to the extraintestinal organs. Collectively, these results emphasize that ceftriaxone-induced disruption of colonization resistance and alteration of mucosal homeostasis facilitate increased intestinal abundance of a limited number of commensals along with enterococci, allowing their translocation and systemic dissemination in a healthy host.

Extracellular SalB Contributes to Intrinsic Cephalosporin Resistance and Cell Envelope Integrity in Enterococcus faecalis.

Enterococci are major causes of hospital-acquired infections. Intrinsic resistance to cephalosporins is a universal trait among clinically relevant enterococci. Cephalosporin resistance enables enterococci to proliferate to high densities in the intestines of patients undergoing cephalosporin treatment, a precursor to the emergence of infection. However, the genetic and biochemical mechanisms of intrinsic cephalosporin resistance in enterococci are not well understood. A two-component signal transduction system, CroR/S, is required for cephalosporin resistance in enterococci. Although the CroR/S regulon is not well defined, one gene reported to be CroR dependent in Enterococcus faecalis JH2-2 encodes an extracellular putative peptidoglycan hydrolase, SalB. To test the hypothesis that SalB is responsible for CroR-dependent cephalosporin resistance, we examined ΔsalB mutants in multiple genetic lineages of E. faecalis, revealing that SalB is required not only for intrinsic cephalosporin resistance but also for maintenance of cell envelope integrity in the absence of antibiotic stress. The N-terminal signal sequence is necessary for SalB secretion, and secretion is required for SalB to promote cephalosporin resistance. Functional dissection revealed that the C-terminal SCP domain of SalB is essential for biological activity and identified three residues within the SCP domain that are required for the stability and function of SalB. Additionally, we found that in contrast to what is seen in E. faecalis JH2-2, SalB is not regulated by the CroR/S two-component system in E. faecalis OG1, suggesting diversity in the CroR/S regulon among distinct lineages of E. faecalis IMPORTANCE Resistance to cephalosporins is universal among clinically relevant enterococci, enabling enterococcal proliferation to high densities in the intestines of patients undergoing cephalosporin treatment, a precursor to the emergence of infection. Disabling cephalosporin resistance could therefore reduce the incidence of enterococcal infections. However, the genetic and biochemical mechanisms of cephalosporin resistance are not well understood. The significance of this work is the identification of a novel extracellular factor (SalB) that promotes cephalosporin resistance in E. faecalis, which could potentially serve as a target for therapeutics that impair enterococcal cephalosporin resistance. Additionally, our work highlights the importance of the C-terminal SCP domain of SalB, including several conserved residues within the SCP domain, for the ability of SalB to promote cephalosporin resistance.

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivoEnterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivoIMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The prevailing model for signaling by PASTA kinases proposes that the extracellular PASTA domains bind ligands to drive kinase dimerization, enhanced autophosphorylation, and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo We show that the PASTA kinase IreK of Enterococcus faecalis responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires the PASTA domains and phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling.

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

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.

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

UNLABELLED: 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. IMPORTANCE: TCSs 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.

Oxidative stress enhances cephalosporin resistance of Enterococcus faecalis through activation of a two-component signaling system.

Enterococcus faecalis is a low-GC Gram-positive bacterium, a normal resident of the gastrointestinal (GI) tract, and an important hospital-acquired pathogen. An important risk factor for hospital-acquired enterococcal infections is prior therapy with broad-spectrum cephalosporins, antibiotics that impair cell wall biosynthesis by inhibiting peptidoglycan cross-linking. Enterococci are intrinsically resistant to cephalosporins; however, environmental factors that modulate cephalosporin resistance have not been described. While searching for the genetic determinants of cephalosporin resistance in E. faecalis, we unexpectedly discovered that oxidative stress, whether from external sources or derived from endogenous metabolism, drives enhanced intrinsic resistance to cephalosporins. A particular source of oxidative stress, H2O2, activates signaling through the CroR-CroS two-component signaling system, a known determinant of cephalosporin resistance in E. faecalis. We find that CroR-CroS is required for adaptation to H2O2 stress and that H2O2 potentiates the activities of cephalosporins against E. faecalis when the CroR-CroS signaling system is nonfunctional. Rather than directly detecting H2O2, our data suggest that the CroR-CroS system responds to cell envelope damage caused by H2O2 exposure in order to promote cell envelope repair and enhanced cephalosporin resistance.

Genetic basis for vancomycin-enhanced cephalosporin susceptibility in vancomycin-resistant enterococci revealed using counterselection with dominant-negative thymidylate synthase.

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 VanYB is both necessary and sufficient to dramatically sensitize E. faecalis to cephalosporins.