RESUMEN
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.
RESUMEN
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.
Asunto(s)
Enterococcus faecalis , Peptidoglicano , Enterococcus faecalis/metabolismo , Peptidoglicano/metabolismo , Resistencia a las Cefalosporinas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fosfotransferasas/metabolismo , Pared Celular/metabolismoRESUMEN
Enterococcus faecalis is an opportunistic pathogen and a major cause of severe nosocomial infections. Treatment options against enterococcal infections are declining due to the resistance of enterococci to numerous antibiotics. A key risk factor for developing enterococcal infections is treatment with cephalosporin antibiotics, to which enterococci are intrinsically resistant. For susceptible organisms, cephalosporins inhibit bacterial growth by acylating the active site of penicillin-binding proteins (PBPs), key enzymes that catalyze peptidoglycan cross-linking. Two specific PBPs of enterococci, Pbp4(5) and PbpA(2b), exhibit low reactivity toward cephalosporins, allowing these PBPs to cross-link peptidoglycan in the presence of cephalosporins to drive resistance in enterococci, but the mechanisms by which these PBPs are regulated are poorly understood. The CroS/R two-component signal transduction system (TCS) is also required for cephalosporin resistance. Activation of CroS/R by cephalosporins leads to CroR-dependent changes in gene expression. However, the specific genes regulated by CroS/R that are responsible for cephalosporin resistance remain largely unknown. In this study, we characterized CroR-dependent transcriptome remodeling by RNA-seq, identifying pbp4(5) as a CroR regulon member in multiple, diverse lineages of E. faecalis. Through genetic analysis of the pbp4(5) and croR promoters, we uncovered a CroR-dependent regulatory motif. Mutations in this motif to disrupt CroR-dependent upregulation of pbp4(5) in the presence of cell wall stress resulted in a reduction of resistance to cephalosporins in E. faecalis, demonstrating that enhanced production of Pbp4(5) and likely other proteins involved in peptidoglycan biogenesis by the CroS/R system drives enterococcal cephalosporin resistance. IMPORTANCE Investigation into molecular mechanisms used by enterococci to subvert cephalosporin antibiotics is imperative for preventing and treating life-threatening infections. In this study, we used genetic means to investigate the functional output of the CroS/R TCS required for enterococcal resistance to cephalosporins. We found that enhanced production of the penicillin-binding protein Pbp4(5) upon exposure to cell wall stress was mediated by CroS/R and was critical for intrinsic cephalosporin resistance of E. faecalis.
Asunto(s)
Resistencia a las Cefalosporinas , Enterococcus faecalis , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Cefalosporinas/farmacología , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/metabolismoRESUMEN
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.
Asunto(s)
Resistencia a las Cefalosporinas , Enterococcus faecalis , Antibacterianos/metabolismo , Antibacterianos/farmacología , Resistencia a las Cefalosporinas/genética , Cefalosporinas/metabolismo , Cefalosporinas/farmacología , Enterococcus faecalis/metabolismo , Expresión Génica , Peptidoglicano/metabolismoRESUMEN
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.
Asunto(s)
Pared Celular , Enterococcus faecalis , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Pruebas de Sensibilidad Microbiana , Fosforilación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
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.
Asunto(s)
Resistencia a las Cefalosporinas/fisiología , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecium/efectos de los fármacos , Proteínas de Unión a las Penicilinas/fisiología , Acilación , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/microbiología , Electroforesis en Gel de Poliacrilamida , Enterococcus faecalis/metabolismo , Enterococcus faecium/metabolismo , Tracto Gastrointestinal/microbiología , Humanos , Immunoblotting , Concentración 50 Inhibidora , Microscopía Electrónica de Transmisión , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/biosíntesisRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Pared Celular/efectos de los fármacos , Enterococcus faecalis/genética , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Proteínas Quinasas/genética , Transactivadores/genética , Resistencia betalactámica/genética , Secuencia de Aminoácidos , Ampicilina/farmacología , Bacitracina/farmacología , Cefalosporinas/farmacología , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/metabolismo , Enterococcus faecium/metabolismo , Eliminación de Gen , Pruebas de Sensibilidad Microbiana , Transducción de Señal/genética , Vancomicina/farmacologíaRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Cefalosporinas/farmacología , Enterococcus faecalis/efectos de los fármacos , Timidilato Sintasa/genética , Resistencia a la Vancomicina/genética , Resistencia a las Cefalosporinas/genética , Evolución Molecular Dirigida/métodos , Sistemas de Liberación de Medicamentos , Enterococcus faecalis/enzimología , Enterococcus faecalis/genética , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Resistencia a las Cefalosporinas/genética , Enterococcus faecalis/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Ceftriaxona/farmacología , Resistencia a las Cefalosporinas/efectos de los fármacos , Medios de Cultivo/química , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/metabolismo , Expresión Génica , Genes Reporteros , Glucosa/metabolismo , Operón Lac , Pruebas de Sensibilidad Microbiana , Mutación , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Proteínas Quinasas/genéticaRESUMEN
As major causes of hospital-acquired infections, antibiotic-resistant enterococci are a serious public health concern. Enterococci are intrinsically resistant to many cephalosporin antibiotics, a trait that enables proliferation in patients undergoing cephalosporin therapy. Although a few genetic determinants of cephalosporin resistance in enterococci have been described, overall, many questions remain about the underlying genetic and biochemical basis for cephalosporin resistance. Here we describe an unexpected effect of specific mutations in the ß subunit of RNA polymerase (RNAP) on intrinsic cephalosporin resistance in enterococci. We found that RNAP mutants, selected initially on the basis of their ability to provide resistance to rifampin, resulted in allele-specific alterations of the intrinsic resistance of enterococci toward expanded- and broad-spectrum cephalosporins. These mutations did not affect resistance toward a diverse collection of other antibiotics that target a range of alternative cellular processes. We propose that the RNAP mutations identified here lead to alterations in transcription of as-yet-unknown genes that are critical for cellular adaption to cephalosporin stress.
Asunto(s)
Resistencia a las Cefalosporinas/genética , ARN Polimerasas Dirigidas por ADN/genética , Enterococcus/efectos de los fármacos , Enterococcus/genética , Antituberculosos/farmacología , Medios de Cultivo , Ligamiento Genético , Pruebas de Sensibilidad Microbiana , Mutación/genética , Mutación/fisiología , Plásmidos/genética , Rifampin/farmacologíaRESUMEN
UNLABELLED: 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. IMPORTANCE: As a major cause of hospital-acquired infections, antibiotic-resistant enterococci represent a serious public health problem. Enterococci are well-known to exhibit intrinsic resistance to broad-spectrum cephalosporin antibiotics, a trait that enables them to proliferate in patients undergoing cephalosporin therapy, thereby predisposing these patients to acquisition of an enterococcal infection. Thus, inhibition of enterococcal cephalosporin resistance could represent an effective new strategy to prevent the emergence of hospital-acquired enterococcal infections. At this time, however, the molecular basis for cephalosporin resistance in E. faecalis is poorly understood. Our results begin to unravel the details of a new phosphorylation-dependent signal transduction system that controls cephalosporin resistance in enterococci. Deeper understanding of the mechanism underlying cephalosporin resistance in E. faecalis may enable the development of new therapeutics designed to reduce the incidence of hospital-acquired enterococcal infections.
Asunto(s)
Proteínas Bacterianas/metabolismo , Resistencia a las Cefalosporinas , Enterococcus faecalis/enzimología , Regulación Bacteriana de la Expresión Génica , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Cefalosporinas/farmacología , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Fosforilación , Proteína Fosfatasa 2/genética , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
One mechanism by which cells post-transcriptionally regulate gene expression is via intercellular and intracellular sorting of mRNA. In Saccharomyces cerevisiae, the localization of ASH1 mRNA to the distal tip of budding cells results in the asymmetric sorting of Ash1p to daughter cell nuclei. Efficient localization of ASH1 mRNA depends upon the activity of four cis-acting localization elements and also upon the activity of trans-factors She2p, She3p, and Myo4p. She2p, She3p, and Myo4p have been proposed to form an ASH1 mRNA localization particle. She2p directly and specifically binds each of the four ASH1 cis-acting localization elements, whereas She3p has been hypothesized to function as an adaptor by recruiting the She2p-mRNA complex to Myo4p, a type V myosin. The Myo4p-She3p-She2p heterotrimeric protein complex has been proposed to localize mRNA to daughter cells using polarized actin cables. Here we demonstrate that whereas the predicted Myo4p-She3p-She2p heterotrimeric complex forms in vivo, it represents a relatively minor species compared with the Myo4p-She3p complex. Furthermore, contrary to a prediction of the heterotrimeric complex model for ASH1 mRNA localization, ASH1 mRNA artificially tethered to She2p is not localized. Upon closer examination, we found that mRNA tightly associated with She2p is transported to daughter cells but is not properly anchored at the bud tip. These results are consistent with a model whereby anchoring of ASH1 mRNA requires molecular remodeling of the Myo4p-She3p-She2p heterotrimeric complex, a process that is apparently altered when mRNA is artificially tethered to She2p.