Cell wall synthesis enzymes and beta-lactam resistance in Enterococcus faecium

Summary

Principal Investigator: Louis B Rice
Abstract: DESCRIPTION (provided by applicant): More than any other human pathogen, Enterococcus. faecium has grown in importance as a result of its resistance to commonly used antimicrobial agents, in particular to the ?-lactams. High-level ? lactam resistance expressed by E. faecium not only compromises therapy of bacterial infections, it promotes gastrointestinal colonization and further dissemination of resistant strains. The hallmark of high-level ?-lactam resistance in E. faecium is the expression of low affinity class B penicillin-binding protein Pbp5. As a class B Pbp with only transpeptidase activity, Pbp5 must coordinate its activities with that of glycosyltransferases to synthesize mature peptidoglycan. Class B Pbps most commonly coordinate with bifunctional (possessing both glycosyltransferase and transpeptidase activities) class A Pbps, which in E. faecium are PbpF, PbpZ and PonA. In work performed during the previous period of this grant, we described auxiliary and parallel mechanisms by which E. faecium expresses resistance to ?-lactam antibiotics. We have deleted the E. faecium class A pbps in every combination and have discovered that deletion of PbpF and PonA results in a heterogeneous susceptibility to ceftriaxone (suggesting that Pbp5 coordinated with either of these class A Pbps to confer resistance to ceftriaxone), from which homogeneous resistance can be selected by growth on ceftriaxone (selection of spontaneous mutants) or induced by exposure to penicillin. Loss of PbpF is also associated with the autolytic phenotype of E. faecium. We have also identified and characterized an L,D-transpeptidase (Ldtfm) that is able to confer ?-lactam and vancomycin resistance in the absence of Pbp5, in concert with the activity of a D,D-carboxypeptidase. The present proposal will continue with this important work in the following manner: 1) We will investigate the mechanisms underlying the class A Pbp deletion phenotypes by characterizing the E. faecium peptidoglycan synthesis complex, targeting a likely alternative glycosyltransferases identified through a genome search and using microarray analysis to analyze the regulatory framework of penicillin-inducible ceftriaxone resistance in the ponApbpF double mutant 2) We will investigate the mechanisms underlying PbpF ceftriaxone-moenomycin synergism vs. E. faecium D344R and the impact of PbpF on the autolytic phenotype through site-directed mutagenesis of PbpF and functional studies of E. faecium autolysins 3) We will explore the molecular basis for the substrate specificity of Ldtfm to understand the surprising pattern of inhibition of the enzyme by ?-lactams 4) We will characterize the physiological aspects of the L,D-transpeptidation pathway using a proteomic approach to identifying partners of Ldtfm in the peptidoglycan polymerization complexes. We will also use transcriptome analysis to identify differentially expressed genes and perform whole genome sequencing to identify all of the mutations leading to expression of high levels of ampicillin and vancomycin resistance through the Ldtfm pathway. These investigations will identify and characterize critical and redundant ?-lactam resistance mechanisms in a bacterial species in which ?-lactam resistance is absolutely essential to its propagation in the hospital setting. They will also enhance our understanding of cell wall synthesis mechanisms in Gram-positive cocci and reveal promising new targets for antibacterial therapy.
Funding Period: 2001-03-01 - 2015-05-31
more information: NIH RePORT

Top Publications

  1. ncbi Aslfm, the D-aspartate ligase responsible for the addition of D-aspartic acid onto the peptidoglycan precursor of Enterococcus faecium
    Samuel Bellais
    INSERM, U655 LRMA, Paris Cedex 06, France
    J Biol Chem 281:11586-94. 2006
  2. pmc Early insights into the interactions of different β-lactam antibiotics and β-lactamase inhibitors against soluble forms of Acinetobacter baumannii PBP1a and Acinetobacter sp. PBP3
    Krisztina M Papp-Wallace
    Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA
    Antimicrob Agents Chemother 56:5687-92. 2012
  3. pmc Analysis of PBP5 of early U.S. isolates of Enterococcus faecium: sequence variation alone does not explain increasing ampicillin resistance over time
    Jessica R Galloway-Peña
    Division of Infectious Disease, Department of Medicine, Center for the Study of Emerging and Re Emerging Pathogens, University of Texas Medical School, Houston, Texas 77030, USA
    Antimicrob Agents Chemother 55:3272-7. 2011
  4. pmc Inactivation kinetics of a new target of beta-lactam antibiotics
    Sébastien Triboulet
    Centre de Recherche des Cordeliers, Laboratoire de Recherche Moleculaire sur les Antibiotiques, Equipe 12, Universite Pierre et Marie Curie, Paris 6, UMR S 872, Paris, F 75006 France
    J Biol Chem 286:22777-84. 2011
  5. pmc The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis
    Diana Panesso
    Department of Internal Medicine, Division of Infectious Diseases, Center for the Study of Emerging and Reemerging Pathogens, Houston, TX, USA
    BMC Microbiol 11:20. 2011
  6. ncbi Progress and challenges in implementing the research on ESKAPE pathogens
    Louis B Rice
    Medical Service, Louis Stokes Cleveland VA Medical Center, 10701 East Boulevard, Cleveland, OH 44106, USA
    Infect Control Hosp Epidemiol 31:S7-10. 2010
  7. ncbi Activation of the L,D-transpeptidation peptidoglycan cross-linking pathway by a metallo-D,D-carboxypeptidase in Enterococcus faecium
    Emmanuelle Sacco
    Centre de Recherche des Cordeliers, LRMA, Equipe 12, Universite Pierre et Marie Curie Paris 6, UMR S 872, Paris, F 75006 France
    Mol Microbiol 75:874-85. 2010
  8. pmc In vitro activity of fosfomycin against blaKPC-containing Klebsiella pneumoniae isolates, including those nonsusceptible to tigecycline and/or colistin
    Andrea Endimiani
    Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
    Antimicrob Agents Chemother 54:526-9. 2010
  9. pmc Role of class A penicillin-binding proteins in the expression of beta-lactam resistance in Enterococcus faecium
    Louis B Rice
    Medical Service 111 W, Louis Stokes Cleveland VA Medical Center, 10701 East Blvd, Cleveland, OH 44106, USA
    J Bacteriol 191:3649-56. 2009
  10. pmc Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USA
    Andrea Endimiani
    Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Research Service, Cleveland, OH 44106, USA
    J Antimicrob Chemother 63:427-37. 2009

Detail Information

Publications19

  1. ncbi Aslfm, the D-aspartate ligase responsible for the addition of D-aspartic acid onto the peptidoglycan precursor of Enterococcus faecium
    Samuel Bellais
    INSERM, U655 LRMA, Paris Cedex 06, France
    J Biol Chem 281:11586-94. 2006
    ..The Aslfm ligase appears as an attractive target for the development of narrow spectrum antibiotics active against multiresistant E. faecium...
  2. pmc Early insights into the interactions of different β-lactam antibiotics and β-lactamase inhibitors against soluble forms of Acinetobacter baumannii PBP1a and Acinetobacter sp. PBP3
    Krisztina M Papp-Wallace
    Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA
    Antimicrob Agents Chemother 56:5687-92. 2012
    ..baumannii. Unraveling the contribution of PBPs to β-lactam susceptibility and resistance brings us one step closer to identifying which PBPs are the best targets for novel β-lactams...
  3. pmc Analysis of PBP5 of early U.S. isolates of Enterococcus faecium: sequence variation alone does not explain increasing ampicillin resistance over time
    Jessica R Galloway-Peña
    Division of Infectious Disease, Department of Medicine, Center for the Study of Emerging and Re Emerging Pathogens, University of Texas Medical School, Houston, Texas 77030, USA
    Antimicrob Agents Chemother 55:3272-7. 2011
    ..Analysis of three other genes encoding cell wall/surface proteins also showed that there are two distinct evolutionary groups for each gene, but with occasional mixing of genes, consistent with a species that evolves by recombination...
  4. pmc Inactivation kinetics of a new target of beta-lactam antibiotics
    Sébastien Triboulet
    Centre de Recherche des Cordeliers, Laboratoire de Recherche Moleculaire sur les Antibiotiques, Equipe 12, Universite Pierre et Marie Curie, Paris 6, UMR S 872, Paris, F 75006 France
    J Biol Chem 286:22777-84. 2011
    ..The methods described in this study will facilitate development of new carbapenems active on extensively resistant M. tuberculosis...
  5. pmc The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis
    Diana Panesso
    Department of Internal Medicine, Division of Infectious Diseases, Center for the Study of Emerging and Reemerging Pathogens, Houston, TX, USA
    BMC Microbiol 11:20. 2011
    ..faecium, although its direct contribution to virulence has not been investigated. Here, we constructed mutants of the hylEfm-region and we evaluated their effect on virulence using a murine peritonitis model...
  6. ncbi Progress and challenges in implementing the research on ESKAPE pathogens
    Louis B Rice
    Medical Service, Louis Stokes Cleveland VA Medical Center, 10701 East Boulevard, Cleveland, OH 44106, USA
    Infect Control Hosp Epidemiol 31:S7-10. 2010
    ....
  7. ncbi Activation of the L,D-transpeptidation peptidoglycan cross-linking pathway by a metallo-D,D-carboxypeptidase in Enterococcus faecium
    Emmanuelle Sacco
    Centre de Recherche des Cordeliers, LRMA, Equipe 12, Universite Pierre et Marie Curie Paris 6, UMR S 872, Paris, F 75006 France
    Mol Microbiol 75:874-85. 2010
    ..The ddc locus displays striking similarities with portions of the van vancomycin resistance gene clusters, suggesting possible routes of emergence of cross-resistance to glycopeptides and beta-lactams in natural conditions...
  8. pmc In vitro activity of fosfomycin against blaKPC-containing Klebsiella pneumoniae isolates, including those nonsusceptible to tigecycline and/or colistin
    Andrea Endimiani
    Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
    Antimicrob Agents Chemother 54:526-9. 2010
    ..Notably, 5 out of 6 extremely drug-resistant (tigecycline and colistin nonsusceptible) KpKPC were susceptible to fosfomycin. Compared to agar dilution, disk diffusion was more accurate than Etest...
  9. pmc Role of class A penicillin-binding proteins in the expression of beta-lactam resistance in Enterococcus faecium
    Louis B Rice
    Medical Service 111 W, Louis Stokes Cleveland VA Medical Center, 10701 East Blvd, Cleveland, OH 44106, USA
    J Bacteriol 191:3649-56. 2009
    ..Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different beta-lactam antibiotics differed as a function of its partner glycosyltransferase...
  10. pmc Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USA
    Andrea Endimiani
    Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Research Service, Cleveland, OH 44106, USA
    J Antimicrob Chemother 63:427-37. 2009
    ..Outbreaks in the Eastern USA have created serious treatment and infection control problems. A comparative multi-institutional analysis of these strains has not yet been performed...
  11. ncbi Transferable capacity for gastrointestinal colonization in Enterococcus faecium in a mouse model
    Louis B Rice
    Medical and Research Services, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA
    J Infect Dis 199:342-9. 2009
    ..These results indicate that E. faecium clinical isolates express transmissible factors other than antimicrobial resistance that promote colonization of the mouse gastrointestinal tract...
  12. pmc Identification of the L,D-transpeptidases for peptidoglycan cross-linking in Escherichia coli
    Sophie Magnet
    INSERM U872 LRMA, Equipe 12, Centre de Recherche des Cordeliers, 15 rue de l Ecole de Medecine, 75270 Paris, Cedex 06, France
    J Bacteriol 190:4782-5. 2008
    ..This activity partially replaces the D,D-transpeptidase activity of penicillin-binding proteins...
  13. ncbi Covalent attachment of proteins to peptidoglycan
    Shaynoor Dramsi
    Unité de Biologie des Bactéries Pathogènes à Gram Positif, Institut Pasteur, Paris, France
    FEMS Microbiol Rev 32:307-20. 2008
    ..This review focuses on the role of enzymes that covalently link surface proteins to the peptidoglycan, the well-known sortases in Gram-positive bacteria, and the recently characterized l,d-transpeptidases in Gram-negative bacteria...
  14. ncbi Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-lactam imipenem
    Jean Luc Mainardi
    INSERM, U872, LRMA Pôle 4, Equipe 12 F 75006 Paris, France
    J Biol Chem 282:30414-22. 2007
    ..Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the LD- and DD-specificities...
  15. pmc Identification of the L,D-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan
    Sophie Magnet
    INSERM, U655 LRMA, Universite Pierre et Marie Curie, Centre de Recherches Biomedicales des Cordeliers, Paris, France
    J Bacteriol 189:3927-31. 2007
    ....
  16. ncbi Specificity of L,D-transpeptidases from gram-positive bacteria producing different peptidoglycan chemotypes
    Sophie Magnet
    INSERM, U655 LRMA F 75006 Paris France
    J Biol Chem 282:13151-9. 2007
    ..Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving L,D-transpeptidases...
  17. pmc Novel mechanism of resistance to glycopeptide antibiotics in Enterococcus faecium
    Julie Cremniter
    INSERM U655 LRMA, Centre de Recherches Biomedicales des Cordeliers, Universite Paris 6, 15 rue de l Ecole de Medecine, Paris 75270, France
    J Biol Chem 281:32254-62. 2006
    ..This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate...
  18. ncbi Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidase
    Sabrina Biarrotte-Sorin
    INSERM, U655 LRMA, Universite Pierre et Marie Curie Paris 6, Universite Paris Descartes, Faculté de Médecine Pitié Salpêtrière and Centre de Recherches Biomédicales des Cordeliers, 91 Boulevard de l Hopital, 75634 Paris Cedex 13, France
    J Mol Biol 359:533-8. 2006
    ..We propose that the two paths to the catalytic residue Cys442 are the binding sites for the acceptor and donor substrates of the L,D-transpeptidase...
  19. pmc Peptidoglycan cross-linking in glycopeptide-resistant Actinomycetales
    Jean Emmanuel Hugonnet
    Centre de Recherche des Cordeliers, LRMA, Equipe 12, Universite Pierre et Marie Curie Paris 6, UMR S 872, Paris, France
    Antimicrob Agents Chemother 58:1749-56. 2014
    ..Since the level of drug production exceeds the level of resistance, we propose that L,D-transpeptidases merely act as a tolerance mechanism in this bacterium...

Research Grants30

  1. Protein-protein interaction essential for bacterial growth and virulence
    Mark A Saper; Fiscal Year: 2013
    ..Moreover, the mode of ac- tion of such an inhibitor would be complementary with, but different from, ?-lactams. ..
  2. Inhibitors of wall teichoic acid biosynthesis of Staphylococcus aureus
    TIMOTHY J OPPERMAN; Fiscal Year: 2013
    ..Aim 3. Validate confirmed inhibitors of WTA synthesis using secondary assays and identify hit series. Aim 4. Verify mechanism of action of selected hit series. ..
  3. Does ampicillin resistance or clade type determine GI colonization by E. faecium?
    KAVINDRA SINGH; Fiscal Year: 2013
    ....
  4. Oklahoma Center for Respiratory and Infectious Diseases
    Lin Liu; Fiscal Year: 2013
    ..The completion of the goals of the present COBRE will have a major impact on research programs on respiratory infectious diseases in the State of Oklahoma. ..
  5. Molecular Targets in Peptidoglycan Synthesis
    Christopher Davies; Fiscal Year: 2013
    ..To understand the functional role of AmiC in peptidoglycan breakdown, but also to pave the way for drug discovery against its two active sites, we will obtain essential structural and biochemical information for AmiC. ..
  6. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..
  7. Molecular Analyses and Interventions for Biodefense and Emerging Pathogens
    Olaf Schneewind; Fiscal Year: 2013
    ..Research and training at the GLRCE is governed by a mechanism involving ongoing review of scientific excellence and translational goals, inter-institutional advisory boards and external scientific advisory bodies. ..
  8. Glycopeptide Complexes with Bacterial Cell Walls by REDOR NMR
    Jacob Schaefer; Fiscal Year: 2013
    ....
  9. Novel Mechanisms of Beta-lactam Resistance in Staph Aureus
    Henry F Chambers; Fiscal Year: 2013
    ..Recombinant GdpP also will be purified and analyzed by x-ray crystallography to identify its critical structural properties. Achieving these aims will increase knowledge of ?-lactam antibiotic effects and mechanisms of resistance. ..
  10. Pacific Southwest RCE for Biodefense &Emerging Infectious Diseases Research
    Alan G Barbour; Fiscal Year: 2013
    ..abstract_text> ..