Genomes and Genes
Cell wall synthesis enzymes and beta-lactam resistance in Enterococcus faecium
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
- Aslfm, the D-aspartate ligase responsible for the addition of D-aspartic acid onto the peptidoglycan precursor of Enterococcus faeciumSamuel 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...
- Early insights into the interactions of different β-lactam antibiotics and β-lactamase inhibitors against soluble forms of Acinetobacter baumannii PBP1a and Acinetobacter sp. PBP3Krisztina 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...
- Analysis of PBP5 of early U.S. isolates of Enterococcus faecium: sequence variation alone does not explain increasing ampicillin resistance over timeJessica 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...
- Inactivation kinetics of a new target of beta-lactam antibioticsSébastien 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...
- The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitisDiana 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...
- Progress and challenges in implementing the research on ESKAPE pathogensLouis 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....
- Activation of the L,D-transpeptidation peptidoglycan cross-linking pathway by a metallo-D,D-carboxypeptidase in Enterococcus faeciumEmmanuelle 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...
- In vitro activity of fosfomycin against blaKPC-containing Klebsiella pneumoniae isolates, including those nonsusceptible to tigecycline and/or colistinAndrea 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...
- Role of class A penicillin-binding proteins in the expression of beta-lactam resistance in Enterococcus faeciumLouis 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...
- Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USAAndrea 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...
- Transferable capacity for gastrointestinal colonization in Enterococcus faecium in a mouse modelLouis 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...
- Identification of the L,D-transpeptidases for peptidoglycan cross-linking in Escherichia coliSophie 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...
- Covalent attachment of proteins to peptidoglycanShaynoor 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...
- Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-lactam imipenemJean 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...
- Identification of the L,D-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycanSophie Magnet
INSERM, U655 LRMA, Universite Pierre et Marie Curie, Centre de Recherches Biomedicales des Cordeliers, Paris, France
J Bacteriol 189:3927-31. 2007....
- Specificity of L,D-transpeptidases from gram-positive bacteria producing different peptidoglycan chemotypesSophie 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...
- Novel mechanism of resistance to glycopeptide antibiotics in Enterococcus faeciumJulie 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...
- Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidaseSabrina 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...
- Peptidoglycan cross-linking in glycopeptide-resistant ActinomycetalesJean 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...
- Protein-protein interaction essential for bacterial growth and virulenceMark A Saper; Fiscal Year: 2013..Moreover, the mode of ac- tion of such an inhibitor would be complementary with, but different from, ?-lactams. ..
- Inhibitors of wall teichoic acid biosynthesis of Staphylococcus aureusTIMOTHY 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. ..
- Does ampicillin resistance or clade type determine GI colonization by E. faecium?KAVINDRA SINGH; Fiscal Year: 2013....
- Oklahoma Center for Respiratory and Infectious DiseasesLin 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. ..
- Molecular Targets in Peptidoglycan SynthesisChristopher 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. ..
- 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. ..
- Molecular Analyses and Interventions for Biodefense and Emerging PathogensOlaf 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. ..
- Glycopeptide Complexes with Bacterial Cell Walls by REDOR NMRJacob Schaefer; Fiscal Year: 2013....
- Novel Mechanisms of Beta-lactam Resistance in Staph AureusHenry 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. ..
- Pacific Southwest RCE for Biodefense &Emerging Infectious Diseases ResearchAlan G Barbour; Fiscal Year: 2013..abstract_text> ..