title: Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium.
abstract: The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as "cephalosporins") is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system.
  
  
  OBJECTIVE
  β-Lactam antibiotics remain our most effective therapies against susceptible Gram-positive bacteria. The intrinsic resistance of Enterococcus faecium to β-lactams, particularly to cephalosporins, therefore represents a major limitation of therapy. Although the primary mechanism of resistance to β-lactams in E. faecium is the presence of low-affinity monofunctional transpeptidase (class B) penicillin-binding protein Pbp5, the interaction of Pbp5 with other proteins is fundamental to maintain a resistant phenotype. The present work identifies a novel, previously uncharacterized, protein that interacts with Pbp5, whose expression increases in conjunction with stimuli that increase resistance to cephalosporins, and that confers increased resistance to cephalosporins when overexpressed. P5AP may represent a promising new target, inhibition of which could restore cephalosporin susceptibility to E. faecium.
authors.affiliation: Department of Medicine, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA.
authors.forename: Charlene
authors.initials: C
authors.lastname: Desbonnet
authors.affiliation: Department of Medicine, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA.
authors.forename: Amelia
authors.initials: A
authors.lastname: Tait-Kamradt
authors.affiliation: Department of Medicine, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA.
authors.forename: Monica
authors.initials: M
authors.lastname: Garcia-Solache
authors.affiliation: Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Dunman
authors.affiliation: Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Coleman
authors.affiliation: INSERM, U1138, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, Paris, France Université Pierre et Marie Curie, UMR S 1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, UMR S 1138, Paris, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
authors.affiliation: Department of Medicine, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA lrice@lifespan.org.
authors.forename: Louis B
authors.initials: LB
authors.lastname: Rice
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018550:Cephalosporin Resistance; D002511:Cephalosporins / Q000494:pharmacology; D016984:Enterococcus faecium / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002511:Cephalosporins; D046915:Penicillin-Binding Proteins; D010770:Phosphotransferases; D010744:Phosphoric Monoester Hydrolases