title: Identification of peptidoglycan-associated proteins as vaccine candidates for enterococcal infections.
abstract: Infections by opportunistic bacteria have significant contributions to morbidity and mortality of hospitalized patients and also lead to high expenses in healthcare. In this setting, one of the major clinical problems is caused by Gram-positive bacteria such as enterococci and staphylococci. In this study we extract, purify, identify and characterize immunogenic surface-exposed proteins present in the vancomycin resistant enterococci (VRE) strain Enterococcus faecium E155 using three different extraction methods: trypsin shaving, biotinylation and elution at high pH. Proteomic profiling was carried out by gel-free and gel-nanoLC-MS/MS analyses. The total proteins found with each method were 390 by the trypsin shaving, 329 by the elution at high pH, and 45 using biotinylation. An exclusively extracytoplasmic localization was predicted in 39 (10%) by trypsin shaving, in 47 (15%) by elution at high pH, and 27 (63%) by biotinylation. Comparison between the three extraction methods by Venn diagram and subcellular localization predictors (CELLO v.2.5 and Gpos-mPLoc) allowed us to identify six proteins that are most likely surface-exposed: the SCP-like extracellular protein, a low affinity penicillin-binding protein 5 (PBP5), a basic membrane lipoprotein, a peptidoglycan-binding protein LysM (LysM), a D-alanyl-D-alanine carboxypeptidase (DdcP) and the peptidyl-prolyl cis-trans isomerase (PpiC). Due to their close relationship with the peptidoglycan, we chose PBP5, LysM, DdcP and PpiC to test their potential as vaccine candidates. These putative surface-exposed proteins were overexpressed in Escherichia coli and purified. Rabbit polyclonal antibodies raised against the purified proteins were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Passive immunization with rabbit antibodies raised against these proteins reduced significantly the colony counts of E. faecium E155 in mice, indicating the effectiveness of these surface-related proteins as promising vaccine candidates to target different enterococcal pathogens.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Freiburg, Germany; EA4655 U2RM Stress/Virulence, University of Caen Lower-Normandy, Caen, France.
authors.forename: Felipe
authors.initials: F
authors.lastname: Romero-Saavedra
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Freiburg, Germany; EA4655 U2RM Stress/Virulence, University of Caen Lower-Normandy, Caen, France.
authors.forename: Diana
authors.initials: D
authors.lastname: Laverde
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: Dominique
authors.initials: D
authors.lastname: Wobser
authors.affiliation: EA4655 U2RM Stress/Virulence, University of Caen Lower-Normandy, Caen, France.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Michaux
authors.affiliation: EA4655 U2RM Stress/Virulence, University of Caen Lower-Normandy, Caen, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Budin-Verneuil
authors.affiliation: Proteogen platform SFR ICORE 4206, University of Caen Lower-Normandy, Caen, France.
authors.forename: Benoit
authors.initials: B
authors.lastname: Bernay
authors.affiliation: EA4655 U2RM Stress/Virulence, University of Caen Lower-Normandy, Caen, France.
authors.forename: Abdellah
authors.initials: A
authors.lastname: Benachour
authors.affiliation: EA4655 U2RM Stress/Virulence, University of Caen Lower-Normandy, Caen, France.
authors.forename: Axel
authors.initials: A
authors.lastname: Hartke
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Freiburg, Germany; Division of Pediatric Infectious Diseases, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University, Munich, Germany; German Center for Infection Research (DZIF), Partnersite Munich, Munich, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Huebner
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D016470:Bacteremia / Q000517:prevention & control*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology; D001428:Bacterial Vaccines / Q000276:immunology; D003429:Cross Reactions; D013293:Enterococcus faecalis / Q000276:immunology*; D016984:Enterococcus faecium / Q000276:immunology*; D005260:Female; D016908:Gram-Positive Bacterial Infections / Q000517:prevention & control*; D051379:Mice; D008807:Mice, Inbred BALB C; D010457:Peptidoglycan / Q000276:immunology*; D011817:Rabbits; D014611:Vaccination
chemical_list: D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D010457:Peptidoglycan