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.
Highlights
Enterococci have emerged as important nosocomial pathogens due to their multiple antibiotic resistances [1]
The ability of this species to survive under a range of adverse environmental conditions, and its dramatic increase in antibiotic resistance worldwide highlights the need for the development of alternative treatment and prevention strategies [8,9]
Aggregation substance (AS) protein and the collagen adhesin Ace have been examined in E. faecalis [21,22] and enterococcal surface protein Esp, secreted antigen protein SagA and two ABC transporters have been tested for antigenicity in E. faecium [23,24,25]
Summary
Enterococci have emerged as important nosocomial pathogens due to their multiple antibiotic resistances [1]. Surface proteins have become attractive targets for drug development [16,17,18] Their ability to interact with the host immune system makes them interesting vaccine candidates, since protein based vaccines may overcome some of the challenges encountered by polysaccharide-based vaccines, like serotype-dependent coverage, high production costs, and low immunogenicity [19,20]. Despite these advantages, only few surface and secreted proteins have been studied in clinically relevant enterococci. Using appropriate in vitro and in vivo models to confirm protective efficacy, only SagA, Ace and an ABC transporter were identified as potential vaccine candidates [10,23]
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