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Identification of peptidoglycan-associated proteins as vaccine candidates for enterococcal infections.

Romero-Saavedra F, Laverde D, Wobser D, Michaux C, Budin-Verneuil A, Bernay B, Benachour A, Hartke A, Huebner J - PLoS ONE (2014)

Bottom Line: The total proteins found with each method were 390 by the trypsin shaving, 329 by the elution at high pH, and 45 using biotinylation.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.

View Article: PubMed Central - PubMed

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.

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.

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Cross-reactivity of the sera against different enterococcal strains.Opsonophagocytic assay used to test the ability to mediate opsonic killing of different enterococcal strains by antibodies raised against the recombinant proteins. A) Opsonophagocytic killing of strains E. faecium E1162 and E. faecalis 12030 by antibodies raised against the recombinant proteins at dilutions between 1∶10 and 1∶100. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compared with the activity of the preimune rabbit serum (NRS, white bar). B) Opsonophagocytic killing in E. faecalis type 2 and E. faecalis type 5 by antibodies raised against the recombinant proteins at dilution 1∶10. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compared with the activity of the preimune rabbit serum (NRS, white bar). Bars represent the mean of data and the error bars represent the standard error of the mean. Statistical significance was determined by ANOVA and Dunnett's Multiple Comparison Test. Comparing killing rates of similar dilutions (i.e. 1∶10, 1∶50 or 1∶100) with the NRS, all comparisons were significant at p<0.001 (indicated by asterisk).
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pone-0111880-g004: Cross-reactivity of the sera against different enterococcal strains.Opsonophagocytic assay used to test the ability to mediate opsonic killing of different enterococcal strains by antibodies raised against the recombinant proteins. A) Opsonophagocytic killing of strains E. faecium E1162 and E. faecalis 12030 by antibodies raised against the recombinant proteins at dilutions between 1∶10 and 1∶100. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compared with the activity of the preimune rabbit serum (NRS, white bar). B) Opsonophagocytic killing in E. faecalis type 2 and E. faecalis type 5 by antibodies raised against the recombinant proteins at dilution 1∶10. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compared with the activity of the preimune rabbit serum (NRS, white bar). Bars represent the mean of data and the error bars represent the standard error of the mean. Statistical significance was determined by ANOVA and Dunnett's Multiple Comparison Test. Comparing killing rates of similar dilutions (i.e. 1∶10, 1∶50 or 1∶100) with the NRS, all comparisons were significant at p<0.001 (indicated by asterisk).

Mentions: To determine if the antibodies directed against the recombinant proteins were able to opsonize different strains, serum dilutions between 1∶10 and 1∶100 were tested in OPAs against E. faecium E1162 and E. faecalis 12030, type 2 and type 5 [46], [53]. The four sera were able to opsonize all strains exhibiting killing above 60% (see Figure 4A and 4B). Passive immunization with antibodies directed against the different proteins promotes clearance of bacteria in mice


Identification of peptidoglycan-associated proteins as vaccine candidates for enterococcal infections.

Romero-Saavedra F, Laverde D, Wobser D, Michaux C, Budin-Verneuil A, Bernay B, Benachour A, Hartke A, Huebner J - PLoS ONE (2014)

Cross-reactivity of the sera against different enterococcal strains.Opsonophagocytic assay used to test the ability to mediate opsonic killing of different enterococcal strains by antibodies raised against the recombinant proteins. A) Opsonophagocytic killing of strains E. faecium E1162 and E. faecalis 12030 by antibodies raised against the recombinant proteins at dilutions between 1∶10 and 1∶100. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compared with the activity of the preimune rabbit serum (NRS, white bar). B) Opsonophagocytic killing in E. faecalis type 2 and E. faecalis type 5 by antibodies raised against the recombinant proteins at dilution 1∶10. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compared with the activity of the preimune rabbit serum (NRS, white bar). Bars represent the mean of data and the error bars represent the standard error of the mean. Statistical significance was determined by ANOVA and Dunnett's Multiple Comparison Test. Comparing killing rates of similar dilutions (i.e. 1∶10, 1∶50 or 1∶100) with the NRS, all comparisons were significant at p<0.001 (indicated by asterisk).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219796&req=5

pone-0111880-g004: Cross-reactivity of the sera against different enterococcal strains.Opsonophagocytic assay used to test the ability to mediate opsonic killing of different enterococcal strains by antibodies raised against the recombinant proteins. A) Opsonophagocytic killing of strains E. faecium E1162 and E. faecalis 12030 by antibodies raised against the recombinant proteins at dilutions between 1∶10 and 1∶100. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compared with the activity of the preimune rabbit serum (NRS, white bar). B) Opsonophagocytic killing in E. faecalis type 2 and E. faecalis type 5 by antibodies raised against the recombinant proteins at dilution 1∶10. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compared with the activity of the preimune rabbit serum (NRS, white bar). Bars represent the mean of data and the error bars represent the standard error of the mean. Statistical significance was determined by ANOVA and Dunnett's Multiple Comparison Test. Comparing killing rates of similar dilutions (i.e. 1∶10, 1∶50 or 1∶100) with the NRS, all comparisons were significant at p<0.001 (indicated by asterisk).
Mentions: To determine if the antibodies directed against the recombinant proteins were able to opsonize different strains, serum dilutions between 1∶10 and 1∶100 were tested in OPAs against E. faecium E1162 and E. faecalis 12030, type 2 and type 5 [46], [53]. The four sera were able to opsonize all strains exhibiting killing above 60% (see Figure 4A and 4B). Passive immunization with antibodies directed against the different proteins promotes clearance of bacteria in mice

Bottom Line: The total proteins found with each method were 390 by the trypsin shaving, 329 by the elution at high pH, and 45 using biotinylation.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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus