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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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Opsonophagocytic assay against the homologous strain E. faecium E155.Opsonophagocytic assay used to test the ability to mediate opsonic killing in the strain E. faecium E155 by antibodies raised against the recombinant proteins at different dilutions. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compare 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) with the NRS, all comparisons were significant at p<0.001 (indicated by asterisk).
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pone-0111880-g002: Opsonophagocytic assay against the homologous strain E. faecium E155.Opsonophagocytic assay used to test the ability to mediate opsonic killing in the strain E. faecium E155 by antibodies raised against the recombinant proteins at different dilutions. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compare 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) with the NRS, all comparisons were significant at p<0.001 (indicated by asterisk).

Mentions: The genes encoding the four candidate proteins were amplified without their signal peptides, cloned into the pQE30 expression vector and transformed into E. coli. The recombinant proteins were then purified under denaturing conditions. The purity of the proteins was assessed by SDS-PAGE and their identity was confirmed by LC-MS/MS (data not shown). New Zeeland white rabbits were immunized with purified proteins and exsanguinated two weeks after the last injection. The obtained polyclonal antibodies raised against the different proteins were tested in an OPA against the corresponding strain E. faecium E155 showing that all the proteins were able to induce opsonic antibodies. Different concentrations were tested to titer out the opsonic activity of the sera. Maximum opsonic activity of the antibodies was between 58–65% of killing with a 1∶10 serum dilution, and a reduction of killing was observed in a dose dependent fashion using increasingly higher dilutions of sera (see figure 2). To verify the specificity of the killing against the respective recombinant protein, opsonophagocytic inhibition assays (OPIA) were carried out by pre-incubating the sera with 100 µg/mL of the corresponding recombinant protein. These sera were then tested in an OPA using E. faecium strain E155 which showed that opsonic killing is inhibited by more than 85% in all cases (see figure 3).


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)

Opsonophagocytic assay against the homologous strain E. faecium E155.Opsonophagocytic assay used to test the ability to mediate opsonic killing in the strain E. faecium E155 by antibodies raised against the recombinant proteins at different dilutions. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compare 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) with the NRS, all comparisons were significant at p<0.001 (indicated by asterisk).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111880-g002: Opsonophagocytic assay against the homologous strain E. faecium E155.Opsonophagocytic assay used to test the ability to mediate opsonic killing in the strain E. faecium E155 by antibodies raised against the recombinant proteins at different dilutions. αPpiC (square grid), αPBP5 (horizontal stripes), αLysM (vertical stripes) and αDdcP (rhombic grid), compare 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) with the NRS, all comparisons were significant at p<0.001 (indicated by asterisk).
Mentions: The genes encoding the four candidate proteins were amplified without their signal peptides, cloned into the pQE30 expression vector and transformed into E. coli. The recombinant proteins were then purified under denaturing conditions. The purity of the proteins was assessed by SDS-PAGE and their identity was confirmed by LC-MS/MS (data not shown). New Zeeland white rabbits were immunized with purified proteins and exsanguinated two weeks after the last injection. The obtained polyclonal antibodies raised against the different proteins were tested in an OPA against the corresponding strain E. faecium E155 showing that all the proteins were able to induce opsonic antibodies. Different concentrations were tested to titer out the opsonic activity of the sera. Maximum opsonic activity of the antibodies was between 58–65% of killing with a 1∶10 serum dilution, and a reduction of killing was observed in a dose dependent fashion using increasingly higher dilutions of sera (see figure 2). To verify the specificity of the killing against the respective recombinant protein, opsonophagocytic inhibition assays (OPIA) were carried out by pre-incubating the sera with 100 µg/mL of the corresponding recombinant protein. These sera were then tested in an OPA using E. faecium strain E155 which showed that opsonic killing is inhibited by more than 85% in all cases (see figure 3).

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