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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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Distribution of E. faecium E155 proteins identified by the different extraction methods.(A) Rate of E. faecium E155 proteins identified with one or more unique peptides in at least two biological replicates by trypsin shaving, elution at high pH and biotinylation and their corresponding subcellular localization predicted by Cellov.2.5 (http://cello.life.nctu.edu.tw) and Gpos-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/Gpos-multi). (B) Venn-diagram of all the proteins identified by the different extraction methods. (B1) Correlation between the proteins extracted by the different extraction methods. (B2) Correlation between the proteins predicted to have both cytoplasmic and extracytoplasmic location by CELLO v.2.5 and Gpos-mPLoc. (B3) Correlation between the Proteins predicted to have exclusively an extracytoplasmic location by CELLO v.2.5 and Gpos-mPLoc.
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pone-0111880-g001: Distribution of E. faecium E155 proteins identified by the different extraction methods.(A) Rate of E. faecium E155 proteins identified with one or more unique peptides in at least two biological replicates by trypsin shaving, elution at high pH and biotinylation and their corresponding subcellular localization predicted by Cellov.2.5 (http://cello.life.nctu.edu.tw) and Gpos-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/Gpos-multi). (B) Venn-diagram of all the proteins identified by the different extraction methods. (B1) Correlation between the proteins extracted by the different extraction methods. (B2) Correlation between the proteins predicted to have both cytoplasmic and extracytoplasmic location by CELLO v.2.5 and Gpos-mPLoc. (B3) Correlation between the Proteins predicted to have exclusively an extracytoplasmic location by CELLO v.2.5 and Gpos-mPLoc.

Mentions: For a more accurate identification of surface proteins in the E. faecium E155 strain, three different approaches were used: trypsin shaving, biotinylation and high pH elution. The number of proteins identified by MS analysis containing at least one unique peptide in at least two sample replicates (see supplementary tables S1 to S3) for the different methods were 390 for trypsin shaving, 309 for elution at high pH, and 45 for biotinylation. We analyzed the sequence of each protein through two Web-server predictors (CELLO v.2.5 and Gpos-mPLoc) to evaluate their sub-cellular localization. For each of the three methods, proteins were then classified in three main groups: a) Inside: If a protein was predicted to have an exclusively cytoplasmic location by both algorithms we considered it to be inside of the cell. b) Both: If one of the algorithms predicted that the subcellular localization of a protein is intracellular (cytoplasmic) and the other predicted that is outside of the cytoplasm (i.e. membrane, cell wall associated and/or extracellular) OR if the algorithms predicts two locations inside and outside of the cytoplasm (Cytoplasm-membrane or cytoplasm-extracellular) at the same time, the protein was considered to be both inside and outside of the cytoplasm. c) Surface-associated: If a protein was predicted to have an exclusively extracytoplasmic location (i.e. membrane, cell wall associated and/or extracellular) by both algorithms we considered these proteins as surface-associated. Among all the proteins identified, 39 (10%), 47 (15%) and 27 (63%) polypeptides were predicted to be extracytoplasmic by trypsin shaving, elution at high pH, and biotinylation, respectively (see figure 1A). On the other hand, we observed that 102 (26%) proteins obtained by trypsin shaving, 85 (27%) by elution at high pH and 4 (9%) by biotinylation were predicted to have both cytoplasmic and extracytoplasmic location. The data were then compared using Venn-diagrams (figure 1B1) to identify proteins classified as surface-associated by more than one method. A total of 552 proteins with at least one unique peptide were uncovered and among them 16 proteins were identified by all three methods; 158 proteins appeared at least in two of the three different extraction procedures. We compared the proteins predicted to have cytoplasmic and extracytoplasmic localizations (see figure 1B2). Three of them were part of those polypeptides identified by all three methods, while 36 appeared at least in two (see supplementary table S4). Finally, we compared the proteins that were predicted to have an extracytoplasmic location (figure 1B3), showing that six of them appeared in all the extraction methods and 23 were identified by at least two of the three methods. Extracytoplasmic proteins identified by more than one method and their subcellular localization are summarized in table 3. Considering these results, we assumed that the six extracytoplasmic proteins identified by all three extraction methods were the most promising candidates to study immunogenicity and protective efficacy. Among the six proteins, we finally decided to focus on four that interact with peptidoglycan (PG) and are more likely to be surface-exposed: (a) the 21.6 kDa peptidoglycan-binding protein LysM (LysM) that has been reported to be non-covalently attached to PG [47]; (b) the 73.7 kDa low-affinity penicillin-binding protein 5 (PBP5) that is involved in polymerization of PG [48]–[50], (c) the 47.7 kDa D-alanyl-D-alanine carboxypeptidase (DdcP) - a low molecular weight penicillin binding protein (LMW-PBP) cross-linking PG chains to form rigid cell walls [49], [51] and (d) the 37.3 kDa PpiC-type peptidyl-prolyl cis-trans isomerase (PpiC) also involved in PG cross-linking [52].


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)

Distribution of E. faecium E155 proteins identified by the different extraction methods.(A) Rate of E. faecium E155 proteins identified with one or more unique peptides in at least two biological replicates by trypsin shaving, elution at high pH and biotinylation and their corresponding subcellular localization predicted by Cellov.2.5 (http://cello.life.nctu.edu.tw) and Gpos-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/Gpos-multi). (B) Venn-diagram of all the proteins identified by the different extraction methods. (B1) Correlation between the proteins extracted by the different extraction methods. (B2) Correlation between the proteins predicted to have both cytoplasmic and extracytoplasmic location by CELLO v.2.5 and Gpos-mPLoc. (B3) Correlation between the Proteins predicted to have exclusively an extracytoplasmic location by CELLO v.2.5 and Gpos-mPLoc.
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pone-0111880-g001: Distribution of E. faecium E155 proteins identified by the different extraction methods.(A) Rate of E. faecium E155 proteins identified with one or more unique peptides in at least two biological replicates by trypsin shaving, elution at high pH and biotinylation and their corresponding subcellular localization predicted by Cellov.2.5 (http://cello.life.nctu.edu.tw) and Gpos-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/Gpos-multi). (B) Venn-diagram of all the proteins identified by the different extraction methods. (B1) Correlation between the proteins extracted by the different extraction methods. (B2) Correlation between the proteins predicted to have both cytoplasmic and extracytoplasmic location by CELLO v.2.5 and Gpos-mPLoc. (B3) Correlation between the Proteins predicted to have exclusively an extracytoplasmic location by CELLO v.2.5 and Gpos-mPLoc.
Mentions: For a more accurate identification of surface proteins in the E. faecium E155 strain, three different approaches were used: trypsin shaving, biotinylation and high pH elution. The number of proteins identified by MS analysis containing at least one unique peptide in at least two sample replicates (see supplementary tables S1 to S3) for the different methods were 390 for trypsin shaving, 309 for elution at high pH, and 45 for biotinylation. We analyzed the sequence of each protein through two Web-server predictors (CELLO v.2.5 and Gpos-mPLoc) to evaluate their sub-cellular localization. For each of the three methods, proteins were then classified in three main groups: a) Inside: If a protein was predicted to have an exclusively cytoplasmic location by both algorithms we considered it to be inside of the cell. b) Both: If one of the algorithms predicted that the subcellular localization of a protein is intracellular (cytoplasmic) and the other predicted that is outside of the cytoplasm (i.e. membrane, cell wall associated and/or extracellular) OR if the algorithms predicts two locations inside and outside of the cytoplasm (Cytoplasm-membrane or cytoplasm-extracellular) at the same time, the protein was considered to be both inside and outside of the cytoplasm. c) Surface-associated: If a protein was predicted to have an exclusively extracytoplasmic location (i.e. membrane, cell wall associated and/or extracellular) by both algorithms we considered these proteins as surface-associated. Among all the proteins identified, 39 (10%), 47 (15%) and 27 (63%) polypeptides were predicted to be extracytoplasmic by trypsin shaving, elution at high pH, and biotinylation, respectively (see figure 1A). On the other hand, we observed that 102 (26%) proteins obtained by trypsin shaving, 85 (27%) by elution at high pH and 4 (9%) by biotinylation were predicted to have both cytoplasmic and extracytoplasmic location. The data were then compared using Venn-diagrams (figure 1B1) to identify proteins classified as surface-associated by more than one method. A total of 552 proteins with at least one unique peptide were uncovered and among them 16 proteins were identified by all three methods; 158 proteins appeared at least in two of the three different extraction procedures. We compared the proteins predicted to have cytoplasmic and extracytoplasmic localizations (see figure 1B2). Three of them were part of those polypeptides identified by all three methods, while 36 appeared at least in two (see supplementary table S4). Finally, we compared the proteins that were predicted to have an extracytoplasmic location (figure 1B3), showing that six of them appeared in all the extraction methods and 23 were identified by at least two of the three methods. Extracytoplasmic proteins identified by more than one method and their subcellular localization are summarized in table 3. Considering these results, we assumed that the six extracytoplasmic proteins identified by all three extraction methods were the most promising candidates to study immunogenicity and protective efficacy. Among the six proteins, we finally decided to focus on four that interact with peptidoglycan (PG) and are more likely to be surface-exposed: (a) the 21.6 kDa peptidoglycan-binding protein LysM (LysM) that has been reported to be non-covalently attached to PG [47]; (b) the 73.7 kDa low-affinity penicillin-binding protein 5 (PBP5) that is involved in polymerization of PG [48]–[50], (c) the 47.7 kDa D-alanyl-D-alanine carboxypeptidase (DdcP) - a low molecular weight penicillin binding protein (LMW-PBP) cross-linking PG chains to form rigid cell walls [49], [51] and (d) the 37.3 kDa PpiC-type peptidyl-prolyl cis-trans isomerase (PpiC) also involved in PG cross-linking [52].

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