Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis
BACKGROUND Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdom...
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Fundação Oswaldo Cruz (FIOCRUZ)
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| Series: | Memorias do Instituto Oswaldo Cruz |
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| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762018000300178&lng=en&tlng=en |
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| author | Thais Gonçalves Ferreira Camilla Nunes dos Reis Trindade Petra Bell André Teixeira-Ferreira Jonas E Perales Rossiane C Vommaro Regina Maria Cavalcanti Pilotto Domingues Eliane de Oliveira Ferreira |
| author_facet | Thais Gonçalves Ferreira Camilla Nunes dos Reis Trindade Petra Bell André Teixeira-Ferreira Jonas E Perales Rossiane C Vommaro Regina Maria Cavalcanti Pilotto Domingues Eliane de Oliveira Ferreira |
| author_sort | Thais Gonçalves Ferreira |
| collection | DOAJ |
| description | BACKGROUND Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein. |
| first_indexed | 2024-03-12T18:04:44Z |
| format | Article |
| id | doaj.art-b2a3e3f104e346c78c4d0a90e14b45b9 |
| institution | Directory Open Access Journal |
| issn | 1678-8060 |
| language | English |
| last_indexed | 2024-03-12T18:04:44Z |
| publisher | Fundação Oswaldo Cruz (FIOCRUZ) |
| record_format | Article |
| series | Memorias do Instituto Oswaldo Cruz |
| spelling | doaj.art-b2a3e3f104e346c78c4d0a90e14b45b92023-08-02T09:29:08ZengFundação Oswaldo Cruz (FIOCRUZ)Memorias do Instituto Oswaldo Cruz1678-8060113317818410.1590/0074-02760170340S0074-02762018000300178Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilisThais Gonçalves FerreiraCamilla Nunes dos Reis TrindadePetra BellAndré Teixeira-FerreiraJonas E PeralesRossiane C VommaroRegina Maria Cavalcanti Pilotto DominguesEliane de Oliveira FerreiraBACKGROUND Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762018000300178&lng=en&tlng=enproteomicsBacteroides fragilisextracellular membrane vesiclesα-enolase |
| spellingShingle | Thais Gonçalves Ferreira Camilla Nunes dos Reis Trindade Petra Bell André Teixeira-Ferreira Jonas E Perales Rossiane C Vommaro Regina Maria Cavalcanti Pilotto Domingues Eliane de Oliveira Ferreira Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis Memorias do Instituto Oswaldo Cruz proteomics Bacteroides fragilis extracellular membrane vesicles α-enolase |
| title | Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis |
| title_full | Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis |
| title_fullStr | Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis |
| title_full_unstemmed | Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis |
| title_short | Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis |
| title_sort | identification of the alpha enolase p46 in the extracellular membrane vesicles of bacteroides fragilis |
| topic | proteomics Bacteroides fragilis extracellular membrane vesicles α-enolase |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762018000300178&lng=en&tlng=en |
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