RESUMO
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.
Assuntos
Bacteroides fragilis/enzimologia , Bacteroides fragilis/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Fosfopiruvato Hidratase , Plasminogênio , Vesículas ExtracelularesRESUMO
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.
Assuntos
Bacteroides fragilis/enzimologia , Vesículas Extracelulares/enzimologia , Fosfopiruvato Hidratase/análise , Bacteroides fragilis/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Vesículas Extracelulares/ultraestrutura , Humanos , Laminina , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Fosfopiruvato Hidratase/metabolismo , PlasminogênioRESUMO
The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100 mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.
Assuntos
Aderência Bacteriana , Bacteroides fragilis/crescimento & desenvolvimento , Proteínas de Transporte/metabolismo , Laminina/metabolismo , Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteroides fragilis/metabolismo , Bacteroides fragilis/ultraestrutura , Matriz Extracelular/metabolismo , Immunoblotting , Microscopia Eletrônica de Transmissão , Oxirredução , Polissacarídeos Bacterianos/metabolismo , Fatores de TempoRESUMO
The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.
Assuntos
Aderência Bacteriana , Bacteroides fragilis/crescimento & desenvolvimento , Proteínas de Transporte/metabolismo , Laminina/metabolismo , Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteroides fragilis/metabolismo , Bacteroides fragilis/ultraestrutura , Matriz Extracelular/metabolismo , Immunoblotting , Microscopia Eletrônica de Transmissão , Oxirredução , Polissacarídeos Bacterianos/metabolismo , Fatores de TempoRESUMO
The effects of subinhibitory concentrations of metronidazole on the general morphology, ultrastructure and charge and hydrophobicity of Bacteroides fragilis were investigated using transmission and scanning electron microscopy, and measurements of zeta-potentials and contact angles. Metronidazole treatment induced morphological alterations in B. fragilis. Cells became filamentous and showed a significant loss of cytoplasm. The surface anionogenicity and hydrophobicity of B. fragilis were enhanced by the drug treatment. Metronidazole, by modifying B. fragilis surface properties, could alter, indirectly, interactions with host cells.