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-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.

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-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.

Ribotypes associated with Clostridium difficile outbreaks in Brazil display distinct surface protein profiles.

Clostridium difficile is a spore-forming anaerobic intestinal pathogen that causes Clostridium difficile infection (CDI). C. difficile is the leading cause of toxin-mediated nosocomial antibiotic-associated diarrhea. The pathogenesis of CDI is attributed to two major virulence factors, TcdA and TcdB toxins, that cause the symptomatic infection. C. difficile also expresses a number of key proteins, including cell wall proteins (CWPs). S-layer proteins (SLPs) are CWPs that form a paracrystalline surface array that coats the surface of the bacterium. SLPs have a role in C. difficile binding to the gastrointestinal tract, but their importance in virulence need to be better elucidated. Here, we describe bottom-up proteomics analysis of surface-enriched proteins fractions obtained through glycine extraction of five C. difficile clinical isolates from Brazil using gel-based and gel-free approaches. We were able to identify approximately 250 proteins for each strain, among them SlpA, Cwp2, Cwp6, CwpV and Cwp84. Identified CWPs presented different amino acid coverage, which might suggest differences in post-translational modifications. Proteomic analysis of SLPs from ribotype 133, agent of C. difficile outbreaks in Brazil, revealed unique proteins and provided additional information towards in depth characterization of the strains causing CDI in Brazil.

Effect of hospital disinfectants on spores of clinical Brazilian Clostridium difficile strains.

The aim of this study was to evaluate the sporicidal activity of hospital disinfectants against spores of two Brazilian Clostridium difficile ribotypes and the BI/NAP1/027. Our results showed that CloroRio(®) and Cidex Opa(®) were the most efficient agents for eliminating spores of C difficile.