Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis

Abstract

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.

Fig. 1. transmission electron microscopy (TEM) of the 638R Bacteroides fragilis strain and the extracellular membrane vesicles (EMV) after negative staining with 4% uranyl acetate and 2% methylcellulose. (A) B. fragilis with EMVs attached to the cells. The asterisks show the EMVs attached to the surface of the bacteria. Bar = 2 μm. (B-C) TEM of the purified EMVs from the 638R strain. (B) Different sized EMVs (arrow heads) Bar = 2 μm; (C) EMVs magnified with the plasma membrane well-defined (arrows). Magnification = 30,000 ×, Bar = 600 nm; (D) immunogold labelling of a preparation of purified EMVs stained with a rabbit anti-Bfp60 antibody and an anti-rabbit colloidal gold conjugate (5 nm). The arrows indicate staining for the surface protein Bfp60 in the EMVs and cell debris. Magnification = 50,000 ×, Bar = 600 nm.

Fig. 2. analysis of the protein profile derived from extracellular membrane vesicles (EMVs) isolated from the Bacteroides fragilis 638R strain; (A) ten micrograms of purified EMV protein were separated on a 12% SDS-PAGE gel followed by Coomassie colloidal staining (G-250). Lane 1 shows the EMV proteins profile; the brace indicates the selected bands excised from the gel and digested with trypsin. Peptides were enriched using Ziptip C18 columns and analysed by mass spectrometry (Maldi-TOF/TOF). MW indicates the molecular weight standards in kDa. (B) Western-blotting of the EMV protein extract showing the recognition of a protein (asterisk) of approximately 50 kDa by the rabbit anti-Bfp60 antibody. MW indicates the molecular weight standards in kDa.

Fig. 3. distribution of the Bacteroides fragilis extracellular membrane vesicle proteins identified based on gene ontology annotations (GO). (A) Molecular function bar chart graphic and (B) Biological process pie diagram. The Blast2Go software was used to classify the proteins.

Fig. 4. distribution of subcellular locations of proteins identified in Bacteroides fragilis extracellular membrane vesicles as determined by PSORTb.

Fig. 5. conservation of enolases. Alignment of enolases from human, mouse, Escherichia coli, and Bacteroides fragilis surface proteins, p46 and p60. The dark shaded residues indicate identity; the light grey residues indicate similarity. Clustal format alignment was made using Kalign.