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

<div><p> 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.</p></div

Sequence coverages of some of the predicted venom gland proteins of <i>S</i>. <i>c</i>. <i>edwardsii</i> as revealed by the combined approach of MALDI and ESI tandem mass spectrometry.

<p>Protein sequences including specific domains are indicted by colored bars; below these, corresponding peptides identified by ESI (black lines) and MALDI (red lines) are indicated.</p

Identification of proteins of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i> not predicted from the transcriptome analysis.

<p>Identification of proteins of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i> not predicted from the transcriptome analysis.</p

2D-PAGE and 2D-WB of <i>Schistosoma mansoni</i> adult worm protein extracts using different IPG strip pH ranges.

<p>A) 2D-PAGE of adult worm total (AW-TOT) and tegumental (AW-TEG) protein extracts using 7 cm, pH 3–10, 3–10NL and 5–8 IPG strips and SDS-PAGE 12%, stained by Colloidal Coomassie Blue G-250. B) Corresponding 2D-WB using pool of <i>S. mansoni</i> infected individuals serum and anti-human Ig's polyvalent antibody HRPO conjugated. The dashed circle indicates a region of strongly stained protein spots in 2D-PAGE that are weakly or not immunoreactive in 2D-WB and the circle indicates a region of protein spots barely visible in 2D-PAGE and highly immunoreactive. C) Schematic representation of immunoreactive spots excised from the corresponding 2D-PAGE to MS/MS identification. Gray circles represent immunogenic spots not identified and black circles represent immunogenic spots identified by MS/MS. All the identified proteins are listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002745#pntd-0002745-t002" target="_blank">Table 2</a>. The figure shows one representative experiment of three replicates.</p

Immunoreactive proteins to the pooled sera of individuals from the schistosomiasis endemic area.

<p>All <i>Smp</i> IDs can be found in schistodb.net. AW-TOT: adult worm total protein extract, AW-TEG: adult worm tegumental protein extract.</p>1<p>: actin was co-extracted from the same spot with reticulocabin, except in pH 5–8 IPG strip;</p>2<p>: annexin was co-extracted from the same spot with one of major egg antigen;</p>3<p>: the same protein corresponding to different spots and protein sequences;</p>4<p>: short-chain dehydrogenase was co-extracted from the same spot with four and A half lim domains;</p>5<p>: gene ID corresponding to Smp_018890 and Smp_187370;</p>6<p>: troponin I from <i>S. japonicum</i>;</p><p>*: proteins selected to <i>in vitro</i> recombinant protein expression.</p

Ang II is promptly metabolized during the invasion assay.

<p>The time course of Ang II metabolism was monitored by mass spectroscopy (MALDI) in the supernatant of infected (•) and non-infected (○) erythrocytes incubated with 10⁻⁵ M Ang II. (A) Ang II, (B) Ang-(1–7) and (C) Ang IV levels. The peak areas for different angiotensin forms (Ang II, Ang-(1–7) and Ang IV) present in the same spectrum (masses 1046.19, 899.02 and 774.92 Da, respectively) were obtained and their sum was arbitrarily assigned as 100%. The results are expressed as means±SE. *Statistically significant compared with the control value (n = 4, <i>P</i><0.05).</p

Sequence alignment of the serine proteinases of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i>.

<p>Different colors indicate the unique peptides identified by tandem mass spectrometry of the corresponding proteins.</p

Indication on the 2D-PAGE of the immunoreactive spots identified in 2D-WB experiments.

<p>Adult worm total (AW-TOT) and tegumental (AW-TEG) protein extracts were separated by 2-DE using 7 cm pH 3–10 IPG strips followed by SDS-PAGE 12%, and stained by Colloidal Coomassie Blue G-250. Protein spots immunoreactive to INF, NE and/or NI pooled sera on the 2D-WB were extracted from the corresponding 2D-PAGE to mass spectrometry identification. The identified protein spots are numbered according to the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002745#pntd-0002745-t003" target="_blank">Table 3</a>. The figure shows one representative experiment of three replicates.</p

<i>In vitro</i> expression and western blotting analysis of recombinant <i>Schistosoma mansoni</i> major egg antigen and hemoglobinase proteins.

<p>A) <i>S. mansoni</i> major egg antigen (MjE) and hemoglobinase precursor (Hem) proteins, lanes 1 and 2 respectively, were expressed using TNT SP6 High-Yield Wheat Germ Protein Expression System and the FluoroTect Green_Lys labeled proteins were analyzed in SDS-PAGE 4–20%. The gel image was obtained with a laser-based fluorescent gel scanner. The negative control using no DNA template was indicated in lane 3 and using no DNA template and no FluoroTect Green_Lys, in lane 4. Lane 5 contains the Precision Plus Protein Kaleidoscope molecular weight marker (BioRad). B) Western blotting analysis of the <i>in vitro</i> recombinant proteins probed with INF (2, 3, 4), NE (5, 6, 7) and NI (8, 9, 10) serum pools. The TNT wheat germ extract using MjE flexi-vector as DNA template was loaded in lanes 2, 5 and 8, and using Hem flexi-vector in lanes 3, 6 and 9. TNT wheat germ extract with no DNA template was loaded in lanes 4, 7 and 10. Rabbit anti-human IgG and anti-rabbit IgG HRP conjugated were used as secondary and tertiary antibodies. The membranes were developed using ECL Plus-Western Blotting Detection System and the proteins were visualized by chemiluminescence detection using a Fujifilm LAS-4000 Imaging System. Pre-stained Precision Plus Protein Kaleidoscope molecular weight marker was loaded in lane 1.</p

2D-WB of <i>Schistosoma mansoni</i> adult worm total and tegumental protein extracts using pooled sera of infected, non-infected from endemic area and non-infected from non-endemic area individuals.

<p>Adult worm total (AW-TOT) and tegumental (AW-TEG) protein extracts were separated by 2-DE using 7 cm pH 3–10 IPG strips and SDS-PAGE 12%. The proteins were blotted onto PVDF membranes and probed with <i>S. mansoni</i> infected (INF), non-infected from endemic area (NE) and non-infected from non-endemic area (NI) serum pools, following an additional incubation with anti-human Ig's polyvalent antibody HRPO conjugated. Circle, arrows and arrowhead indicate immunogenic protein spots which reacted exclusively with INF serum pool, while dotted circle indicates immunogenic spot which reacted exclusively with NE pooled sera. The figure shows one representative experiment of three replicates.</p