MEM INST OSWALDO CRUZ, RIO DE JANEIRO, Vol. 113 | 2018
PAGES: DOI: 10.1590/0074-02760170340 Full paper
Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis

Thais Gonçalves Ferreira1, Camilla Nunes dos Reis Trindade1, Petra Bell6, André Teixeira-Ferreira2,3, Jonas E Perales2,3, Rossiane C Vommaro4, Regina Maria Cavalcanti Pilotto Domingues1,+, Eliane de Oliveira Ferreira1,5

1Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Departamento de Microbiologia Médica, Laboratório de Biologia de Anaeróbios, Rio de Janeiro, RJ, Brasil
2Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Toxinologia, Rio de Janeiro, RJ, Brasil
3Rede Proteômica do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
4Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Laboratório de Ultraestrutura Celular Hertha Meyer, Rio de Janeiro, RJ, Brasil
5Universidade Federal do Rio de Janeiro, Duque de Caxias, RJ, Brasil
6University of Leeds, Faculty of Biological Sciences, School of Biology, Leeds, UK

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.

Financial support: FAPERJ, CNPq
+ Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.
Received 31 August 2017
Accepted 17 November 2017

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