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Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

ABSTRACT: The only recognized virulence factor of enterotoxigenic Bacteroides fragilis (ETBF) that accompanies bloodstream infections is the zinc-dependent non-lethal metalloprotease B. fragilis toxin (BFT). The isolated toxin stimulates intestinal secretion, resulting in epithelial damage and necrosis. Numerous publications have focused on the interrelation of BFT with intestinal inflammation and colorectal neoplasia, but nothing is known about the mechanism of its secretion and delivery to host cells. However, recent studies of gram-negative bacteria have shown that outer membrane vesicles (OMVs) could be an essential mechanism for the spread of a large number of virulence factors. Here, we show for the first time that BFT is not a freely secreted protease but is associated with OMVs. Our findings indicate that only outer surface-exposed BFT causes epithelial cell contact disruption. According to our in silico models confirmed by Trp quenching assay and NMR, BFT has special interactions with outer membrane components such as phospholipids and is secreted during vesicle formation. Moreover, the strong cooperation of BFT with polysaccharides is similar to the behavior of lectins. Understanding the molecular mechanisms of BFT secretion provides new perspectives for investigating intestinal inflammation pathogenesis and its prevention.

SUBMITTER: Zakharzhevskaya NB

PROVIDER: S-EPMC5240029 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

Zakharzhevskaya Natalya B NB Tsvetkov Vladimir B VB Vanyushkina Anna A AA Varizhuk Anna M AM Rakitina Daria V DV Podgorsky Victor V VV Vishnyakov Innokentii E IE Kharlampieva Daria D DD Manuvera Valentin A VA Lisitsyn Fedor V FV Gushina Elena A EA Lazarev Vassili N VN Govorun Vadim M VM

Frontiers in cellular and infection microbiology 20170117

The only recognized virulence factor of enterotoxigenic Bacteroides fragilis (ETBF) that accompanies bloodstream infections is the zinc-dependent non-lethal metalloprotease B. fragilis toxin (BFT). The isolated toxin stimulates intestinal secretion, resulting in epithelial damage and necrosis. Numerous publications have focused on the interrelation of BFT with intestinal inflammation and colorectal neoplasia, but nothing is known about the mechanism of its secretion and delivery to ...[more]

PMID: 28144586

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Project description: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.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.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).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.Although the Bfp60 protein was not detected by proteomics, ?-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.

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Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

Publications

Interaction of <i>Bacteroides fragilis</i> Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

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