Shiga toxin A subunit mutant ofEscherichia coliO157:H7 releases outer membrane vesicles containing the B-pentameric complex

Abstract

Shiga toxins (STx) are secreted extracellularly through the outer membrane vesicles (OMVs) of Escherichia coli O157:H7. In an attempt to produce STxAdeficient OMVs from E. coli O157:H7, site-specific deletions of the stx1A and stx2A subunit genes were carried out. The STxA-deficient phenotype of the stx1A/stx2A mutant was confirmed by Vero cell cytotoxicity and VTEC-RPLA s assay. Western blot analyses showed that the B (STxB) subunits were present without coupling to STxA in the OMVs of the STxA-deficient mutant. Furthermore, STxB was located in its homo-pentameric complexes, as revealed by immunoprecipitation and immunoblotting with anti-STxB antibodies. These results suggest that STxB alone can be oligomerized into the B pentamer in the periplasm, and subsequently entrapped into the OMVs. Determination of the median lethal dose concentration for the OMV preparations suggests that the STxA-deficient OMVs containing STxB complex could be safely used as vaccine delivery vehicles.

Membrane vesicles of Clostridium perfringens type A strains induce innate and adaptive immunity

“…It has been observed that bacterial toxins components could be associated with either OMVs in Gram-negative bacteria (Chatterjee and Chaudhuri, 2011; Donato et al, 2012; Kim et al, 2010; Rompikuntal et al, 2012) or MVs in Gram-positive bacteria (Rivera et al, 2010; Thay et al, 2013). As an important potential biological weapon, Bacillus anthracis produces membrane vesicles containing biologically active toxins such as lethal factor (LF) and edema factor (EF) toxins, and serve as an important vesicular transport system for anthrax toxins and some other virulence factors (Rivera et al, 2010).…”

Mouse model for hemolytic uremic syndrome induced by outer membrane vesicles ofEscherichia coliO157:H7

“…Outer membrane vesicles (OMVs) are nano-sized spherical membrane blebs released from the outer membrane of Gram-negative bacteria during their growth (Kuehn & Kesty, 2005). As STx toxins have been known to be present within the OMVs (Kolling & Matthews, 1999;Kim et al, 2010), it is plausible that OMVs of STEC O157:H7 serve as natural vehicles for delivering STx toxins and O157-LPS to patients with D + HUS.…”

Protection from Hemolytic Uremic Syndrome by Eyedrop Vaccination with Modified Enterohemorrhagic E. coli Outer Membrane Vesicles

“…MsbB- and STxA-deficient mutants of EHEC O157:H7 (Sakai-DM/ stx1A / stx2A ) [11] were used as parental strains for producing mOMVs. The preparation of OMVs has been described previously [12].…”

“…Four weeks after eyedrop immunization with mOMVs, 1.5X LD 50 of wild-type EHEC O157:H7 OMVs (LD 50 0.274 mg/kg) [11] were injected intraperitoneally for the challenge experiment. Body weight changes were monitored daily for 8 days after injection.…”

“…However, up until now, an effective and safe OMV vaccine for protection from EHEC O157:H7 infection and sequelae HUS has not been reported, presumably because OMVs generated from EHEC O157:H7 are intrinsically toxic due to presence of STx exotoxin and LPS endotoxin, which are two major virulence factors that contribute towards the development of HUS. In order to overcome the toxicity of the EHEC O157-OMVs, we used a detoxified OMV (produced from MsbB- and STxA-deficient mutant) that was characterized previously for use as a vaccine [5], [11]. Moreover, we generated waaJ-mOMVs, which were composed of a truncated version of O157-LPS lacking the O-antigen side chains, to test whether the absence of O-antigen in LPS would affect immunogenicity of mOMVs administered via an ocular-mucosal route.…”