Shiga toxin A subunit mutant of Escherichia coli O157:H7 releases outer membrane vesicles containing the B-pentameric complex.
Shiga toxins (STx) are secreted extracellularly through the outer membrane vesicles (OMVs) of Escherichia coli O157:H7. In an attempt to produce STxA-deficient 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 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.
Kim
Sang-Hyun
SH
The National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Cheongwon, Korea.
Lee
Sang-Rae
SR
Kim
Keun-Su
KS
Ko
Ara
A
Kim
Ekyune
E
Kim
Yong-Hwan
YH
Chang
Kyu-Tae
KT
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D002522:Chlorocebus aethiops; D019453:Escherichia coli O157 / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D021122:Protein Subunits; D022142:Secretory Vesicles / Q000737:chemistry / Q000502:physiology*; D022621:Shiga Toxin / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D022603:Shiga Toxins / Q000235:genetics / Q000633:toxicity; D014709:Vero Cells
D021122:Protein Subunits; D022603:Shiga Toxins; C493898:Shiga toxin subunit A; C077239:stxB toxin; D022621:Shiga Toxin