Abstract
Shiga toxins (Stxs) are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC) infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY) from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection.
Highlights
Enterohemorrhagic Escherichia coli (EHEC) causes a spectrum of human diseases, including diarrhea, hemorrhagic colitis, disordered consciousness, renal failure and hemolytic uremic syndrome (HUS) [1,2]
Coomassie Brilliant Blue (CBB) staining of SDS-PAGE gels loaded with Highly purified Stx-1 (hpStx-1) revealed bands at 32.2 kDa and 6.1 kDa, indicating the Shiga toxins (Stxs)-1 A subunit and monomeric B subunit, respectively
Visualization of the Stx B subunits by CBB staining suggested that a sufficient amount of antigen was present in the blotted lane for detection of any antibody reactive against monomeric B subunit
Summary
Enterohemorrhagic Escherichia coli (EHEC) causes a spectrum of human diseases, including diarrhea, hemorrhagic colitis, disordered consciousness, renal failure and hemolytic uremic syndrome (HUS) [1,2]. EHEC colonizes the large intestine and produces Shiga toxins (Stxs) as major virulence factors that are involved in the pathogenicity of EHEC infections. EHEC produces two types of Stxs, Stx-1 and Stx-2. Stxs released into the intestinal lumen enter the systemic circulation and reach target organs, where they manifest their toxicity [3,4]. Stxs are multimeric proteins that consist of an A subunit (32 kDa) and a pentamer of identical B subunits (7.7 kDa each). The B pentamer is responsible for the toxin binding to target cell-surface glycolipid receptors such as galabiosyl ceramide (Gb2-Cer) and globotriaosyl ceramide (Gb3Cer) [5,6]. The toxin is internalized into the cells, and the A subunit is cleaved into A1 (28 kDa) and A2 (4 kDa) fragments. The A1 fragment exerts RNA N-glycosidase activity, which results in inhibition of protein synthesis by inactivation of 28S rRNA [7,8]
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