Tumour necrosis factor alpha is not an essential component of verotoxin 1-induced toxicity in mice.

Previous studies have shown that tumour necrosis factor alpha (TNF-alpha) gene transcription is induced in the mouse kidney in response to Shiga-like toxin 1 (Stx 1, or Verotoxin 1, VT1) administration, suggesting that local TNF-alpha expression plays a role in renal pathogenesis caused by the toxin. Further, TNF-alpha neutralizing antibody pretreatment of mice orally infected with VT-producing Escherichia coli (VTEC) protected the animals from disease development and death. We examined the role of TNF-alpha release in response to parenteral challenge with purified VT1. Mice injected with 10- and 100-fold the 50% lethal dose (LD(50)) of VT1 showed a weak, transient elevation of serum TNF-alpha only at the higher toxin dose. TNF-alpha was not detected in the urine of mice at either dose. Treatment of BALB/c mice with a neutralizing anti-TNF-alpha antibody prior to administration of 3 LD(50) of toxin failed to protect the mice from VT1-mediated toxicity. Further, TNF-alpha knock-out mice administered 3 LD(50) of VT1 were not protected against the lethal effects of the toxin relative to the wild-type animals. These findings suggest that VT1 is a poor inducer of TNF-alpha in vivo and that the low levels of the cytokine released in response to toxin challenge do not play a direct role in potentiating the toxicity of VT1 in mice. Strong toxin accumulation in the kidney but not in the brain was demonstrated by immunohistochemistry after intraperitoneal administration of VT1. Tubular damage and extensive apoptosis in the kidney, together with a 10-fold increase in levels of blood urea nitrogen, suggest that mice died of acute renal failure.

A mutational analysis of the globotriaosylceramide-binding sites of verotoxin VT1.

Escherichia coli verotoxin, also known as Shiga-like toxin, binds to eukaryotic cell membranes via the glycolipid Gb(3) receptors which present the P(k) trisaccharide Galalpha(1-4)Galbeta(1-4)Glcbeta. Crystallographic studies have identified three P(k) trisaccharide (P(k)-glycoside) binding sites per verotoxin 1B subunit (VT1B) monomer while NMR studies have identified binding of P(k)-glycoside only at site 2. To understand the basis for this difference, we studied binding of wild type VT1B and VT1B mutants, defective at one or more of the three sites, to P(k)-glycoside and pentavalent P(k) trisaccharide (pentaSTARFISH) in solution and Gb(3) presented on liposomal membranes using surface plasmon resonance. Site 2 was the key site in terms of free trisaccharide binding since mutants altered at sites 1 and 3 bound this ligand with wild type affinity. However, effective binding of the pentaSTARFISH molecule also required a functional site 3, suggesting that site 3 promotes pentavalent binding of linked trisaccharides at site 1 and site 2. Optimal binding to membrane-associated Gb(3) involved all three sites. Binding of all single site mutants to liposomal Gb(3) was weaker than wild type VT1B binding. Site 3 mutants behaved as if they had reduced ability to enter into high avidity interactions with Gb(3) in the membrane context. Double mutants at site 1/site 3 and site 2/site 3 were completely inactive in terms of binding to liposomal Gb(3,) even though the site 1/site 3 mutant bound trisaccharide with almost wild type affinity. Thus site 2 alone is not sufficient to confer high avidity binding to membrane-localized Gb(3). Cytotoxic activity paralleled membrane glycolipid binding. Our data show that the interaction of verotoxin with the Gb(3) trisaccharide is highly context dependent and that a membrane environment is required for biologically relevant studies of the interaction.