Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin.

AB(5) toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target-cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB(5) toxin secreted by Shiga toxigenic Escherichia coli (STEC), which causes serious gastrointestinal disease in humans. SubAB causes haemolytic uraemic syndrome-like pathology in mice through SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesized in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite lack of Neu5Gc biosynthesis in humans, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, and the lack of Neu5Gc-containing body fluid competitors in humans, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxin's receptor is generated by metabolic incorporation of an exogenous factor derived from food.

AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP.

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.

A new family of potent AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli.

The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is unrelated to any bacterial toxin described to date. It is the prototype of a new family of AB(5) toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits. The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis. The B subunit is related to a putative exported protein from Yersinia pestis, and binds to a mimic of the ganglioside GM2. Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes (subA and subB), which, in strain 98NK2, are located on a large, conjugative virulence plasmid. Homologues of the genes are present in 32 out of 68 other STEC strains tested. Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver. Oral challenge of mice with E. coli K-12-expressing cloned subA and subB resulted in dramatic weight loss. These findings suggest that the toxin may contribute to the pathogenesis of human disease.

Reduced virulence of an fliC mutant of Shiga-toxigenic Escherichia coli O113:H21.

The contribution of flagellin to the virulence of the O113:H21 Shiga-toxigenic Escherichia coli (STEC) strain 98NK2 was investigated in the streptomycin-treated mouse model. Groups of mice were challenged with either the wild-type STEC or a fliC deletion derivative thereof. There was no difference in the level of gut colonization by the two strains, but the fliC mutant was significantly less virulent than its parent; the overall survival rates were 43.7% and 81.2%, respectively (P < 0.025). This is the first report of a nontoxic accessory virulence factor contributing to a fatal outcome of STEC infection in this model. Although H21 FliC is known to be a potent inducer of CXC chemokines, including interleukin 8, there was no obvious difference in the recruitment of polymorphonuclear leukocytes to the intestinal epithelium of mice challenged with either strain. However, immunofluorescence microscopy suggested that the fliC mutant was less capable of forming a close association with the colonic epithelium. This may have reduced the uptake of Stx2 by mice infected with the mutant.

Protective immunization of mice with an active-site mutant of subtilase cytotoxin of Shiga toxin-producing Escherichia coli.

We have recently described a novel AB(5) cytotoxin produced by certain Shiga toxin-producing Escherichia coli strains. The A subunit of this toxin is a subtilase-like serine protease, while the B pentamer mediates binding to host cell glycolipid receptors. The subtilase cytotoxin is lethal for mice, causing extensive microvascular thrombosis as well as necrosis in the brain, kidneys, and liver. In the present study, we have immunized mice with a purified derivative of the toxin with a Ser272 --> Ala mutation in the A subunit which abolishes cytotoxicity. This elicited strong antibody responses, as judged by enzyme-linked immunosorbent assay, which conferred protection against intraperitoneal challenge with purified toxin. Immunized mice were also protected from weight loss resulting from oral challenge with an E. coli K-12 clone expressing the active toxin.