SUMMARY Since their initial recognition 20 years ago, Shiga toxin-producing Escherichia coli (STEC) strains have emerged as an important cause of serious human gastrointestinal disease, which may result in life-threatening complications such as hemolytic-uremic syndrome. Food-borne outbreaks of STEC disease appear to be increasing and, when mass-produced and mass-distributed foods are concerned, can involve large numbers of people. Development of therapeutic and preventative strategies to combat STEC disease requires a thorough understanding of the mechanisms by which STEC organisms colonize the human intestinal tract and cause local and systemic pathological changes. While our knowledge remains incomplete, recent studies have improved our understanding of these processes, particularly the complex interaction between Shiga toxins and host cells, which is central to the pathogenesis of STEC disease. In addition, several putative accessory virulence factors have been identified and partly characterized. The capacity to limit the scale and severity of STEC disease is also dependent upon rapid and sensitive diagnostic procedures for analysis of human samples and suspect vehicles. The increased application of advanced molecular technologies in clinical laboratories has significantly improved our capacity to diagnose STEC infection early in the course of disease and to detect low levels of environmental contamination. This, in turn, has created a potential window of opportunity for future therapeutic intervention.

We recently described a novel megaplasmid-encoded adhesin produced by certain Shiga toxigenic Escherichia coli (STEC) strains that lack the locus for enterocyte effacement (LEE) pathogenicity island. This adhesin, designated Saa (STEC autoagglutinating adhesin), may be a marker for a subset of LEE-negative STEC strains capable of causing severe gastrointestinal and systemic diseases in humans. In this study, we developed a pentavalent PCR assay for the detection of saa as well as other proven and putative STEC virulence genes (stx 1 , stx 2 , eae, and ehxA). The five primer pairs used in the assay do not interfere with each other and generate amplification products of 119, 180, 255, 384, and 534 bp.

Protein folding by the endoplasmic reticulum (ER) is physiologically critical, while its disruption causes ER stress and augments disease. ER stress activates the unfolded protein response (UPR) to restore homeostasis. If stress persists, the UPR induces apoptotic cell death, but the mechanisms remain elusive. Here we find that unmitigated ER stress promotes apoptosis through cell-autonomous, UPR-controlled activation of death receptor 5 (DR5). ER stressors induced DR5 transcription via the UPR mediator CHOP; however, the UPR sensor IRE1α transiently catalyzed DR5 mRNA decay, allowing time for adaptation. Persistent ER stress built up intracellular DR5 protein, driving ligand-independent DR5 activation and apoptosis engagement via caspase-8. Thus, DR5 integrates opposing UPR signals to couple ER stress and apoptotic cell fate.

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 AB5 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.

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.