In this study, the cytotoxicity of the recently described subtilase variant SubAB 2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB 1 and the chromosome-encoded SubAB 2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Shiga toxin-producing Escherichia coli (STEC) strains are zoonotic bacterial pathogens causing a variety of symptoms in humans, ranging from relatively mild forms, such as diarrhea, to hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome (HUS) (1). Besides Shiga toxin, the best-characterized pathogenicity factor for the development of serious diseases is the locus of enterocyte effacement (LEE), encoding a type III secretion system and associated effector proteins (2, 3). Other pathogenicity factors can be involved in the development of human disease (4, 5). An example is the subtilase cytotoxin (SubAB), which is located on a 163-kb plasmid of STEC E. coli O113:H21 strain 98NK2 (6). This strain was isolated from a case of HUS in the south of Australia. The subtilase cytotoxin was shown to cause HUS-like symptoms in mice and apoptosis in human epithelial cells (7-9). Subtilase cytotoxin genes were detected in a variety of LEE-negative STEC strains of human, ovine, and game origin (10-14). Furthermore, strains from human origin were shown to cause symptoms in humans ranging from watery diarrhea to fully developed HUS (15, 16).The SubAB toxin is a typical AB 5 toxin, composed of an enzymatically active A subunit (SubA) and a B pentamer (SubB), which mediates the uptake of the toxin into target cells by binding to a specific surface sialic acid, namely, N-glycolylneuraminic acid (Neu5Gc) (17). Neu5Gc is present in most mammals but is not synthesized by human cells due to a 92-bp deletion in the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene (18). However, it was shown that human cells are able to incorporate Neu5Gc from external sources (19). Inside the eukaryotic cell, the enzymatic active A subunit acts as a subtilisinlike serine protease. SubA cleaves the cellular endoplasmic reticulum chaperone GRP78/BiP at an L-L motif on position 416 between the N-terminal ATPase domain and the C-terminal protein binding domain (20). This highly specific protease activity is unique within AB 5 toxins and leads to the accumulation of unfolded proteins in the endoplasmic reticulum. This accumulation induces the unfolded protein response (UPR), which in the end results in the death of the affected cell (21). Yahiro and coworkers demonstrated that binding of SubAB to one of four different cell surface receptors, namely, NG2, hepatocyte growth factor receptor (Met), L1 cell adhesion molecule (CAM), and ß1 integrin (ITG), induces signals which also result in apoptosis of HeLa cells without internalization of SubAB (22). However, the underlying mechanism is not completely u...