Infection of children with Shiga toxin (Stx)-producing Escherichia coli (STEC) is the leading cause of hemolyticuremic syndrome (HUS). Stx2, one of two toxins liberated by the bacteria, is directly linked with HUS. We have previously shown that Stx2-specific human monoclonal antibodies (HuMAbs) protect mice and piglets from fatal systemic complications of Stx2. The present study investigates the mechanisms by which our most efficacious A-and B-subunit-specific HuMAbs neutralize the cytotoxic effects of Stx2 in vitro. Whereas the B-subunit-specific HuMAb 5H8 blocked binding of Stx2 to its receptor on the cell surface, the A-subunit-specific HuMAb 5C12 did not interfere with the toxin-receptor binding. Further investigations revealed that 5C12 did not block endocytosis of Stx2 by HeLa cells as both Stx2 and 5C12 colocalized with early endosomes. However, 5C12 blocked the retrograde transport of the toxin into the Golgi and the endoplasmic reticulum, preventing the toxin from entering the cytosol where the toxin exerts its cytotoxic effect. The endocytosed 5C12/Stx2 complexes appear to be rapidly transported to the plasma membrane and/or to the slow recycling perinuclear compartments, followed by their slow recycling to the plasma membrane, and release into the extracellular environment.Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) can become life threatening if it induces systemic complications, mainly hemolytic-uremic syndrome (HUS), the leading cause of acute renal failure in children (2,11,21,25). Of Stx1 and Stx2, the two immunologically distinct Stxs produced by STEC, strains producing only Stx2 are more frequently associated with HUS (10, 27). Stx1 and Stx2 are similar in basic structure, binding specificity, and mode of action (9). The Stx molecule consists of an A-subunit monomer and a B-subunit pentamer. The pentameric B subunit binds to its cell surface receptor CD77, also called globotriaosylceramide (Gb 3 ). This triggers endocytosis of the holotoxin, mainly through clathrin-coated pits (16). Internalized Stx is then delivered to the trans-Golgi network, where it is carried by retrograde transport to the endoplasmic reticulum (ER), and then to the cytosol (28). During this process, the A subunit is nicked by the membrane bound protease furin, generating a catalytically active N-terminal A1 fragment, while a C-terminal A2 fragment remains linked by a disulfide bond (28). This disulfide bond is subsequently reduced to release the active A1 component. The released A1 fragment has RNA N-glycosidase activity that removes a single adenine from the 28S rRNA, thereby inhibiting protein synthesis in the intoxicated cells (7).Several therapeutic approaches that attempted to neutralize Stx either in the gut or in the circulation include the use of synthetic Gb 3 analogues, genetically manipulated bacteria expressing Gb 3 , and Stx-specific neutralizing antibodies (32). The systemic administration of Stx-specific neutralizing antibodies, we believe, is currently the most promising approach for...