Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2 subtypes also differ in potency, but these differences have not been well documented for purified toxin. The relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved) Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1.

The food-borne pathogen, Escherichia coli O157:H7, has been associated with gastrointestinal disease and the life-threatening sequela hemolytic uremic syndrome. The genes for the virulence factor, Shiga toxin 2 (Stx2), in E. coli O157:H7 are encoded on a temperate bacteriophage under the regulation of the late gene promoter. Induction of the phage lytic cycle is required for toxin synthesis and release. We investigated the hypothesis that nonpathogenic E. coli could amplify Stx2 production if infected with the toxin-encoding phage. Toxin-encoding phage were incubated with E. coli that were either susceptible or resistant to the phage. The addition of phage to phage-susceptible bacteria resulted in up to 40-fold more toxin than a pure culture of lysogens, whereas the addition of phage to phage-resistant bacteria resulted in significantly reduced levels of toxin. Intestinal E. coli isolates incubated with Shiga toxin-encoding phage produced variable amounts of toxin. Of 37 isolates, 3 produced significantly more toxin than was present in the inoculum, and 1 fecal isolate appeared to inactivate the toxin. Toxin production in the intestine was assessed in a murine model. Fecal toxin recovery was significantly reduced when phage-resistant E. coli was present. These results suggest that the susceptibility of the intestinal flora to the Shiga toxin phage could exert either a protective or an antagonistic influence on the severity of disease by pathogens with phage-encoded Shiga toxin. Toxin production by intestinal flora may represent a novel strategy of pathogenesis.

The T-cell-mediated resolution of herpes simplex virus type 2 (HSV-2) genital infections is not fully understood. In these studies, the mechanisms by which CD8 ؉ T cells clear virus from the genital epithelium were examined. Ovalbumin (OVA)-specific CD8 ؉ T cells from OT-I transgenic mice cleared a thymidine kinase-deficient, ovalbumin-expressing HSV-2 virus (HSV-2 tk ؊ OVA) from the genital epithelium of recipient mice, and clearance was abrogated by in vivo neutralization of gamma interferon (IFN-␥). Further, CD8؉ OT-I T cells deficient in IFN-␥ were unable to clear HSV-2 tk ؊ OVA from the vaginal epithelium. The requirement for cytolytic mechanisms in HSV-2 tk ؊ OVA clearance was tested in radiation chimeras by adoptive transfer of wild-type or perforin-deficient OT-I T cells to irradiated Fas-defective or wild-type recipients. Although a dramatic decrease in viral load was observed early after challenge with HSV-2 tk ؊ OVA, full resolution of the infection was not achieved in recipients lacking both perforin-and Fas-mediated cytolytic pathways. These results suggest that IFN-␥ was responsible for an early rapid decrease in HSV-2 virus titer. However, either perforin-or Fas-mediated cytolytic mechanisms were required to achieve complete clearance of HSV-2 from the genital epithelium.Herpes simplex virus type 2 (HSV-2) infects epithelial cells in the genital mucosa, spreads to the sensory ganglia via retrograde transport, and establishes a lifelong latent infection in sensory neurons (50). The virus periodically reactivates and descends sensory neurons via anterograde transport, resulting in development of recurrent lesions at or near the site of primary infection or in shedding of infectious virus in the absence of disease symptoms. The primary and recurrent lesions of immunocompetent individuals are generally self limiting and are resolved primarily by cell-mediated immune mechanisms. Recurrent disease is less well controlled in immunocompromised individuals, resulting in more frequent recurrences and sometimes severe mucocutaneous disease manifestations. Studies of HSV infection in human immunodeficiency virus (HIV)-infected individuals suggested that the severity of HSV disease could be inversely correlated with the number of HSV-specific CD8 ϩ T cells (39). Studies of recurrent HSV lesions in immunocompetent humans have demonstrated the early infiltration of CD4 ϩ T cells and macrophages, local production of IFN-␥, and late arrival of CD8ϩ T cells at the site of HSV infection. Both CD4 ϩ and CD8 ϩ T lymphocytes capable of IFN-␥ secretion and HSVspecific cytolysis have been isolated from human herpetic lesions (10) and clearance of infectious virus, and resolution of lesions has been correlated with the detection of HSV-specific cytolytic T-lymphocyte activity (10, 22-23). However, the role for these cytolytic and noncytolytic immune mechanisms in resolution of HSV-2 genital infections is not well understood.Murine models of HSV-2 genital infection have also demonstrated the importance of cell-mediated imm...

The presence of commensal flora reduced colonization of Escherichia coli O157:H7 and production of Shiga toxin (Stx) in the murine intestine. Stx production was not detected in mice colonized with E. coli that were resistant to the Shiga toxin phage, but it was detected in mice colonized with phage-susceptible E. coli.