Promoter Sequence of Shiga Toxin 2 (Stx2) Is Recognized In Vivo , Leading to Production of Biologically Active Stx2

Abstract

Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfected in vitro with the stx2 gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx2 gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection.

Role of bacteriophages in STEC infections: new implications for the design of prophylactic and treatment approaches

“…In previous reports, we have showed that the native phage promoter controlling Stx expression is active in eukaryotic cells as demonstrated both in vitro 1 and in vivo 2 . Based on these results and the reports previously described, we sought to evaluate whether bacteriophages could be considered a target for treating STEC infections.…”

“…The stx gene is present in the genome of prophages, which are similar to the bacteriophage lambda found in the lysogenic form of various E. coli strains. Previously we reported that the native promoter of the Stx-encoding gene can drive expression of the toxin in eukaryotic cells in both in vivo and in vitro conditions 1, 2 .…”

Role of Shiga Toxins in Cytotoxicity and Immunomodulatory Effects of Escherichia coli O157:H7 during Host-Bacterial Interactions in vitro

“…While regulation of Stx expression has been extensively investigated in bacterial broth cultures, it remains poorly understood how toxin production is regulated in the complex environment of the human gut. We have previously demonstrated that biologically active Stx2 is produced in vivo after pStx2 transfection, driven by its own stx 2 promoter [20]. This evidence raised the question about which is the involvement of host eukaryotic cells in Stx2 production and tissue injury during EHEC infections.…”

“…We have previously reported the ability of eukaryotic cells to recognize putative promoter-like sequences on stx 2 driving Stx2 expression by cell lines [19]. Moreover, mouse in vivo transfection with stx 2 cloned into a prokaryotic plasmid (pStx2) showed stx 2 mRNA in the liver and Stx2 biological toxicity [20]. Therefore, in this work we analyzed the hypothesis that human cell lines participate in Stx2 production after infection with EHEC strains.…”

Animal Models of EnterohemorrhagicEscherichia coliInfection

“…Finally, a recent report suggests that uptake of Stxencoding genetic material by host cells, rather than the toxic protein, may contribute to Stx-induced pathology in mice (119,120). These studies raise the question of whether Stx-coding bacteriophages, which are released along with Stx following bacterial lysis in the intestine, play a role in Stx dissemination (121).…”