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 pre-viously described Stx2 expression by eukaryotic cells after they were transfected in vitrowith the stx2 gene cloned into a prokary-otic 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, percent-age 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 mRNAwas recovered from the liver, and Stx2 cytotoxic activity was ob-served in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, andmarkers 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 in