Shiga-like toxin-based high-efficiency and receptor-specific intracellular delivery system for a protein.

The cell-specific cytosolic delivery of functional macromolecules with high efficiency is of great significance in molecular medicine and biotechnology. Herein, we present a Shiga-like toxin II-based high-efficiency and receptor-specific intracellular delivery system. We designed and constructed the Shiga-like toxin-based carrier (STC) to comprise the targeting and translocation domains, and used it for delivering a protein cargo. The STC was shown to deliver a protein cargo into the cytosol with high efficiency in a receptor-specific manner, exhibiting much higher efficiency than the most widely used cell-penetrating peptide. The general utility of the STC was demonstrated by modulating the targeting domain. The present delivery platform can be widely used for the intracellular delivery of diverse biomolecules in a receptor-specific and genetically encodable manner.

Mouse in vivo neutralization of Escherichia coli Shiga toxin 2 with monoclonal antibodies.

Shiga toxin-producing Escherichia coli (STEC) food contaminations pose serious health concerns, and have been the subject of massive food recalls. STEC has been identified as the major cause of the life-threatening complication of hemolytic uremic syndrome (HUS). Besides supportive care, there currently are no therapeutics available. The use of antibiotics for combating pathogenic E. coli is not recommended because they have been shown to stimulate toxin production. Clearing Stx2 from the circulation could potentially lessen disease severity. In this study, we tested the in vivo neutralization of Stx2 in mice using monoclonal antibodies (mAbs). We measured the biologic half-life of Stx2 in mice and determined the distribution phase or t(1/2) α to be 3 min and the clearance phase or t(1/2) ß to be 40 min. Neutralizing mAbs were capable of clearing Stx2 completely from intoxicated mouse blood within minutes. We also examined the persistence of these mAbs over time and showed that complete protection could be passively conferred to mice 4 weeks before exposure to Stx2. The advent of better diagnositic methods and the availability of a greater arsenal of therapeutic mAbs against Stx2 would greatly enhance treatment outcomes of life threatening E. coli infections.

Shiga toxin type 2 (Stx2), a potential agent of bioterrorism, has a short distribution and a long elimination half-life, and induces kidney and thymus lesions in rats.

Shiga toxin type 2, a major virulence factor produced by the Shiga toxin-producing Escherichia coli, is a potential toxin agent of bioterrorism. In this study, iodine-125 (125I) was used as an indicator to describe the in vivo Stx2 biodistribution profile. The rats were injected intravenously (i.v.) with 125I-Stx2 at three doses of 5.1-127.5 µg/kg body weight. Stx2 had a short distribution half-life (t (1/2)α, less than 6 min) and a long elimination half-life in rat. The toxicokinetics of Stx2 in rats was dose dependent and nonlinear. Stx2 concentrations in various tissues were detected at 5-min, 0.5-h, and 72-h postinjection. High radioactivity was found in the lungs, kidneys, nasal turbinates, and sometimes in the eyes, which has never been reported in previous studies. In a preliminary assessment, lesions were found in the kidney and thymus.

Differential tissue targeting and pathogenesis of verotoxins 1 and 2 in the mouse animal model.

BACKGROUND: Both verotoxin (VT)1 and VT2 share the same receptor, globotriaosyl ceramide (Gb(3)). Although VT1 is slightly more cytotoxic in vitro and binds Gb(3) with higher affinity, VT2 is more toxic in mice and may be associated with greater pathology in human infections. In this study we have compared the biodistribution of iodine 125 ((125)I)-VT1 and (125)I-VT2 versus pathology in the mouse. METHODS: (125)I-VT1 whole-body autoradiography defined the tissues targeted. VT1 and VT2 tissue distribution, clearance, and tissue binding sites were compared. The effect of a soluble receptor analogue, adamantylGb(3), on VT2/Gb3 binding and in vivo pathology was assessed. RESULTS: (125)I-VT1 autoradiography identified the lungs and nasal turbinates as major, previously unrecognized, targets, while kidney cortex and the bone marrow of the spine, long bones, and ribs were also significant targets. VT2 did not target the lung, but accumulated in the kidney to a greater extent than VT1. The serum half-life of VT1 was 2.7 minutes with 90% clearance at 5 minutes, while that of VT2 was 3.9 minutes with only 40% clearance at 5 minutes. The extensive binding of VT1, but not VT2, within the lung correlated with induced lung disease. Extensive hemorrhage into alveoli, edema, alveolitis and neutrophil margination was seen only after VT1 treatment. VT1 targeted lung capillary endothelial cells. Identical tissue binding sites (subsets of proximal/distal tubules and collecting ducts) for VT1 and VT2 were detected by toxin overlay of serial frozen kidney sections. Glucosuria was found to be a new marker of VT1- and VT2-induced renal pathology and positive predictor of outcome in the mouse, consistent with VT-staining of proximal tubules. Lung Gb3 migrated on thin-layer chromatography (TLC) faster than kidney Gb(3), suggesting a different lipid composition. AdamantylGb(3), a soluble Gb(3) analogue, competed effectively for Gb3 binding by VT1 and VT2 in vitro. However, the effect in the mouse model (only measured against VT2, due to the lower LD(50), a concentration required for 50% lethality) was to increase, rather than reduce, pathology and further reduce the VT2 serum clearance rate. Additional renal pathology was seen in VT2 + adamantylGb(3)-treated mice. CONCLUSIONS: The lung is a preferential (Gb(3)) "sink" for VT1, which explains the relatively slower clearance of VT2 and subsequent increased VT2 renal targeting and VT2 mortality in this animal model.

Oedema disease is associated with metabolic acidosis and small intestinal acidosis.

Limited information is available about the pathogenesis and pathophysiology of oedema disease (OD). Oedema disease is caused by specific enterotoxemic Escherichia coli (SLTIIv-toxin producing) strains; however, the same strains are also found in non-afflicted pigs. Furthermore, it is unclear how the 80 kDa SLTIIv-toxin can pass the intestinal barrier. In the present paper, piglets showing signs of acute OD were anaesthetised, instrumented and cardiovascular and intestinal parameters were determined at 0, 1, 2 and 3 hours. Healthy piglets from the same herd were used as a control. Cardiac output, blood pH and bicarbonate, small intestinal intramucosal pH, and (pulmonary) blood pressure were significantly lower in OD-pigs than in control pigs. It is concluded that OD is associated with metabolic and intestinal acidosis. Intestinal acidosis is known to increase macromolecular permeability. This suggests that once OD has developed, influx of SLTIIv-toxin into the blood stream is facilitated, thus perpetuating the disease. Since intestinal permeability appears to be central in OD, it is argued that post-weaning events increase intestinal permeability and predispose individuals to OD.

Adsorption effect of activated charcoal on enterohemorrhagic Escherichia coli.

The adsorption property of activated charcoal on verotoxin (VT)-producing Escherichia coli (VTEC) was examined using E. coli O157:H7. In the present study, E. coli O157:H7 strains were effectively adsorbed by activated charcoal. Adsorption was dose-dependent, and the maximum adsorption occurred within 5 min. At 10 mg of activated charcoal, bacteria tested were completely adsorbed. Activated charcoal also had the capacity to adsorb toxin (verotoxin 2) activity from the bacterial extract. Furthermore, the adsorption efficiency of activated charcoal for the normal bacterial flora in the intestine was assessed using Enterococcus faecium, Bifidobacterium thermophilum, and Lactobacillus acidophilus. Activated charcoal showed lower binding capacity to the normal bacterial flora tested than that to E. coli O157:H7 strains. These results suggest that activated charcoal could be a good adsorbent system for the removal of VTEC and verotoxin.