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1.
Article in English | MEDLINE | ID: mdl-27747196

ABSTRACT

Seizures and neurologic involvement have been reported in patients infected with Shiga toxin (Stx) producing E. coli, and hemolytic uremic syndrome (HUS) with neurologic involvement is associated with more severe outcome. We investigated the extent of renal and neurologic damage in mice following injection of the highly potent form of Stx, Stx2a, and less potent Stx1. As observed in previous studies, Stx2a brought about moderate to acute tubular necrosis of proximal and distal tubules in the kidneys. Brain sections stained with hematoxylin and eosin (H&E) appeared normal, although some red blood cell congestion was observed. Microglial cell responses to neural injury include up-regulation of surface-marker expression (e.g., Iba1) and stereotypical morphological changes. Mice injected with Stx2a showed increased Iba1 staining, mild morphological changes associated with microglial activation (thickening of processes), and increased microglial staining per unit area. Microglial changes were observed in the cortex, hippocampus, and amygdala regions, but not the nucleus. Magnetic resonance imaging (MRI) of Stx2a-treated mice revealed no hyper-intensities in the brain, although magnetic resonance spectroscopy (MRS) revealed significantly decreased levels of phosphocreatine in the thalamus. Less dramatic changes were observed following Stx1 challenge. Neither immortalized microvascular endothelial cells from the cerebral cortex of mice (bEnd.3) nor primary human brain microvascular endothelial cells were found to be susceptible to Stx1 or Stx2a. The lack of susceptibility to Stx for both cell types correlated with an absence of receptor expression. These studies indicate Stx causes subtle, but identifiable changes in the mouse brain.


Subject(s)
Disease Models, Animal , Nervous System/drug effects , Nervous System/pathology , Shiga Toxin/toxicity , Amygdala/drug effects , Amygdala/pathology , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Calcium-Binding Proteins , Cell Culture Techniques , Cerebral Cortex/drug effects , Cerebral Cortex/pathology , DNA-Binding Proteins , Endothelial Cells/drug effects , Endothelial Cells/pathology , Erythrocytes/drug effects , Escherichia coli/metabolism , Escherichia coli/pathogenicity , Hemolytic-Uremic Syndrome/microbiology , Hemolytic-Uremic Syndrome/pathology , Hippocampus/drug effects , Hippocampus/pathology , Humans , Kidney/drug effects , Kidney/pathology , Magnetic Resonance Imaging/methods , Male , Mice , Microfilament Proteins , Microglia/drug effects , Microglia/pathology , Phosphocreatine/analysis , Rabbits , Repressor Proteins , Shiga Toxin/administration & dosage , Shiga Toxin 2/administration & dosage , Shiga Toxin 2/toxicity , Spectrum Analysis/methods , Thalamus/chemistry , Toxicity Tests/methods , Tumor Necrosis Factor-alpha/pharmacology , Weight Gain/drug effects , Weight Loss/drug effects
2.
J Bacteriol ; 198(11): 1621-1630, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27002129

ABSTRACT

UNLABELLED: Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness, including the life-threatening complication hemolytic-uremic syndrome. The German outbreak in 2011 resulted in nearly 4,000 cases of infection, with 54 deaths. Two forms of Stx, Stx1 and Stx2, differ in potency, and subtype Stx2a is most commonly associated with fatal human disease. Stx is considered to be an AB5 toxin. The single A (enzymatically active) subunit inhibits protein synthesis by cleaving a catalytic adenine from the eukaryotic rRNA. The B (binding) subunit forms a homopentamer and mediates cellular association and toxin internalization by binding to the glycolipid globotriaosylceramide (Gb3). Both subunits are essential for toxicity. Here we report that unlike other AB5 toxin family members, Stx is produced by STEC as unassembled A and B subunits. A preformed AB5 complex is not required for cellular toxicity or in vivo toxicity to mice, and toxin assembly likely occurs at the cell membrane. We demonstrate that disruption of A- and B-subunit association by use of A-subunit peptides that lack enzymatic activity can protect mice from lethal doses of toxin. Currently, no treatments have been proven to be effective for hemolytic-uremic syndrome. Our studies demonstrate that agents that interfere with A- and B-subunit assembly may have therapeutic potential. Shiga toxin (Stx) produced by pathogenic Escherichia coli is considered to be an AB5 heterohexamer; however, no known mechanisms ensure AB5 assembly. Stx released by E. coli is not in the AB5 conformation and assembles at the receptor interface. Thus, unassembled Stx can impart toxicity. This finding shows that preventing AB5 assembly is a potential treatment for Stx-associated illnesses. IMPORTANCE: Complications due to Shiga toxin are frequently fatal, and at present, supportive care is the only treatment option. Furthermore, antibiotic treatment is contraindicated due to the ability of antibiotics to amplify bacterial expression of Shiga toxin. We report, contrary to prevailing assumptions, that Shiga toxin produced by STEC circulates as unassembled A and B subunits at concentrations that are lethal to mice. Similar to the case for anthrax toxin, assembly occurs on receptors expressed on the surfaces of mammalian target cells. Disruption of Shiga toxin assembly by use of A-subunit peptides that lack enzymatic activity protects mice from lethal challenge with Shiga toxin, suggesting a new approach for development of therapeutics.


Subject(s)
Gene Expression Regulation, Bacterial/physiology , Shiga Toxin 2/toxicity , Shiga-Toxigenic Escherichia coli/physiology , Amino Acid Sequence , Animals , Catalytic Domain , Male , Mice , Models, Molecular , Protein Biosynthesis , Protein Conformation , Protein Subunits , Shiga Toxin 2/genetics , Shiga Toxin 2/metabolism
3.
PLoS One ; 8(7): e69823, 2013.
Article in English | MEDLINE | ID: mdl-23875002

ABSTRACT

Shiga toxin (Stx), the main virulence factor of Shiga toxin producing Escherichia coli, is a major public health threat, causing hemorrhagic colitis and hemolytic uremic syndrome. Currently, there are no approved therapeutics for these infections; however manganese has been reported to provide protection from the Stx1 variant isolated from Shigella dysenteriae (Stx1-S) both in vitro and in vivo. We investigated the efficacy of manganese protection from Stx1-S and the more potent Stx2a isoform, using experimental systems well-established for studying Stx: in vitro responses of Vero monkey kidney cells, and in vivo toxicity to CD-1 outbred mice. Manganese treatment at the reported therapeutic concentration was toxic to Vero cells in culture and to CD-1 mice. At lower manganese concentrations that were better tolerated, we observed no protection from Stx1-S or Stx2a toxicity. The ability of manganese to prevent the effects of Stx may be particular to certain cell lines, mouse strains, or may only be manifested at high, potentially toxic manganese concentrations.


Subject(s)
Escherichia coli Infections/drug therapy , Manganese/toxicity , Manganese/therapeutic use , Shiga Toxin/toxicity , Shiga-Toxigenic Escherichia coli/drug effects , Animals , Chlorocebus aethiops , Male , Mice , Vero Cells
4.
Infect Immun ; 79(3): 1329-37, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21199911

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Shiga Toxins/genetics , Shiga Toxins/toxicity , Adult , Amino Acid Sequence , Animals , Cell Line , Child , Chlorocebus aethiops , Flow Cytometry , Humans , Male , Mice , Molecular Sequence Data , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/toxicity , Protein Structure, Quaternary , Sequence Homology, Amino Acid , Shiga Toxins/chemistry , Vero Cells
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