AB ₅ Preassembly Is Not Required for Shiga Toxin Activity

Abstract

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 AB 5 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 AB 5 toxin family members, Stx is produced by STEC as unassembled A and B subunits. A preformed AB 5 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 AB 5 heterohexamer; however, no known mechanisms ensure AB 5 assembly. Stx released by E. coli is not in the AB 5 conformation and assembles at the receptor interface. Thus, unassembled Stx can impart toxicity. This finding shows that preventing AB 5 assembly is a potential treatment for Stx-associated illnesses. IMPORTANCEComplications 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.

Augmenting the Detection of Verotoxin-Producing Escherichia coli Through use of Fluorescently-labeled DNA Oligonucleotides

“…VT2 was seen to cleave adenosine without reduction (Fraser et al, 2006). Additionally, VT has been shown to be released from VTEC not as holotoxins, but primarily as individual subunits (Pellino et al, 2016). This suggested that VT2 A subunits could be enzymatically active on smaller DNA substrates in solution.…”

“…Unlike other AB5 toxins VT is not secreted as holotoxin, but is released as individual subunits through lytic activity of the VT bacteriophage (Neely and Friedman, 1998). Combination of subunits is facilitated by B subunit interaction with Gb3 receptors (Pellino et al, 2016), with the A subunit subsequently binding to form holotoxin ( Figure 1). The B subunits bind to Gb3 cell receptors and cause internalization of the holotoxin.…”

“…The B subunits bind to Gb3 with multiple binding sites per B monomer in a sequential cooperative model (Yung et al, 2003). Binding of the B monomers increased local concentration and enhances B pentamer formation (Pellino et al, 2016). The binding strength of the pentamer has been shown to have a greatly increased binding affinity (4.7 x 10 9 M -1 ) compared to monomers (~10 3 M -1 ) (St. Hilaire et al, 1994;Fuchs et al, 1986).…”

“…The binding strength of the pentamer has been shown to have a greatly increased binding affinity (4.7 x 10 9 M -1 ) compared to monomers (~10 3 M -1 ) (St. Hilaire et al, 1994;Fuchs et al, 1986). This association allows the subunits to combine and further stabilize the holotoxin, with evidence suggesting that the A subunit has a role in enhancing this association (Pellino et al, 2016).…”

“…VT is primarily secreted with phage particles through viral cell lysis. The subunits can then combine to produce toxicity (Pellino et al, 2016).…”