Bimodal Response to Shiga Toxin 2 Subtypes Results from Relatively Weak Binding to the Target Cell

Abstract

There are two major antigenic forms of Shiga toxin (Stx), Stx1 and Stx2, which bind the same receptor and act on the same target but nonetheless differ in potency. Stx1a is more toxic to cultured cells, but Stx2 subtypes are more potent in animal models. To understand this phenomenon in cultured cells, we used a system that combines flow cytometry with a fluorescent reporter to monitor the Stx-induced inhibition of protein synthesis in single cells. We observed that Vero cells intoxicated with Stx1a behave differently than those intoxicated with Stx2 subtypes: cells challenged with Stx1a exhibited a population-wide loss of protein synthesis, while cells exposed to Stx2a or Stx2c exhibited a dose-dependent bimodal response in which one subpopulation of cells was unaffected (i.e., no loss of protein synthesis). Cells challenged with a hybrid toxin containing the catalytic subunit of Stx1a and the cell-binding subunit of Stx2a also exhibited a bimodal response to intoxication, while cells challenged with a hybrid toxin containing the catalytic subunit of Stx2a and the cell-binding subunit of Stx1a exhibited a population-wide loss of protein synthesis. Other experiments further supported a primary role for the subtype of the B subunit in the outcome of host-Stx interactions. Our collective observations indicate that the bimodal response to Stx2 subtypes is due to relatively weak binding between Stx2 and the host cell that reduces the total functional pool of Stx2 in comparison to that of Stx1a. This explains, in part, the molecular basis for the differential cellular toxicity between Stx1a and Stx2 subtypes.

Core and Accessory Genome Comparison of Australian and International Strains of O157 Shiga Toxin-Producing Escherichia coli

“…Stx1a has been shown to have antagonistic effects against Stx2a toxicity, shown in both in vitro and in vivo models, where Stx2a alone appears to be more potent (Petro et al, 2019). This reduction in Stx2a toxicity is potentially due to the stronger receptor binding affinity to the globotriaosylceramide (Gb3) receptor of the B subunit of Stx1a, blocking out the binding of Stx2a to the Gb3 receptor (Head et al, 1991;Tesh et al, 1993;Zumbrun et al, 2010;Karve and Weiss, 2014;Russo et al, 2014;Cherubin et al, 2019). The reduced toxicity of Stx2a in the presence of Stx1a has also been linked epidemiologically, reporting a reduced risk of HUS from STEC strains that possess an stx1a and stx2a genotype (Ostroff et al, 1989;Brandal et al, 2015;Tarr et al, 2019).…”

Switching Shiga Toxin (Stx) Type from Stx2d to Stx2a but Not Stx2c Alters Virulence of Stx-Producing Escherichia coli (STEC) Strain B2F1 in Streptomycin (Str)-Treated Mice

“…This means that in BALB/c mice, activation of Stx2d is not required for virulence. Finally, because of our finding that mice infected with B2F1Stx2a had lower than expected levels of Stx2a in the stool, and because the Stx2d and Stx2c B subunits exhibit apparently reduced binding to Gb3 relative to Stx2a, at least in vitro [ 10 ], it may be that Stx2a binds to the intestine at greater levels, and therefore is shed into the feces at reduced levels, and perhaps trafficked to the kidney at lower levels, which would result in reduced virulence.…”

Stx1 and Stx2 subtyping and antimicrobial resistance in Shiga toxin-producing Escherichia coli (STEC) isolates from cattle and sheep feces in the Southeastern region of the State of Goiás, Brazil

“…Although they bind to the same receptor, Stx2a is more toxic in mice, while Stx1a is more cytotoxic in cell culture (Russo et al 2014). Stx1a is ten times more cytotoxic to Vero cells than Stx2a; however, the reverse is seen in mice: Stx1a is 100 to 400 times less lethal than Stx2a, even though the toxins exhibit equivalent enzymatic activities (Melton-Celsa 2014, Cherubin et al 2019, Petro et al 2019. According to Scheutz et al (2012), the subtypes Stx1a and Stx2a are the most frequently associated with HUS in humans.…”

“…They pose a serious public health threat, as they are frequently associated with severe illness and outbreaks in humans, which are transmitted from animals (Veneti et al 2019). STEC is a diverse group of bacteria characterized by the production of potent cytotoxins, called Shiga toxin 1 and Shiga toxin 2 (Stx1 and Stx2), which bind to the same receptor and act on the same target in the cell but differ in the level of cytotoxicity (Russo et al 2014, Cherubin et al 2019. Ruminants, especially cattle and sheep, are the main reservoirs of STEC, shedding these bacteria in the feces, which get directly or indirectly transmitted to humans, thereby causing diseases (Ferreira et al 2014, Yang et al 2017.…”

“…The simultaneous expression of more than one subtype can increase the in-vivo toxicity of the non-O157 strain, making it as virulent as the O157: H7 strain (Scheutz et al 2012, Jajarmi et al 2017. Stx1a is more cytotoxic to Vero cells than Stx2a, but Stx2a has a lower 50% lethal dose (LD 50 ) in mice (Russo et al 2014, Cherubin et al 2019. Epidemiological data suggest that infections with STEC isolates that produce only Stx2a, progress more frequently to HUS than isolates that produce only Stx1a or produce both Stx1a and Stx2a (Petro et al 2019).…”