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

Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) strain B2F1 produces Stx type 2d, a toxin that becomes more toxic towards Vero cells in the presence of intestinal mucus. STEC that make Stx2d are more pathogenic to streptomycin (Str)-treated mice than most STEC that produce Stx2a or Stx2c. However, purified Stx2d is only 2- or 7-fold more toxic by the intraperitoneal route than Stx2a or Stx2c, respectively. We hypothesized, therefore, that the toxicity differences among Stx2a, Stx2c, and Stx2d occur at the level of delivery from the intestine. To evaluate that hypothesis, we altered the toxin type produced by stx2d+ mouse virulent O91:H21 clinical isolate B2F1 to Stx2a or Stx2c. Because B2F1 encodes two copies of stx2d, we did these studies in a derivative of B2F1 in which stx2d1 was deleted. Although the strains were equivalently virulent to the Str-treated mice at the 1010 dose, the B2F1 strain that produced Stx2a was attenuated relative to the ones that produced Stx2d or Stx2c when administered at 103 CFU/mouse. We next compared the oral toxicities of purified Stx2a, Stx2c, and Stx2d. We found that purified Stx2d is more toxic than Stx2a or Stx2c upon oral administration at 4 µg/mouse. Taken together, these studies suggest that Stx2 toxins are most potent when delivered directly from the bacterium. Furthermore, because Stx2d and Stx2c have the identical amino acid composition in the toxin B subunit, our results indicate that the virulence difference between Stx2a and Stx2d and Stx2c resides in the B or binding subunit of the toxins.

Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli

“…The panel strains carry either one or two ΦStx-prophages featuring suballeles stx 1a , stx 2a , and stx 2d ( Figures 5 , 6 ). Two suballeles have been associated with elevated cytotoxicity, stx 2a ( Fuller et al, 2011 ; Hauser et al, 2020 ; Pinto et al, 2021 ) and stx 2d ( McNichol et al, 2021 ). Suballele stx 2a was found alone (BAA-2219 O121:H19) or in combination with stx 1a (BAA-2196 O26:H11, BAA-2440 O111:H8).…”

“…STEC can harbor one or multiple Stx-bacteriophages featuring different combinations of stx -suballeles ( Krüger and Lucchesi, 2015 ; Rusconi et al, 2016 ) that can also form hybrid toxins ( Skinner et al, 2014 ). The most potent cytopathic toxins, Stx 2a and Stx 2d ( Fuller et al, 2011 ; Hauser et al, 2020 ; McNichol et al, 2021 ), are prevalent in the Big Six serogroups ( Jinnerot et al, 2020 ), and a strain’s Stx-status is shaped by the dynamic Stx-phage acquisition, rather than by a common evolutionary history ( Cowley et al, 2019 ; Nyong et al, 2020 ). Mobilization of Stx-prophages is triggered by diverse abiotic and biotic cues ( Pacheco et al, 2012 ; Pacheco and Sperandio, 2012 ), and is required to produce toxin causing adverse toxigenic effects in murine STEC models ( Nguyen and Sperandio, 2012 ; Tyler et al, 2013 ; Baumler and Sperandio, 2016 ; Balasubramanian et al, 2019 ; Rodríguez-Rubio et al, 2021 ).…”