Comparisons of Native Shiga Toxins (Stxs) Type 1 and 2 with Chimeric Toxins Indicate that the Source of the Binding Subunit Dictates Degree of Toxicity

Lisa M Russo,Angela R Melton-Celsa,Michael J Smith,Alison D O'Brien,Nicholas J Mantis

doi:10.1371/journal.pone.0093463

Lisa M Russo, Angela R Melton-Celsa + Show 3 more

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https://doi.org/10.1371/journal.pone.0093463

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Abstract

Shiga toxin (Stx)-producing E. coli (STEC) cause food-borne outbreaks of hemorrhagic colitis. The main virulence factor expressed by STEC, Stx, is an AB5 toxin that has two antigenically distinct forms, Stx1a and Stx2a. Although Stx1a and Stx2a bind to the same receptor, globotriaosylceramide (Gb3), Stx2a is more potent than Stx1a in mice, whereas Stx1a is more cytotoxic than Stx2a in cell culture. In this study, we used chimeric toxins to ask what the relative contribution of individual Stx subunits is to the differential toxicity of Stx1a and Stx2a in vitro and in vivo. Chimeric stx1/stx2 operons were generated by PCR such that the coding regions for the A2 and B subunits of one toxin were combined with the coding region for the A1 subunit of the heterologous toxin. The toxicities of purified Stx1a, Stx2a, and the chimeric Stxs were determined on Vero and HCT-8 cell lines, while polarized HCT-8 cell monolayers grown on permeable supports were used to follow toxin translocation. In all in vitro assays, the activity of the chimeric toxin correlated with that of the parental toxin from which the B subunit originated. The origin of the native B subunit also dictated the 50% lethal dose of toxin after intraperitoneal intoxication of mice; however, the chimeric Stxs exhibited reduced oral toxicity and pH stability compared to Stx1a and Stx2a. Taken together, these data support the hypothesis that the differential toxicity of the chimeric toxins for cells and mice is determined by the origin of the B subunit.

Highlights

Shiga toxin (Stx)-producing E. coli (STEC) are Gram-negative, enteric pathogens with an estimated infectious dose of less than 50 organisms [1]
Similar toxicity results were obtained on HCT-8 cells, the overall activity of the toxins was 10-fold lower than observed for Vero cells as expected (Figure 1B). (HCT8 cells exhibit reduced sensitivity to Stx due to decreased expression of the toxin receptor, Gb3, on the cell surface [28])
When the chimeric toxins were examined for the capacity to bind Gb3 by enzyme-linked immunosorbent assay (ELISA), we found that the Gb3-binding capacity of the chimeric toxins was again dependent on the origin of the Bsubunit

Summary

Introduction

Shiga toxin (Stx)-producing E. coli (STEC) are Gram-negative, enteric pathogens with an estimated infectious dose of less than 50 organisms [1]. Among the multiple serotypes associated with disease, O157:H7 is responsible for more than 63,000 of the 175,000 total estimated STEC cases each year [2]. Especially cattle, are the natural carriers of STEC, and these bacteria most commonly enter the food chain during beef processing [3,4,5,6]. Outbreaks and sporadic cases of STEC infection have been attributed to contaminated fresh produce, personto-person spread, and environmental sources [6,7]. Upon E. coli O157:H7 STEC infection of humans, the most common disease manifestation is hemorrhagic colitis. A more severe sequela, the hemolytic uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute kidney failure, occurs in 5–15% of E. coli O157:H7-infected individuals [8,9,10,11]

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