Supplementary MaterialsFigure S1: Stx subunits are stained in a linear manner

Supplementary MaterialsFigure S1: Stx subunits are stained in a linear manner by Oriole fluorescent stain. against fluorescent PI4KB intensity (arbitrary models).(TIF) pone.0093463.s001.tif (157K) GUID:?46A7D9AD-A1DB-4CD5-B0E5-3A20FB68BF07 Abstract Shiga toxin (Stx)-producing (STEC) cause food-borne outbreaks of Epirubicin Hydrochloride manufacturer hemorrhagic colitis. The main virulence factor expressed by STEC, Stx, is an AB5 toxin that has two antigenically unique forms, Stx1a and Stx2a. Although Stx1a and Stx2a bind to the same receptor, globotriaosylceramide (Gb3), Stx2a is usually more potent than Stx1a in mice, whereas Stx1a is usually 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 usually to the differential toxicity of Stx1a and Stx2a and 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 decided on Vero and HCT-8 cell lines, while polarized HCT-8 cell monolayers produced on permeable supports were used to follow toxin translocation. In all 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 this differential toxicity of the chimeric toxins Epirubicin Hydrochloride manufacturer for cells and mice is determined by the origin of the B subunit. Introduction Shiga toxin (Stx)-generating (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]. Ruminants, especially cattle, are the natural service providers of STEC, and these bacteria most commonly enter the food chain during beef processing [3]C[6]. Outbreaks and sporadic cases of STEC contamination have also been attributed to contaminated new produce, person-to-person spread, and environmental sources [6], [7]. Upon O157:H7 STEC contamination of humans, the Epirubicin Hydrochloride manufacturer most common disease manifestation is usually hemorrhagic colitis. A more severe sequela, the hemolytic uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute kidney failure, occurs in 5C15% of O157:H7-infected individuals [8]C[11]. Stx is the main virulence factor associated with disease caused by STEC. This group of organisms may encode for Stx1a and/or Stx2a, two biologically similar, though antigenically unique toxins with analogous crystal structures and identical modes of action [examined in [12]]. The Stxs are AB5 toxins; the A subunit, which has a protease sensitive site near the C-terminus, is usually cleaved into two parts. The A1 subunit is responsible for the catalytic activity of the toxin molecule, and the A2 peptide, which threads through the center of the B pentamer, links the binding moiety to the catalytic subunit [13], [14]. The homopentameric B subunit binds to the host cell receptor, globotriaosylceramide (Gb3) [15], a glycolipid that that is primarily expressed on endothelial cells. After Stx binds Gb3, the toxin undergoes retrograde transport through the Golgi network to the endoplasmic reticulum [16]. The A1 subunit is usually released into the cytoplasm from your endoplasmic reticulum and depurinates a single adenine residue from your 28 s RNA of the 60 S ribosome, an injury that halts protein synthesis and prospects to cell death [17], [18]. Although Stx1a and Stx2a are biologically comparable, Stx2a is usually associated with an increased quantity of outbreaks and more severe disease [19]C[24]. These latter observations correlate with reports that Stx2a has a parenteral 50% lethal dose (LD50) for mice that is 100-fold lower than is usually Stx1a [25], [26]. Conversely, Stx1a has a 50% cytototoxic dose (CD50) for Vero cells that is 10 times lower than is usually Stx2a [26]. Multiple methods and approaches have been used to study the paradox of the differential (on Vero cells) and (in mice) toxicities of Stx1a and Stx2a. There is no difference in the enzymatic activity between Stx1a and Stx2a in a cell-free rabbit reticulocyte lysate assay of protein synthesis inhibition [26], [27]; therefore, the A subunit is not responsible for the differential toxicity of Stx1a and Stx2a, at least at the level of the ribosome. Multiple studies propose that the biological differences between Stx1a and Stx2a are specific to the B-subunit. For example, Stx1a binds Gb3 with a greater affinity than does Stx2a [25], [26], [28], [29], a finding that may explain the greater toxicity.