title: Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1.
abstract: Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.
authors.affiliation: Dipartimento di Patologia Sperimentale, Università di Bologna, Bologna, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Brigotti
authors.forename: Domenica
authors.initials: D
authors.lastname: Carnicelli
authors.forename: Valentina
authors.initials: V
authors.lastname: Arfilli
authors.forename: Laura
authors.initials: L
authors.lastname: Rocchi
authors.forename: Francesca
authors.initials: F
authors.lastname: Ricci
authors.forename: Pasqualepaolo
authors.initials: P
authors.lastname: Pagliaro
authors.forename: Pier Luigi
authors.initials: PL
authors.lastname: Tazzari
authors.forename: Antonio González
authors.initials: AG
authors.lastname: Vara
authors.forename: Matteo
authors.initials: M
authors.lastname: Amelia
authors.forename: Francesco
authors.initials: F
authors.lastname: Manoli
authors.forename: Sandra
authors.initials: S
authors.lastname: Monti
mesh_terms: D000225:Adenine / Q000737:chemistry; D001427:Bacterial Toxins / Q000378:metabolism; D002942:Circular Dichroism; D042783:Endothelial Cells / Q000166:cytology; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000494:pharmacology; D006463:Hemolytic-Uremic Syndrome / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000166:cytology* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012276:Ricin / Q000737:chemistry; D022621:Shiga Toxin; D022622:Shiga Toxin 1 / Q000378:metabolism*; D022603:Shiga Toxins / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D001427:Bacterial Toxins; D005456:Fluorescent Dyes; D022622:Shiga Toxin 1; D022603:Shiga Toxins; D022621:Shiga Toxin; D012276:Ricin; D000225:Adenine