title: Biochemical and biological characterization of Escherichia coli STb His12 to Asn variant.
abstract: We identified a variant of Escherichia coli STb toxin by PCR amplification of clinical isolates obtained from diseased pigs. The variant differed by only one amino acid at position 12 from His to Asn. This change was observed in 23 of the 100 randomly selected enterotoxigenic E. coli (ETEC) isolates tested. There was a positive correlation between the presence of the STa enterotoxin and the STb variant. As the variant represented a high percentage of the ETEC strains tested, we were interested in determining if the single amino acid change results in altered biological characteristics of the toxin. Circular dichroism analysis revealed that the secondary structure of the variant was similar to wildtype and that their thermal stabilities were similar. Surface plasmon resonance showed that the variant and the wildtype toxins possessed similar binding affinities for sulfatide but the variant exhibited a reduced binding capacity. A flow cytometry-based internalization assay showed that the variant toxin is more internalized into epithelial intestinal cells than the wildtype strain. However, this difference was minor. Overall, our results indicate that while wildtype STb and the variant share similar structural properties, modest differences exist in their internalization.
authors.affiliation: Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 rue Sicotte, Saint-Hyacinthe, Québec, Canada J2S 7C6.
authors.forename: Christine
authors.initials: C
authors.lastname: Taillon
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hancock
authors.forename: Michaël
authors.initials: M
authors.lastname: Mourez
authors.forename: J Daniel
authors.initials: JD
authors.lastname: Dubreuil
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry*; D002460:Cell Line; D002942:Circular Dichroism / Q000379:methods; D004269:DNA, Bacterial / Q000302:isolation & purification*; D054307:Enterotoxigenic Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D004768:Enterotoxins / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006639:Histidine / Q000737:chemistry; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA; D013433:Sulfoglycosphingolipids / Q000378:metabolism; D013552:Swine / Q000382:microbiology
chemical_list: D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D013433:Sulfoglycosphingolipids; C034151:heat stable toxin (E coli); D006639:Histidine; D001216:Asparagine