Binding to sulfatide and enterotoxicity of various Escherichia coli STb mutants.
Binding of the 48 amino acid polypeptide of the mature heat-stable Escherichia coli enterotoxin b (STb) to the functional receptor sulfatide (SFT) constitutes the first step in inducing secretory diarrhoea in the intestinal lumen of animals. The NMR structure of this toxin dictated the choice of amino acids for site-directed mutagenesis to delineate the binding site of STb to SFT. Amino acids facing the solvent either in the loop or the hydrophobic alpha-helix were selected. Seventeen site-specific mutants of STb toxin were produced and purified by high-pressure liquid chromatography. Enterotoxicity of the 17 mutants was determined using a rat loop assay and binding was evaluated using a microtitre plate binding assay. Both hydrophobic and electrostatic interactions are important for STb attachment. When mutations (F37K, I41S and M42S) were introduced into the hydrophobic alpha-helix to lessen hydrophobicity, binding activity and enterotoxicity decreased by more than sixfold. The loop defined by C21 and C36 also made specific contributions. Mutants generated at basic residues (K22, K23 and R29) within this region exhibited both reduced binding activities and reduced toxic activities. For all STb mutants constructed and analysed, when binding to SFT was reduced, a reduction in toxicity equivalent or greater was noted, indicating that binding to SFT is a step that precedes the toxic effect observed for STb toxin. Significantly, when the negatively charged D30 was substituted for either alanine or valine, the binding to SFT was about twice that of native STb, whereas the enterotoxicity was reduced by half.
Labrie
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Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Sicotte, CP 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6.
Beausoleil
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HE
Harel
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Dubreuil
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JD
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D001665:Binding Sites; D002942:Circular Dichroism; D004768:Enterotoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism / Q000382:microbiology; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000378:metabolism*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); C412037:sulfatide receptor; D014867:Water; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled