Extracellular secretion of STa heat-stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide.
The mature 19-amino acid STa heat-stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1-19) and Pro (aa 20-53) sequences, proposed to be essential for extracellular toxin release by this host. The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E. coli heat-labile enterotoxin B-subunit (LTB). Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E. coli independently of natural STa neighboring genetic sequences. Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretion in this fashion might be extended to other disulfide bond-rich small peptides, the 13 amino acid conotoxin GI and a non-enterotoxic STa-related decapeptide were cloned. None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteine-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. coli.
Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, Mexico.
J
J
Sanchez
R M
RM
Solorzano
J
J
Holmgren
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D020916:Conotoxins; D004269:DNA, Bacterial; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000378:metabolism; D010456:Peptides, Cyclic / Q000235:genetics; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; D020916:Conotoxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008978:Mollusk Venoms; D010456:Peptides, Cyclic; D021382:Protein Sorting Signals; C034151:heat stable toxin (E coli); C029960:conotoxin GI