Extracellular DsbA-insensitive Folding of Escherichia coli Heat-stable Enterotoxin STa in Vitro

Abstract

To study the folding of human Escherichia coli heatstable enterotoxin STh, we used the major protein subunit of CS31A fimbriae (ClpG) as a marker of STh secretion and a provider of a signal peptide. We established that STh genetically fused to the N or C terminus of ClpG was able to mobilize ClpG to the culture supernatant while still retaining full enterotoxicity. These features indicate that the STh activity was not altered by the chimeric structure and suggest that spatial conformation of STh in the fusion is close to that of the native toxin, thus permitting recognition and activation of the intestinal STh receptor in vivo. In contrast to other studies, we showed that disulfide bond formation did not occur in the periplasm through the DsbA pathway and that there was no correlation between DsbA and secretion, folding, or activity. This discrepancy was not attributable to the chimeric nature of STh since there was no effect of dsbA or dsbB mutations on secretion and activity of recombinant STh from which ClpG had been deleted. Periplasmic and lysate fractions of dsbA ؉ and dsbA ؊ cells did not have any STh activity. In addition, the STh chimera was exclusively found in an inactive reduced form intracellularly and in an active oxidized form extracellularly, irrespective of the dsbA background. Subsequently, a time course experiment in regard to the secretion of STh from both dsbA ؉ and dsbA ؊ cells indicated that the enterotoxin activity (proper folding) in the extracellular milieu increased with time. Overall, these findings provide evidence that STa toxins can be cell-released in an unfolded state before being completely disulfide-bonded outside the cell.Escherichia coli heat-stable enterotoxin STa (also referred to as STI) is responsible for serious and sometimes fatal diarrheal disease in both humans and domestic animals (1). STa causes intestinal fluid secretion by binding to its cellular receptor, transmembrane guanylyl cyclase C (2), thus activating guanylyl cyclase C and leading to an increase in intracellular mucosal cGMP. This increase in cGMP adversely affects electrolyte flux in the intestine; sodium absorption is inhibited, and chloride secretion is stimulated. These ion flux changes result in the secretory diarrhea characteristic of enterotoxigenic E. coli infection (3, 4). The receptor binding and enterotoxic properties of STa have been mapped to a highly conserved 13-amino acid C-terminal core sequence essential for toxicity and the heatstable nature of the toxin. Six cysteine residues are present within this domain, and the three intramolecular disulfide bonds formed between the cysteine residues are necessary for toxicity of the molecule (5). The two species of STa, STp and STh, from porcine (or bovine) and human strains of enterotoxigenic E. coli, respectively, are typical extracellular peptides consisting of 18 (STp) or 19 (STh) amino acid residues (6, 7). STa is synthesized in the cytoplasm as a 72-amino acid precursor consisting of pre-, pro-, and mature STa regions (8, 9). The precursor i...

Contribution of defined amino acid residues to the immunogenicity of recombinantEscherichia coliheat-stable enterotoxin fusion proteins

“…1. Plasmids pEH838, pEHSTC22 [12,13] and pEHProSTC28 [12,13] derived from pEH524 [14], a vector carrying the clp gene cluster that codes for the ClpG export machinery required for the biogenesis of CS31A fimbriae [14] and the secretion of ClpG‐STh fusion proteins by E. coli DH5 α [13]; the wild‐type clpG gene from pEH524 was replaced either by the mutated clpG gene from pHPCO838 [12,13], giving pEH838, or by the clpG :: sth fusion genes from pSTC22 and pProSTC28, leading to pEHSTC22 and pEHProSTC28, respectively. Plasmid pSTCDM1 was made by inserting a double‐stranded oligonucleotide coding for the entire mature STh with glycine and leucine simultaneously substituted for Pro 13 and Ala 14 amino acid residues, between the Hpa I and Xba I sites of pHPCO838.…”

“…Plasmid pSTCDM1 was made by inserting a double‐stranded oligonucleotide coding for the entire mature STh with glycine and leucine simultaneously substituted for Pro 13 and Ala 14 amino acid residues, between the Hpa I and Xba I sites of pHPCO838. Because of many unsuccessful attempts to subclone the fusion genes from pSTC17 [12,13] and pSTCDM1 into pEH524, we trans‐complemented pSTC17 and pSTCDM1 with pDSPH524 [15], which is pEH524 with clpG deleted, for permitting secretion of fusion proteins. Gene fusions were checked by sequencing.…”

Cure and Curse: E. coli Heat-Stable Enterotoxin and Its Receptor Guanylyl Cyclase C

“…Other results are in conflict with this view as deletion of the pro sequence or the STa peptide leads to detection of STa or pro peptide in the supernatant of cells, clearly indicating that the pro region can cross the outer membrane [38]. Other studies showed that various fusion proteins consisting of STa and the major subunit ClpG of E. coli CS31A fimbriae antigen are secreted across the outer membrane and that the disulfide bonds in the bioactive STa were formed extracellularly by a DsbA-independent mechanism possibly involving molecular oxygen [39,40]. However, these authors also showed that the export of their fusion proteins was dependent on the CS31A secretion pathway and might differ substantially from the secretion of native STa.…”

Contribution of defined amino acid residues to the immunogenicity of recombinant Escherichia coli heat-stable enterotoxin fusion proteins

“…1. Plasmids pEH838, pEHSTC22 [12,13] and pEHProSTC28 [12,13] derived from pEH524 [14], a vector carrying the clp gene cluster that codes for the ClpG export machinery required for the biogenesis of CS31A ¢mbriae [14] and the secretion of ClpG-STh fusion proteins by E. coli DH5 K [13]; the wild-type clpG gene from pEH524 was replaced either by the mutated clpG gene from pHPCO838 [12,13], giving pEH838, or by the clpG: :sth fusion genes from pSTC22 and pProSTC28, leading to pEHSTC22 and pEHProSTC28, respectively. Plasmid pSTCDM1 was made by inserting a double-stranded oligonucleotide coding for the entire mature STh with glycine and leucine simultaneously substituted for Pro 13 and Ala 14 amino acid residues, between the HpaI and XbaI sites of pHPCO838.…”

“…Plasmid pSTCDM1 was made by inserting a double-stranded oligonucleotide coding for the entire mature STh with glycine and leucine simultaneously substituted for Pro 13 and Ala 14 amino acid residues, between the HpaI and XbaI sites of pHPCO838. Because of many unsuccessful attempts to subclone the fusion genes from pSTC17 [12,13] and pSTCDM1 into pEH524, we trans-complemented pSTC17 and pSTCDM1 with pDSPH524 [15], which is pEH524 with clpG deleted, for permitting secretion of fusion proteins. Gene fusions were checked by sequencing.…”