Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation

Abstract

The Escherichia coli heat-stable enterotoxin STp is synthesized as a precursor consisting of pre, pro and mature regions. Mature STp is released into the culture supernatant and is composed of 18-amino-acid residues which contain three intramolecular disulfide bonds. The involvement of DsbA in the formation of the disulfide bonds of STp was examined in this study. A dsbA mutant was transformed with a plasmid harboring the STp gene, and the ST activity was significantly lower than that of the parent strain harboring the same plasmid. Furthermore, purified DsbA induced the conversion of synthetic STp peptide (inactive form) to the active form and increased the ST activity of the culture supernatant derived from the dsbA transformants. These results showed that DsbA directly catalyzes the formation of the disulfide bonds of STp. DsbA is located in periplasmic space, where STp is released as an intermediate form consisting of the pro and mature regions. To examine the effect of the pro region on the action of DsbA, we replaced the cysteine residue at position 39 and tested the effect in vivo. The substitution caused a significant decrease of ST activity in the culture supernatant, the accumulation of inactive ST in periplasmic space, and an alteration in the cleavage site of the intermediate of STp. We conclude that Cys-39 is important for recognition by the processing enzymes required for the maturation of STp.

Functional Analysis of Paralogous Thiol-disulfide Oxidoreductases in Streptococcus gordonii

“…Nevertheless, there are examples of disulfide bond-containing proteins produced by Gram-positive species, such as diphtheria and tetanus toxins, botulinum neurotoxin A, and streptococcal pyrogenic exotoxin (17,18,52,53). In Gramnegative pathogens, DsbA forms disulfide bonds in secreted toxins, but the machinery that forms these bonds in Grampositive species has not been identified (54,55).…”

Heat-Stable Enterotoxin of EnterotoxigenicEscherichia colias a Vaccine Target

“…Outer membrane (OM) translocation is accompanied by cleavage of the propeptide (STh amino acids 20 to 53; STp, 20 to 54), generating the mature ST (STh, 54 to 72; STp, 55 to 72); OM translocation is believed to require the TolC channel (67,68), though this has not been directly demonstrated. The periplasmic thiol:disulfide interchange protein DsbA is required for the formation of the three disulfide bridges found in the mature ST (66).…”

Carboxy-Terminal Region Involved in Activity of Escherichia coli TolC

“…These STs are synthesized as precursor proteins with an amino-terminal signal sequence and are translocated across the inner membrane via the Sec machinery (8,14). In contrast to the secretory molecules which cross two membranes in a single step without stagnating in the periplasm, these STs, after being released into the periplasm, remain for a short period in this location (4,13,22). After being processed in the periplasm, these STs translocate across the outer membrane through the action of TolC.…”