Secretion of the ST_A3 heat‐stable enterotoxin of Escherichia coli: extracellular delivery of Pro‐ST_A is accomplished by either Pro or ST_A

Abstract

Summary The methanol‐soluble, heat‐stable enterotoxin of Escherichia coli is a protease‐resistant extracellular peptide which is synthesized as a 72‐amino‐acid precursor Pre‐Pro‐STA3. The specific roles of Fre (19 amino acids), Pro (34 amino acids) and STA3 (19 amino acids) in the secretion process were studied by functionally deleting each of the three domains. Deletion of the Pre signal sequence resulted in a short‐lived cell‐associated molecule with an Mr equivalent to that of Pro‐STA3. Deletion of Pro (i.e., Pre‐STA3) resulted in the rapid extracellular accumulation of STA3 the periplasmic intermediate found in the secretion of the wild‐type toxin was undetected. Deletion of the STA3 domain resulted in a cell‐associated Pre‐Pro peptide; with time this form converted to periplasmic Pro which later became extracellular. When DNA encoding either STA3, by itself, or Pro‐STA3 (lacking the signal peptide) was expressed, these peptides were degraded intracellularly, with no periplasmic or extracellular forms detected. The results presented demonstrate that the signal peptide (Pre) is essential even for the export of small peptides to the periplasm, and that its absence causes the STAS domain to become susceptible to intracellular proteases. The rapid degradation of intracetlular STA3 indicates that its proteolytic resistance is acquired in a compartment other than the cytoplasm. The results also show that after the Pre domain is proteolytically cleaved from Pre‐STA3 and Pre‐Pro, the STA3 and Pro peptides can exit to the culture supernatant. Since STA3 and Pro have neither sequence nor conformational homology, it appears

Extracellular secretion of STa heat‐stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide

“…However, no solid data on the role of this precursor was provided until recently when Okamoto and Takahara [7] duly showed that deletion of the intermediate 33 amino acid residues prior to mature STa (designated Pro sequence) did not prevent folding and secretion of the toxin by E. coli. Those findings have now been confirmed by others [20]. In Pro deletion mutants the so-called Pre-sequence [7] as well as sequences flanking the STa gene, including the natural STa promoter, have been left intact.…”

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

“…5 and 6). We showed that the pro region of STp can promote the translocation of the nascent mature STp region from the cytoplasm to the periplasmic space (34 (36). This retardation is thought to be favorable for the intermediate of STp to be processed correctly in the periplasmic space.…”

“…Rasheed et al (25) and Yang et al (36) hypothesized from pulse-labeling experiments that the three disulfide bridges of ST are formed at the exterior of the cells. Our observation was in conflict with their hypothesis.…”

“…They analyzed the STs synthesized in vivo with the T7 expression system, which required a shift of the incubation temperature to 42°C. Furthermore, rifampin must be incorporated into the cells (25,36). It is likely that these events change the permeability of the E. coli membrane, resulting in leakage of small peptides such as STp.…”

“…SDS-PAGE was performed as described by Laemmli (14 regions (20,34). As the intermediates are soon translocated into culture medium, they are difficult to quantify (36). Therefore, we used fusion proteins containing the amino-terminal region of STp (pre and pro regions) and the nuclease A mature region (the entire fusion protein was referred to as pre-pronuclease A).…”