Secretion of methanol-insoluble heat-stable enterotoxin (STB): energy- and secA-dependent conversion of pre-STB to an intermediate indistinguishable from the extracellular toxin

Abstract

The methanol-insoluble, heat-stable enterotoxin of Escherichia coli synthesized by clinical strains or strains that harbor the cloned gene was shown to be an extraceilular polypeptide. The toxin (STB) was first detected as an 8,100-M, precursor (pre-STB) that was converted to a transiently cell-associated 5,200-Mr form.Proteolytic conversion of pre-STB to STB was shown to be inhibited by the proton motive force uncoupler carbonyl cyanide m-chlorophenylhydrazone and did not occur in a secA background. After STB was detected as a cell-associated molecule, an extracellular form with identical electrophoretic mobility became apparent. The results suggest that there is no proteolytic processing during the mobilization of STB from the periplasm to the culture supernatant. The determined amino acid sequence of STB coincides fully with the 48 carboxy-terminal amino acids inferred from the DNA sequence. The 23 amino-terminal residues inferred from the DNA sequence were absent in the mature toxin.Enterotoxigenic Escherichia coli synthesizes the heatlabile and the heat-stable (ST) families of enterotoxins; these toxins have been shown to be responsible for secretory diarrhea in humans and animals (reviewed in references 1 and 30). STs have been classified as methanol soluble (STA) and methanol insoluble (STB) (3, 36), and these subdivisions correlate well with the inferred or known amino acid compositions of the toxins (18,21,27). The toxic activity of STA is resistant to proteases (6, 33), while STB is inactivated upon trypsin treatment (35). STAs are 18-or 19-amino-acid extracellular enterotoxins that result from two independent proteolytic cleavages on a 72-amino-acid precursor (prepro-STA); the first cleavage yields a periplasmic 53-aminoacid pro-STA that is extracellularly processed to mature STA (29a). The three disulfide bridges formed by the six cysteine residues of STA are sine qua non for toxic activity (6,10,33). A structural model based on proton nuclear magnetic resonance has been proposed for this toxin (11,24). It is also known that STA interacts with an enterocyte receptor that activates guanylate cyclase and results in increased intestinal secretion (8); in contrast to STA, very little is known about the mechanism of action and the export-secretion pathway of methanol-insoluble STB; its gene (estB) has been sequenced (21, 27), and the 71-codon open reading frame, when translated, is very different from the 72 residues of the precursor of STA (reviewed in reference 18). The first 23 amino-terminal amino acids inferred from the estB sequence have properties compatible with a signal peptide (21, 27), and it has been proposed that mature STB is a 48-residue molecule; it was unclear, however, whether STB is an extracellular polypeptide secreted into the medium like STA (16) or whether it is a periplasmic enterotoxin like heat-labile enterotoxin (26). In this communication, we show that mature STB is a 48-amino-acid extracellular polypeptide that corresponds to the previously inferred carboxy-terminal * Correspo...

Escherichia coli STb enterotoxin

“…It now seems that failure to demonstrate a response to STb in species other than the pig could reflect the presence of one or more intraluminal factors, such as, for example, intrinsic protease activity, which precluded a response. A subsequent study (Whipp, 1990) showed that STb evoked a dose-dependent secretory response in infant mice and jejunal loops of rats as long as endogenous protease activity was blocked with soybean trypsin inhibitor. Variation in sensitivity to STb was noted for different animal species.…”

“…Thus, the STb promoter is capable of binding RNA polymerase, but it is a poor initiator of transcription so very little STb is produced. Lawrence et al (1990) indicated that cloning the estB gene into a high-expression vector downstream to the strong bacteriophage lambda pL promoter increased by 10-20-fold the mRNA produced, but the amount of STb enterotoxin, as revealed by the bioassay, was nor: increased.…”

“…The pathways by which these bonds are formed have been established. Kupersztoch et al (1990) polypeptide. This molecule is a transient periplasmic species and a single processing event on a precursor results in it becoming extracellular.…”

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines

“…STb is synthesized as a pre-polypeptide of 71 amino acids [5,6] which is cleaved in the periplasmic space to yield a mature peptide of 48 amino acids with a molecular weight of 5.2 kDa [7]. Mature STb is comprised of two -helices [8] joined by two disulfide bonds [9], a glycine-rich extended coil region [10], and a C-terminal oligomerization domain [11].…”

Amino‐Acid Residues Involved in the Expression of the Activity of Escherichia coli TolC

“…They are then transported across the inner membrane via the Sec machinery and appear in periplasm. During their stay in the periplasm, they are processed into a mature form (15,42). The mature ST is moved across the outer membrane by TolC.…”