The Escherichia coli heat-stable enterotoxin II (STII) is a typical extracellular toxin consisting of 48 amino acid residues, of which 4 are cysteine. There are two disulfide bonds, one between Cys-10 and Cys-48 and one between Cys-21 and Cys-36. We examined the involvement of DsbA in the formation of the disulfide bonds of STII and the role of each in the secretion of STII. A dsbA mutant was transformed with a plasmid harboring the STII gene, and STII was not detected either in the cells or in the culture supernatant. Reducing the level of STII brought about the dsbA mutation restored by introducing the wild-type dsbA gene into the mutant strain. These results showed that DsbA is involved in forming the disulfide bonds of STII and that STII without these disulfide bonds is degraded during secretion. We substituted these four cysteine residues in vivo by oligonucleotide-directed site-specific mutagenesis. The amino acid sequence of the purified STII(C48S) and pulse-chase studies revealed that two intermolecular disulfide bonds must be formed to be efficiently secreted and that cleavage between amino acid residues 14 and 15 is probably the first step in the proteolytic degradation of STII.Enterotoxigenic Escherichia coli strains produce two heatstable enterotoxins (STs), which cause intestinal secretion and diarrhea (5). One is termed STI (also referred to as STa), and the other is termed STII (also referred to as STb). STI is an 18-or 19-amino-acid acidic peptide containing three disulfide bonds and a protease-stable peptide (1,19,25). STI is active in the suckling-mouse assay. On the other hand, STII was reported to be active only in the weaned-pig ligated intestinal loop assay (26). Because of the lack of a convenient assay, STII was not purified for some time after its detection. We and Whipp found that STII is positive in the mouse intestinal loop assay in the presence of a protease inhibitor (9,11,27). We purified STII and confirmed that the mature form released into culture supernatant is composed of 48 amino acid residues that contain disulfide bonds between Cys-10 and Cys-48 and between Cys-21 and Cys-36 (9).The gene encoding STII (estII) has been isolated and the DNA sequence has been determined (15, 21). The sequence indicates that STII is synthesized as a 71-amino-acid-residue peptide. The 48-amino-acid sequence that we determined is identical to that of the 48 carboxy-terminal amino acids of STII predicted from the DNA sequence. This means that STII is synthesized as a precursor consisting of 71 amino acid residues and that the 23 amino-terminal amino acids consist of a conventional leader peptide which is cleaved by signal peptidase after the STII mRNA translation is initiated. This cleavage allows the resulting peptide to translocate through the inner membrane to the periplasm (13). To be converted into active STII, the intramolecular disulfide bonds must be correctly formed. However, the pathway in which the disulfide bond is formed has not been elucidated.A periplasmic protein, termed DsbA, which...

Escherichia coli heat-stable enterotoxin II (STII) is composed of 48 amino acid residues. Among these, one histidine, two arginine, and six lysine residues are basic. Isoelectric focusing showed that the isoelectric point of STII is 9.7, indicating that the side chains of some of these basic amino acid residues project outside the molecule. To understand the role that these basic amino acid residues play in toxicity, STII was chemically modified with ethoxyformic anhydride, maleic anhydride, and phenylglyoxal, which alter the side chains of basic amino acid residues in proteins. Maleic anhydride, which modifies the E amino group, caused a significant loss of enterotoxic activity, but the other two modifiers did not. This indicated that lysine residues play an important role in the expression of the enterotoxic activity of STII and that the contribution of the other basic amino acid residues to the toxicity is relatively low. To confirm this hypothesis, we substituted these nine basic amino acid residues by oligonucleotide-directed site-specific mutagenesis and examined the enterotoxicity of these purified mutant STIls. The enterotoxic activity was reduced when the lysine residues at positions 18, 22, 23, and 46 were substituted. In particular, the substitution at positions 22 and 23 induced a remarkable reduction. These results demonstrate that the lysine residues at positions 22 and 23 are very important in the expression of the enterotoxic activity of STII. Enterotoxigenic Escherichia coli strains produce two kinds of heat-stable enterotoxin (STs), which cause intestinal secretion and diarrhea (2, 10). One is termed STI (also referred to as STa), and the other is STII (also referred to as STb). STI is an 18or 19-amino-acid acidic peptide containing three disulfide

Stock strains of Eschericia coli isolated from patients with traveller's diarrhea were examined for production of heat-stable enterotoxin II (STII). Of 400 strains examined, 3 were found to produce STII. The nucleotide sequence of the STII gene of these human strains was shown to be identical to that of porcine strains. Cultured cells of these strains induced fluid accumulation in ligated mouse intestinal loops and the activity was neutralized by anti-STII antiserum. These results suggest that STII-produciing enterotoxigenic E. coli can cause human diarrhea.

Enterotoxigenic Escherichia coli isolated from diarrhea stools of chickens were examined for production of heat‐stable enterotoxin II which is considered to be implicated only in diarrhea of pigs. Seven out of 38 strains examined were found to contain heat‐stable enterotoxin II gene, determined by colony hybridization and the polymerase chain reaction. The culture supernatants of these strains caused fluid accumulation in the mouse intestinal loop test. This fluid accumulation activity was not lost by heating at 100°C and was neutralized by anti‐heat‐stable enterotoxin II antiserum. Purified heat‐stable enterotoxin II caused fluid accumulation in the chicken intestinal loop assay. These results indicate that STII‐producing E. coli is implicated in chicken diarrhea.