Enterotoxin STp (E. coli)

Background: Enterotoxigenic E. coli (ETEC) is an important cause of diarrhoea among children in developing countries. The burden of ETEC infection was investigated for the first time in children in Malawi. Methods: Faecal samples obtained from children < 5 years of age hospitalised with diarrhoea in Blantyre, Malawi over a 10-year period (1997-2007) before introduction of the rotavirus vaccine were examined by PCR for ETEC heat-labile (LT) and heat-stable (STh and STp) enterotoxins. Children hospitalised without diarrhoea were enrolled over a 2-year period as a comparison group. Results: ETEC was detected in 205 (10.6%) of 1941 children with diarrhoea. The most prevalent toxin type was STh (6·6%), followed by LT (2·1%) and STp (0·9%). ETEC infection was most prevalent in infants aged 6-11 months. Co-infection with rotavirus was common. ETEC was detected in 37 (7·3%) of 507 children without diarrhoea. Conclusion: The burden of ETEC infection in young Malawian children is substantial and should become a focus of diarrhoea prevention efforts in the post-rotavirus vaccine era.

Escherichia coli heat-stable enterotoxin STp is presumed from its DNA sequence to be synthesized in vivo as a 72-amino-acid residue precursor that is cleaved to generate mature STp consisting of the 18 carboxy-terminal amino acid residues. There are two methionine residues in the inferred STp sequence in addition to the methionine residue at position 1. In order to confirm production of the STp 72-amino-acid residue precursor, we substituted the additional methionine residues by oligonucleotide-directed site-specific mutagenesis. Since these substitutions did not cause a significant change in STp production, it can be concluded that STp is normally synthesized as the 72-amino-acid residue precursor. The length of the STp precursor indicated the existence of a pro sequence between the signal peptide and the mature protein. In order to identify the pro sequence and determine its role in protein secretion, deletion and fusion proteins were made. A deletion mutant in which the gene fragment encoding amino acid residues 22 to 53 of STp was removed was made. STp activity was found in the culture supernatant of cells. Amino acid sequence analysis of the purified STp deletion mutant revealed that the pro sequence encompasses amino acid residues 20 to 54. A hybrid protein consisting of STp amino acids 1 to 53 fused in frame from residue 53 to nuclease A was not secreted into the culture supernatant. These results indicate that the pro sequence does not function to guide periplasmic protein into the extracellular milieu.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea among children in developing countries and in travelers to areas of ETEC endemicity. ETEC strains isolated from humans may produce a heat-labile enterotoxin (LT) and two types of the heat-stable enterotoxin STa, called STh and STp, encoded by the estA gene. Two commonly used assay methods for the detection of STa, the infant mouse assay or different enzyme-linked immunosorbent assays, are unable to distinguish between the two subtypes of ST. Different genotypic methods, such as DNA probes or PCR assays, may, however, allow such discrimination. Using gene probes, it has recently been reported that ETEC strains producing STp as the only enterotoxin are not associated with diarrhea. In this study, we have used highly specific PCR methods, including newly designed primers for STh together with previously described STp primers, to compare the relative distribution of STh and STp in ETEC isolated from children with diarrhea in three different geographically distinct areas, i.e., Bangladesh, Egypt, and Guatemala, and from travelers to Mexico and Guatemala. It was found that ETEC strains producing STp were as commonly isolated from cases of diarrhea as strains producing STh both in Egypt and Guatemala, whereas STp strains were considerably less common in Bangladesh. No difference was found in the relative distribution of STh and STp in ETEC strains isolated from travelers with diarrhea and from asymptomatic carriers. Irrespective of ST genotype, the disease symptoms were also similar in both children and travelers.