Amino‐Acid Sequence of a Heat‐Stable Enterotoxin Produced by Human Enterotoxigenic Escherichia coli

Abstract

A heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse-phase high-performance liquid chromatography. The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng. The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-G Cys-Tyr. This sequence was identical to that deduced from the nucleotide sequence encoding a human heatstable enterotoxin, reported by Moseley et al., except for the C-terminal Tyr residue.Enterotoxigenic Escherichia coli produces two types of enterotoxins that are responsible for diarrhea : a high-molecular-weight heat-labile enterotoxin (LT) and a low-molecularweight heat-stable enterotoxin (ST) [l]. It has been reported that several distinct ST molecules are produced by enterotoxigenic E. coli. Burgess et al. [2] reported two types of ST: the first, ST,, is methanol-soluble and active in neonatal piglets and infant mice, but inactive in weaned pigs; the second, STb, is methanol-insoluble and active in weaned pigs, but inactive in infant mice. This finding was confirmed by several investigators [3,4]. Moreover, recent reports by So and her coworkers [5,6] purified the STh by chromatography on cation and anion exchangers and high-performance liquid chromatography (HPLC) on a reverse-phase column and determined its amino acid sequence by a combination of fast atom bombardment (FAB) mass spectrometry and carboxypeptidase digestion and Edman degradation. The sequence was found to be the same as that deduced from the nucleotide sequence encoding STh 191, except for the C-terminal amino acid residue. The amino acid sequence of STh determined clearly differed from that of ST reported by Chan and Giannella [8]. MATERIALS A N D METHODS Materials and ApparatusThe chemicals used were reagent grade from several suppliers and were purified before use. SP-Sephadex C-50 and DEAE-Sephadex A-25 were purchased from Pharmacia Japan. Carboxypeptidase B and (Staphylococcus uureus) protease V 8 were from Boehringer (Mannheim, FRG) and Miles (Indiana, USA), respectively. Zorbax ODS columns (5 pm, 4 . 6~ 250 mm) were obtained from DuPont (USA). The high-performance liquid chromatography equipment consisted of a Shimadzu HPLC LC-3A (Japan) fitted with a solvent programmer (GRE-2B) and a data processor chromatopac C-RIA. Reversed-phase columns were packed with Lichrosorb RP-8 (5 pm, Merck) in our laboratory. Column eluates were monitored for absorbance at 220 nm using a variable-wavelength flow-through spectrophotometer and fractions were collected in silicon-coated glass tubes. Bacterial Strain and Culture ConditionsEscherichia coli SK-1 was used throughout. This strain was obtained by genetic labeling of E. coli 53402 A-1, an STproducing strain from a human patient, by Kan' transposon (Tn5) [12]. The cells were grow...

Amino acid sequence of a heat‐stable enterotoxin isolated from enterotoxigenic Escherichia coli strain 18D

“…The crude toxin was chromatographed successively on SP-Sephadex C-50 (H+ form) and DEAE-Sephadex A-25 (acetate form) as in [6]. In each case, the main fraction with biological activity was collected for further purification.…”

“…Recently, we isolated and purified STh from ETEC of human patients [5,6] and determined its amino acid sequence of 19 amino acid residues [6]. The sequence was different from that deduced from the DNA sequence encoding ST,, but identical to that deduced from the DNA sequence of STh, except for the C-terminal amino acid residue.…”

Genetic fusion of a non‐toxic heat‐stable enterotoxin‐related decapeptide antigen to cholera toxin B‐subunit

“…The STa heat-stable enterotoxin of E. coli has six cysteine residues in its primary structure which are known to form three intra-chain disulfide bridges [5]. The cysteine residues are contained within a 15 amino acid stretch of the protein and this region is not only identical between the two different variants of STa molecules known (STa I and STa II) but it also shares extensive homology with the heat-stable enterotoxins from non-01 V. cholerae and Yersinia enterocolitica [20,21].…”

“…Two kinds of STa molecules which vary only slightly have been identified; an STa subtype (STaI) produced by ETEC isolated from both humans and pigs which is 18 amino acids long and another (STaII) only isolated from strains infecting humans which is 19 amino acids in length [4,5]. Both enterotoxins have six cysteines which are known to form intra-chain disulfide bridges [5]. The importance of the disulfide bridges for the toxicity of STa is a subject of great interest because of its bearance on the design of safe vaccines for protection against ETEC diarrhea.…”

The significance of Ser1029 of the heat‐stable enterotoxin receptor (STaR): Relation of STa‐mediated guanylyl cyclase activation and signaling by phorbol myristate acetate

“…The heat-stable enterotoxin (STa) of Escherichia coli is an 18 or 19 amino acid peptide toxin (STp or STh) that causes diarrhea in humans and domestic animals [1,2]. STa binds to a cell surface receptor (STaR/GC-C) [3][4][5][6] in the intestine, which subsequently leads to activation of a guanylyl cyclase (GC) catalytic domain resulting in an accumulation of intracellular cyclic GMP (cGMP).…”