title: Structural and functional features of modified heat-stable toxins produced by enteropathogenic Klebsiella cells.
abstract: Heat-stable enterotoxins (STs) are 18- or 19-amino acid peptides (STa or ST1) produced by enteropathogenic bacteria with small differences in their amino acid sequence and a highly conserved carboxy terminus. All STs contain a core of three disulfide bridges whose integrity is believed to be necessary for full biologic activity. We previously reported that strains of Klebsiella pneumoniae transformed by the plasmid pSLM004 produce a modified toxin not recognized by MAb raised against genuine Escherichia coli ST. Investigation of the chemical structure of the modified toxins revealed that three new toxins were present. These were purified to homogeneity by a series of sequential chromatography on reverse-phase columns using guanylate cyclase to monitor the enterotoxic activity during purification procedures. The sequence of the modified toxins was obtained by a combination of Edman degradation and mass spectrometry, showing that they are proteolytically processed forms of E. coli ST1b. In particular, toxin A-2 lacks the cysteine at position 18 and then is not able to form the disulfide bridge cysteine-10-cysteine-18. All three toxins showed the ability to stimulate guanylate cyclase and to elicit chloride secretion in Caco-2 cell monolayers mounted in Ussing chambers. Toxin A-1 and toxin B demonstrated greatly reduced immunoreactivity whereas toxin A-2 was not recognized at all in the ELISA. It is likely that the three modified toxins were generated by Klebsiella specific proteolytic processing of the original pretoxin. These results have important implications for the diagnosis and prevention of heat-stable toxin-induced diarrhea.
authors.affiliation: Department of Pediatrics, University Federico II, Naples, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Albano
authors.forename: M R
authors.initials: MR
authors.lastname: Thompson
authors.forename: S
authors.initials: S
authors.lastname: Orrú
authors.forename: A
authors.initials: A
authors.lastname: Scaloni
authors.forename: A
authors.initials: A
authors.lastname: Musetta
authors.forename: P
authors.initials: P
authors.lastname: Pucci
authors.forename: A
authors.initials: A
authors.lastname: Guarino
mesh_terms: D000907:Antibodies, Bacterial; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D018938:Caco-2 Cells; D004768:Enterotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000378:metabolism; D006801:Humans; D007709:Klebsiella / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D014170:Transformation, Genetic; D014774:Virulence
chemical_list: D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C034151:heat stable toxin (E coli); D006162:Guanylate Cyclase