title: Identification of a CHO cell-elongating factor produced by Vibrio cholerae O1.
abstract: Vibrio cholerae strains with all known toxin genes deleted or inactivated still cause diarrhoea in some volunteers, suggesting the presence of an unknown virulence factor or factors. Lysozyme-EDTA treated cells of JBK70, a genetically manipulated cholera toxin negative strain of Vibrio cholerae O1, biotype El Tor, release a factor that causes elongation of Chinese hamster ovary (CHO) cells. CHO cell-elongating toxin (Cef) was purified by FPLC chromatography (anion exchange; Q Sepharose High Performance) followed by 2D electrophoresis (isoelectric focusing gel, IEF; pH 3-9 and SDS-PAGE, 8-25% gradient gel). Partly purified toxin (anion exchange or IEF-eluted concentrate) caused fluid accumulation in sealed infant mice suggesting that Cef shows some properties of an enterotoxin. On SDS-PAGE (8-25%) and IEF (pH 2.5-5.0) gels, CHO cell activity was associated with a single band at 85 kDa and a pI of 3.8, respectively. A unique amino terminal sequence, XGDETNSSGASTEVVYESYIQQ, was determined by automated Edman degradation of gel-purified protein. The unique molecular mass, N-terminal sequence and activity on CHO cells indicate that this factor is not zonula occludens toxin (Zot) or accessory cholera enterotoxin (Ace) or the Hly A haemolysin. Partly purified Cef did not increase cyclic AMP or prostaglandin E(2)levels in CHO cells which suggests that its mechanism of action differs from that of cholera toxin.
authors.affiliation: U.S. Food and Drug Administration, Division of Virulence Assessment, 200 C. St. SW, Washington, DC 20204, USA. bmccarde@bangate.fda.gov
authors.forename: B A
authors.initials: BA
authors.lastname: McCardell
authors.forename: M H
authors.initials: MH
authors.lastname: Kothary
authors.forename: R H
authors.initials: RH
authors.lastname: Hall
authors.forename: V
authors.initials: V
authors.lastname: Sathyamoorthy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000833:Animals, Suckling; D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D016466:CHO Cells; D048429:Cell Size; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000737:chemistry / Q000302:isolation & purification; D007525:Isoelectric Focusing; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D014734:Vibrio cholerae / Q000737:chemistry / Q000378:metabolism / Q000472:pathogenicity*; D014774:Virulence
chemical_list: D001427:Bacterial Toxins; C412287:cell-elongating factor, Vibrio cholerae; D000242:Cyclic AMP; D004950:Esterases; D015232:Dinoprostone