Purification and characterization of an enterotoxin from Bacteroides fragilis.
An enterotoxin produced by Bacteroides fragilis was purified to homogeneity and characterized as to its biological activity and basic molecular properties. Toxin preparations were prepared by growing B. fragilis VPI 13784 in brain heart infusion broth to early stationary phase, immediately precipitating the culture supernatant fluid with 70% ammonium sulfate, and stabilizing the precipitate with the protease inhibitor TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone). The toxin was sequentially purified by anion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on phenyl-agarose, and high-resolution ion-exchange chromatography on Mono Q. The toxin appeared homogeneous as judged by polyacrylamide gel electrophoresis. The estimated molecular weight of the highly purified toxin as determined by gel filtration chromatography on Superose-12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 19,000. It has an isoelectric point of approximately 4.5 and is stable at pHs 5 to 10. The purified toxin is stable at -20 and 4 degrees C and upon freeze-drying, but it is unstable at temperatures above 55 degrees C. It is sensitive to proteinase K and Streptomyces protease but is resistant to trypsin and chymotrypsin. The activity of the purified toxin is neutralized by antiserum to a toxigenic strain of B. fragilis but not by antiserum to nontoxigenic strains. N-terminal amino acid analysis reveal an unambiguous sequence of Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly- Ser-Thr . The highly purified toxin induced a strong fluid accumulation response in the lamb ileal-loop assay as well as a cytotoxic response (cell rounding) on HT-29 colon carcinoma cells. Thus, the purified toxin can cause both enterotoxic and cytotoxic activities.
Van Tassell
R L
RL
Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Lyerly
D M
DM
Wilkins
T D
TD
D000818:Animals; D001441:Bacteroides fragilis; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D006358:Hot Temperature / Q000009:adverse effects; D006863:Hydrogen-Ion Concentration; D007082:Ileum / Q000187:drug effects; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009500:Neutralization Tests; D011817:Rabbits; D012756:Sheep
D004768:Enterotoxins