title: Inhibition of anthrax lethal toxin-induced cytolysis of RAW264.7 cells by celastrol.
abstract: BACKGROUND
  Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized.
  
  
  RESULTS
  Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation.
  
  
  CONCLUSIONS
  This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Sarah
authors.initials: S
authors.lastname: Chapelsky
authors.forename: Sarah
authors.initials: S
authors.lastname: Batty
authors.forename: Mia
authors.initials: M
authors.lastname: Frost
authors.forename: Jeremy
authors.initials: J
authors.lastname: Mogridge
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000633:toxicity; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000633:toxicity; D015153:Blotting, Western; D002460:Cell Line; D006868:Hydrolysis; D008264:Macrophages / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D053978:Pentacyclic Triterpenes; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D029757:Tripterygium / Q000737:chemistry; D014315:Triterpenes / Q000494:pharmacology*
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D053978:Pentacyclic Triterpenes; D014315:Triterpenes; C030325:anthrax toxin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C050414:celastrol