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.Methodology/Principal FindingsHere, 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/SignificanceThis work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.

Efficient approaches for characterizing ubiquitinated proteins

“…This work resulted in data indicating that several proteasomal subunits are substrates for ubiquitination, a fact that corresponds well with results from a study where the purification strategy was based on the use of anti-ubiquitin antibody [36]. The use of the S5a UIMs can further be exemplified by several studies, such as the detection of higher levels of ubiquitinated proteins in dilated cardiomyopathy [37], the implication of ubiquitin in protein turnover as a control of synapse remodelling and signalling [38] and to probe the mechanism of action of the anthrax lethal toxin inhibitor, celastrol [39]. In a recent proteomic study performed in plants, regions from two Arabidopsis proteins containing triple UIMs (Arabidopsis S5a orthologue) and double UBAs (Arabidopsis UBP14) respectively were used to pull down ubiquitinated substrates in Arabidopsis thaliana [40].…”

Proteasome Inhibitors Prevent Caspase-1-Mediated Disease in Rodents Challenged with Anthrax Lethal Toxin

“…4,7,13,16 We also demonstrated that LT-mediated caspase-1 activation and subsequent necrosis in susceptible murine macrophages are controlled by proteasome activity. [5][6][7]17 In fact, proteasome inhibitors are the most efficient inhibitors of LT/caspase-1-mediated macrophage killing identified. [5][6][7]17,18 As observed in mice, the susceptibility to LT in rats is strain-dependent.…”

“…[5][6][7]17 In fact, proteasome inhibitors are the most efficient inhibitors of LT/caspase-1-mediated macrophage killing identified. [5][6][7]17,18 As observed in mice, the susceptibility to LT in rats is strain-dependent. Rats are divided into susceptible strains that are rapidly killed after LT challenge, and strains that are resistant to rapid disease progression.…”

Anthrax and the inflammasome

“…Examples include reports showing a heat shock-induced trapping of pro-caspase-1 in a large complex, therefore preventing its autoproteolytic activation [45], celesterol-induced inhibition of proteasome activity [60], auranofin-mediated inhibition of caspase-1 activity [47], as well as a role of idebenone, which acts as an inhibitor of potassium channels, and synergizes with auranofin for increased protection [47]. Inhibition of cathepsin B activity with drugs such as CA-074Me can also inhibit LT's activation of the Nlrp1 inflammasome and cell death [61,46].…”