Prevention of anthrax cytopathicity by inhibition of spore germination and caspase-1 activation
Squires, Raynal C.
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Spores of Bacillus anthracis are the etiological agents of anthrax disease in mammals. Spore germination into vegetative cells and subsequent release of the bacterium's potent toxins are essential steps in anthrax pathogenicity. Lethal toxin (LT), the main virulence factor released, kills host antigen-presenting cells (APCs), leading to unchecked bacterial growth and eventual morbidity. Here we show that we have identified compounds that block spore germination as well as gained critical insight into the mechanism of cytopathicity caused by LT.;Germination of B. anthracis spores allows the transition to the vegetative form of the bacterium. This process can be triggered in vitro by the common germinants inosine and alanine. As inosine is critical for germination, we screened inosine analogs for the ability to interfere with this process in B. anthracis spores. We identified seven analogs that efficiently blocked germination in vitro and one analog, 6-thioguanosine (6-TG), that also efficiently blocked spore germination in macrophages and prevented killing of these cells mediated by B. anthracis spores. 6-TG therefore shows potential as an anti-anthrax therapeutic agent.;Following germination, vegetative bacteria release the virulence factor LT, which kills host APCs. In the animal model for anthrax disease, mice and their APCs differ in their response to LT in a strain-specific manner. The most striking response of APCs to LT is rapid, caspase-1-mediated necrosis governed by specific isoforms of the inflammasome protein Nalp1b. We found that high extracellular potassium and caspase-1 inhibitors efficiently block this necrosis and that caspase-1 inhibitors could reverse damage to metabolic function and membrane integrity in LT-treated macrophages. Remarkably, proteasome inhibitors also facilitated this reverse. Unlike caspase-1 inhibitors however, proteasome inhibitors could not block caspase-1 activation and necrosis triggered by lipopolysaccharide and nigericin, suggesting that the proteasomal control of caspase-1 activity in LT killing is indirect. Caspase-l-mediated necrosis therefore appears to be differentially regulated depending on the source of caspase-1 induction.;Taken together, we identified several compounds that prevent killing of murine macrophages by blocking either germination of B. anthracis spores or by preventing caspase-1 activation in LT-treated cells. The therapeutic potential of these agents remains to be determined.