Bacillus anthracis is the etiological agent of anthrax disease. The life cycle of this endospore-forming bacterium fluctuates between metabolically inactive sporulation phase and metabolically active vegetative phase. The sporulation phase is characterized by the high resistance of endospores to very harsh conditions. These endospores are taken up by the host where they germinate into the vegetative form of the bacterium, an essential step to mount a systemic infection. Germination of the anthrax bacterium requires binding of specific germinants to specific germination receptors present on the surface of the endospores. While germinants that trigger germination of B. anthracis endospores within the infected host remain to be identified, powerful germinants have been identified that trigger germination of endospores in cell-free systems. For example, it has been shown that a combination of specific nucleosides and amino acids, i.e. inosine and alanine, efficiently germinate B. anthracis spores in vitro. We screened nucleoside analogues and identified the inosine analog 6-thioguanosine (6-TG) that efficiently blocked in vitro germination of B. anthracis spores. We further demonstrated that 6-thioguanosine also prevented macrophage killing mediated by B. anthracis spores. As germination is the initial step required for a systemic infection, the inosine analog has therefore therapeutic potential as an early intervention agent.;After germination inside the host phagocytic vacuoles, Bacillus anthracis produces a number of virulence factors that enables the bacterium to establish a systemic infection resulting in disease progression of the infected host. Anthrax lethal toxin (LT) is the main virulence factor of B. anthracis linked to morbidity and mortality of systemic anthrax. In fact, rodents injected with LT develop similar disease symptoms as those infected with B. anthracis spores. The mechanism of how LT triggers disease is still unclear. However, it has been shown that LT kills specific target in a strain-dependent fashion. The LT susceptibility of murine cells is controlled by nucleotide oligomerization domain (NOD)-like receptors (NLRs). Mice expressing a specific allele of the NLR protein Nalp1b are highly susceptible to LT killing. In recent years it has been shown that multiple microbial pathogens activate specific NLRs resulting in caspase-1 activation. In fact, LT killing of susceptible macrophages is caspase-1-dependent. However, it is unclear how activation of NLRs results in cell death and disease progression by microbial pathogens. Several NLRs have been described to form high-molecular complexes, a so-called inflammasome complex, following stimulation by microbial components. In order to analyze processes triggered by LT, we tested whether the anthrax toxin also stimulated the formation of an inflammasome complex. In this study, we combined biochemical and cell biology tools to identify the molecular size, proteins composition, and subcellular localization of a putative inflammasome complex in LT-treated macrophages. We found that in unstimulated murine macrophages, caspase-1 is part of a ∼200 kDa complex and Nalp1b part of a of a high-molecular (∼800 kDa) complex. We demonstrated that LT treatment of these cells resulted in caspase-1 recruitment to the high-molecular (Nalp1b)-containing complex, concurrent with processing of cytosolic caspase-1 substrate IL-18. Using subcellular fractionation techniques we further demonstrated that Nalp1b and caspase-1 are membrane associated. Intriguingly, both caspase-1 and Nalp1b were membrane-associated, while the caspase-1 substrate interleukin-18 was cytosolic. Consistent with formation of an inflammasome complex, we showed that Nalp1b and caspase-1 are able to directly interact with each other. Caspase-1-associated inflammasome components included, besides Nalp1b, the pro-inflammatory caspase-11 and the caspase-1 substrate, alpha-enolase. Taken together, our findings suggest that LT triggers the formation of a membrane-associated inflammasome complex in murine macrophages resulting in cleavage of cytosolic caspase-1 substrates and cell death. Moreover, we optimized the conditions for reconstituting the minimal required components for Nalp1b inflammasome formation, procaspase-1 and Nalp1b, in 293T cells. We found that Anthrax lethal toxin is not interacting directly with Nalp1b and did not alone enhance the reconstituting inflammasome complex formation in 293T cells. Therefore, we assume that LT induces an inflammasome complex via cleavage of a macrophage protein acting as an endogenous dangerous signal in the susceptible murine macrophages.
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