Subtilase cytotoxin from Shiga-toxigenic Escherichia coli impairs the inflammasome and exacerbates enteropathogenic bacterial infection

Non-canonical inflammasome activation analysis in a mouse model of Citrobacter rodentium infection

“…For example, when we investigated the pathogenicity of the virulence factor (VF) subtilase cytotoxin (SubAB), we used the less susceptible C57BL/6J mice (female, 8 weeks old) and administered SubAB-expressing C. rodentium (Cr-SubABwt) at a high dose of 5 × 10 9 CFUs ( Tsutsuki et al., 2022 ). In another report, a dose of 2 × 10 9 CFUs per mouse was used when investigating a host immune response with specific knockout mice bred in a C57BL/6 background ( Guo et al., 2015 ).…”

“… Timing: 7 days Preparation of an expression vector containing the VF of interest. Note: This section introduces preparation of C. rodentium (Cr) transformants for infection of mice as reported in a previous publication in which we used C57BL/6J mice and Cr-SubABwt (wild-type SubAB), Cr-SubABmt (catalytically inactive mutant SubAB), and Cr-empty (vector control Cr transformants) ( Tsutsuki et al., 2022 ). Here, we provide a protocol that uses SubAB as a representative VF.…”

“…Here, we provide a protocol detailing the approaches for non-canonical inflammasome analysis in a mouse model of C. rodentium infection, including preparation of bacteria, oral administration of bacteria to mice, counting colony-forming units to quantify bacterial colonization, and analysis of expression and activation of inflammasome-related factors. For complete details on the use and execution of this protocol, please refer to Tsutsuki et al. (2022) .…”

“…For complete details on the use and execution of this protocol, please refer to Tsutsuki et al. (2022) .…”

Controlled Delivery of an Anti-Inflammatory Toxin to Macrophages by Mutagenesis and Nanoparticle Modification

“…After translocation into the ER, SubAB cleaves BiP and results in ER stress-induced cytotoxicity through activation of ER stress sensors protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6) that mediate a unique signaling pathway including eukaryotic initiation factor 2α (eIF2α) phosphorylation and C/EBP homologous protein (CHOP) expression [30,31]. Several studies have demonstrated that SubAB induces cytotoxicity, stress granule formation [31], inhibition of autophagy [32] and inflammasome [33] in vitro, and lethal severe hemorrhagic inflammation and enteropathogenic bacterial infection in mice [34]. Additionally, SubAB inhibits lipopolysaccharide (LPS)-induced nitric oxide (NO) production in macrophages through suppression of nuclear factor kappa-B (NF-κB) activation and subsequent inducible NO synthase (iNOS) expression [35].…”