Salmonella virulence factor sptP activates the NLRP3/Caspase-1 pathway to induce pyroptosis and exacerbate intestinal...

Salmonella enterica serovar Enteritidis (SE) is a major foodborne pathogen causing severe intestinal diseases in humans and animals, posing significant global public health and economic burdens. The Salmonella protein tyrosine phosphatase (sptP), a type III secretion system effector and critical SE virulence factor, contributes to pathogenesis, but its precise role in SE-induced intestinal injury remains unclear. Specifically, whether sptP modulates the NLRP3/Caspase-1 inflammasome (a central inflammatory regulator) is undefined. To address this, we constructed an sptP gene deficient SE strain (sptP^-) via CRISPR/Cas9, verified by PCR and sequencing. Given that MCC950 is a selective NLRP3 inflammasome inhibitor, we designed an animal study to examine the contribution of sptP to SE pathogenicity in chicks and its potential interplay with NLRP3 activation. Chicks were randomly divided into 5 groups (n = 10/group): SE sptP⁺ (wild-type), sptP⁻ (mutant), sptP⁺+MCC950 (NLRP3 inhibitor), sptP⁻+MCC950, and PBS control. Ileal tissues were collected 24 h post-infection. Histopathological injury was scored via H&E staining. Bacterial burdens in the ileum and spleen were quantified by plate counting. Cytokine levels (IL-1β, IL-18) were measured using ELISA and RT‑PCR. Protein expression (pro‑caspase‑1, cleaved‑caspase‑1, GSDMD, GSDMD‑N) was analyzed by Western blot, and pyroptosis was quantified via LDH release assay. The sptP mutant (sptP^-) was successfully generated without unintended mutations. The sptP⁻ infection reduced IL-1β/IL-18 expression, cleaved-caspase-1 and GSDMD-N levels, and significantly diminished pyroptosis. MCC950 pretreatment markedly attenuated pathology in sptP⁺-infected chicks, including reduced Caspase-1/GSDMD cleavage, lower inflammatory cytokines, decreased pyroptosis, and alleviated intestinal damage. Notably, no significant differences in pathology, cytokines, or pyroptosis markers were observed between sptP⁻ and sptP⁻+MCC950 groups. Importantly, bacterial loads did not differ across groups, excluding colonization differences as a confounding factor. These findings demonstrate that sptP promotes SE-induced intestinal injury by activating the host NLRP3/Caspase-1 inflammasome pathway, driving inflammatory cytokine release and pyroptosis. This study clarifies a previously undefined mechanism by which sptP exacerbates SE-associated intestinal damage, addressing the knowledge gap regarding its regulation of the NLRP3 inflammasome.