Mechanistic Insights into Cronobacter Sakazakii-Induced Neonatal Meningitis Via TLR4-NLRP3-Mediated Pyroptosis Pathway

Background: Cronobacter sakazakii (C. sakazakii) is a Gram-negative pathogen that invades the gastrointestinal tract and spreads hematogenously, leading to bacterial meningitis in premature and low-birth-weight neonates, posing a significant threat to neonatal health. However, the exact mechanisms by which C. sakazakii breaches the blood-brain barrier (BBB) and induces inflammation remain poorly understood. This study aims to elucidate the signaling pathways underlying C. sakazakii-induced neonatal meningitis using in vitro and in vivo models. Methods: We utilized human brain microvascular endothelial cells (HBMECs), BV2 microglia cells, C57BL/6 neonatal mice, and NLRP3-/- mice to establish models of C. sakazakii-induced meningitis. In vitro assays comprised cell adhesion and invasion experiments, transmembrane resistance measurements, immunofluorescence to assess tight junctions, flow cytometry to evaluate HBMEC cell viability, and Western blot to examine changes in pyroptosis-related proteins in BV2 cells. In vivo experiments involved assessing C. sakazakii penetration through the BBB by cerebrospinal fluid (CSF) plating, immunohistochemistry for Neun protein expression in brain tissue, and Western blot for changes in pyroptosis-related proteins in brain tissue. Results: Our in vitro findings demonstrated that C. sakazakii adheres to and invades HBMECs, reducing transmembrane resistance and disrupting tight junctions. This bacterial action induced pyroptosis in BV2 cells. In vivo experiments showed that C. sakazakii crosses the BBB, colonizes the CSF, and induces meningitis in neonatal mice. The mechanism proposed involves C. sakazakii-stimulated expression of TLR4 on the cell surface, activation of MyD88, and subsequent nuclear translocation of NF-κB, which induces the expression of NLRP3 inflammasome-related proteins. This leads to the activation of GSDMD, induces pyroptosis, and the subsequent release of the inflammatory cytokine IL-1β, culminating in the development of meningitis. Conclusion: This study reveals that C. sakazakii disrupts tight junction proteins and breaches the BBB, activating the TLR4-NLRP3-mediated pyroptosis pathway in the brain. This mechanism induces pyroptosis and the release of the inflammatory cytokine IL-1β, ultimately leading to the induction of neonatal meningitis.