Extracellular pneumolysin enhances the activation of cytosolic pattern-recognition receptor NOD2 through its...

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, meningitis, and sepsis. This bacterium produces pneumolysin, a cholesterol-dependent cytolysin that forms oligomeric transmembrane pores in the host cells. Although pneumolysin is known to exhibit proinflammatory properties, the mechanisms by which it activates innate immune responses remain to be investigated. Here, we show that extracellular pneumolysin enhances the activation of nucleotide-binding oligomerization domain 2 (NOD2), a cytosolic receptor that recognizes bacterial peptidoglycans. Experiments using HEK-Blue cell lines expressing specific pattern recognition receptors revealed that pneumolysin does not directly activate Toll-like receptors or other innate immune receptors. In contrast, our findings suggest that pneumolysin-generated membrane pores may facilitate the cytosolic entry of peptidoglycan, thereby contributing to an elevated activation of NOD2. Furthermore, pneumolysin enhances NOD1 activation in cells stimulated with a NOD1 ligand, suggesting a broader role for pore-forming toxins in innate immunity. These findings shed light on S. pneumoniae-modulated immune modulation and highlight toxin-induced immune pathways as potential therapeutic targets.IMPORTANCEThe mechanisms by which the pneumococcal pore-forming toxin pneumolysin activates innate immune responses have not been fully understood. Specifically, it remains unclear whether pneumolysin is directly sensed by Toll-like receptor 4 (TLR4) or activates the NLRP3 inflammasome. Here, we show that pneumolysin is not a direct ligand for pattern-recognition receptors. Instead, pneumolysin forms membrane pores that increase plasma membrane permeabilization, thereby amplifying innate immune signaling through multiple pathways. These pores may provide a route for the cytosolic entry of pneumococcal peptidoglycan, contributing to enhanced activation of the cytosolic receptor nucleotide-binding oligomerization domain 2 (NOD2). Additionally, membrane pores may promote the extracellular release of damage-associated molecular patterns, such as high mobility group box 1 (HMGB1), providing a mechanistic explanation for previously reported pneumolysin-induced TLR4 activation. Furthermore, pore-induced ion efflux provides a framework to explain previously reported NLRP3 inflammasome activation. Together, our findings establish membrane permeabilization as a central mechanism by which pneumolysin modulates innate immune sensing during pneumococcal infection.