Journal of oral microbiology · 2025
Recognition of Porphyromonas gingivalis lipopolysaccharide by human caspase-4 depends on lipopolysaccharide purity and guanylate-binding protein 1.
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Domae Eisuke, Mori Taiki, Hanaoka Mariko, Into Takeshi
Аннотация
BACKGROUND: Porphyromonas gingivalis, a key pathogen in chronic periodontitis, produces heterogeneous lipopolysaccharides (LPS) that are weakly recognized by Toll-like receptor 4 (TLR4). Human cells detect cytosolic LPS via caspase-4 (CASP4); however, whether P. gingivalis LPS (PgLPS) can directly activate CASP4 remains unclear. This study aimed to investigate the CASP4-activating properties of purified PgLPS.
METHODS: Standard-grade (ST) and ultrapure-grade (UP) purified PgLPS were tested for CASP4 activation using recombinant CASP4 in fluorogenic and IL-18 assays. PgLPS-CASP4 interactions were examined by pull-down. In HSC-2 cells, IL-18 maturation, gasdermin D cleavage, pyroptosis, and dependence on CASP4 or TLR4 were assessed after cytosolic LPS delivery. Guanylate-binding protein-1 (GBP1) involvement was evaluated by siRNA knockdown. Effects of outer membrane vesicles (OMVs) were also tested.
RESULTS: ST-PgLPS directly activated recombinant CASP4, while UP-PgLPS did not. Intracellular delivery of ST-PgLPS induced weak pyroptosis, while UP-PgLPS had no effect. Responses were abrogated by CASP4 inhibition but not by TLR4 inhibition. IFN-γ priming enhanced responses: ST-PgLPS induced moderate activation, whereas UP-PgLPS triggered robust, GBP1-dependent activation. OMVs activated CASP4 in IFN-γ-primed cells.
CONCLUSION: CASP4 activation depends on PgLPS purity and host cell priming. GBP1 contributes to PgLPS sensing, and OMVs serve as vehicles for PgLPS delivery to the cytosol.
METHODS: Standard-grade (ST) and ultrapure-grade (UP) purified PgLPS were tested for CASP4 activation using recombinant CASP4 in fluorogenic and IL-18 assays. PgLPS-CASP4 interactions were examined by pull-down. In HSC-2 cells, IL-18 maturation, gasdermin D cleavage, pyroptosis, and dependence on CASP4 or TLR4 were assessed after cytosolic LPS delivery. Guanylate-binding protein-1 (GBP1) involvement was evaluated by siRNA knockdown. Effects of outer membrane vesicles (OMVs) were also tested.
RESULTS: ST-PgLPS directly activated recombinant CASP4, while UP-PgLPS did not. Intracellular delivery of ST-PgLPS induced weak pyroptosis, while UP-PgLPS had no effect. Responses were abrogated by CASP4 inhibition but not by TLR4 inhibition. IFN-γ priming enhanced responses: ST-PgLPS induced moderate activation, whereas UP-PgLPS triggered robust, GBP1-dependent activation. OMVs activated CASP4 in IFN-γ-primed cells.
CONCLUSION: CASP4 activation depends on PgLPS purity and host cell priming. GBP1 contributes to PgLPS sensing, and OMVs serve as vehicles for PgLPS delivery to the cytosol.