Porphromonas gingivalis infection induces gingipain-dependent changes in the brain vasculature of zebrafish larvae

Recent studies link infections with blood-brain barrier (BBB) dysfunctions, neuroinflammation and subsequent neurodegeneration. Here, we employed a zebrafish larval model to study the impact of systemic infection with an oral pathogen Porphyromonas gingivalis (Pg) and its major virulence factors - gingipains, on BBB integrity and cerebral vasculature. We demonstrated that systemic infection with wild-type Pg W83 significantly increased BBB permeability in zebrafish larvae, as evidenced by the extravasation of tracers into the brain parenchyma. This effect was absent in larvae infected with a gingipain-deficient mutant bacteria (ΔK/R-ab), indicating a pivotal role for gingipains in BBB disruption. Immunohistochemical analysis revealed a marked reduction in the expression of tight junction (TJ) proteins: Claudin-5 and Zo-1 on the cerebral vessels of Pg W83-infected larvae, while expression of genes encoding TJ proteins (cldn5a/b and tjp1a/b) was not changed, suggesting post-translational degradation as the primary mechanism. To verify the individual contributions of gingipains, larvae were injected with purified arginine- (RgpA and RgpB) or lysine- (Kgp) specific proteases. In larvae treated with RgpB and Kgp, but not those treated with RgpA, there was increased BBB permeability, loss of TJ proteins, and cerebrovascular changes. Among these, Kgp exerted the most pronounced effects, emphasizing its dominant role in Pg-mediated vascular destruction. Our findings for the first time provide in vivo evidence that Pg compromises BBB integrity via gingipain-dependent degradation of tight junction proteins on the cerebral vessels. Zebrafish larvae offer a robust model for studying Pg-BBB interactions and may support the development of therapeutic strategies targeting gingipains to preserve cerebrovascular integrity in neurodegenerative disease.