Porphyromonas gingivalis induces neuroinflammation in a gingipain-dependent manner in zebrafish larvae

Porphyromonas gingivalis (Pg), a keystone pathogen in the development of periodontitis, has been implicated in neurodegenerative diseases such as Alzheimer's disease (AD). The gingipains, Pg crucial virulence factors, have been detected in the brains of AD patients and suggested to be involved in neuroinflammation, which leads to neuronal death. However, the mechanisms underlying Pg brain invasion and its inflammatory effect remain poorly understood. In this study, using a zebrafish larval model, we investigated the gingipain-dependent effects of systemic and local hindbrain infection with Pg, on: (i) neuroinflammation and cell death, (ii) Pg phagocytosis and persistence in the brain, (iii) activation of microglia/macrophages, (iv) cerebral vasculature integrity, and (v) larval behavioural changes. Systemic infection with wild-type Pg W83, but not with the gingipain-null mutant (ΔK/R-ab), resulted in increased bacterial survival in the brain and upregulation of the expression of pro-inflammatory genes both in the brain and periphery. Furthermore, Pg W83 also induced microglia/macrophages activation, as indicated by morphological changes and upregulation of activation marker expression, however number of microglia was reduced upon Pg W83 systemic infection. Finally, alterations in cerebral vasculature and larval locomotor activity were also observed. In contrast, the ΔK/R-ab mutant was rapidly cleared and did not induce inflammatory responses, underscoring the pivotal role of gingipains in Pg survival, microglia activation and neuroinflammation. Interestingly, the direct hindbrain inoculation of Pg induced only a mild, transient inflammation, indicating that systemic dissemination and potentially peripheral inflammation is crucial in the affecting blood-brain barrier and neuroinflammation. In contrast to systemic infection, local infection with Pg W83 or injection of purified gingipains led to increased microglia/macrophages numbers but similarly to systemically administered bacteria, Pg W83 was more effective in activating the microglia/macrophages than the ΔK/R-ab mutant, which was rapidly phagocytosed and cleared. Moreover, during local infection Pg W83, but not ΔK/R-ab, was able to induce cell death in the brains of infected larvae. Collectively, these findings highlight the gingipain-dependent mechanisms of Pg-induced neuroinflammation and emphasize the importance of further investigation into the role of the oral-brain axis in neurodegenerative diseases. In addition, this study highlights the utility of zebrafish larvae as a powerful tool to investigate host immunity-pathogen interactions in the brain.