Secreted gingipains from Porphyromonas gingivalis increase permeability in human cerebral microvascular endothelial cells through intracellular degradation of tight junction proteins

• Published in: Neurochemistry international Vol. 154; p. 105282
• Main Authors: Nonaka, Saori, Kadowaki, Tomoko, Nakanishi, Hiroshi
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.03.2022
• Subjects: Adhesins, Bacterial - metabolism; Endothelial Cells - metabolism; Gingipain; Gingipain Cysteine Endopeptidases; hCMEC/D3 cell; Humans; Occludin; Outer membrane vesicles; Permeability; Porphyromonas gingivalis; Porphyromonas gingivalis - metabolism; Tight Junction Proteins; Zonula occludens-1; BBB; Zonula occludens-1; Na–F; Occludin; P. gingivalis; ANOVA; OD; FBS; OMVs; DIC; Porphyromonas gingivalis; PBS; AD; SDS; Gingipain; MOI; Outer membrane vesicles; PAGE; MMP; ERK1/2; ZO-1; PAR2; Kgp; HA; hCMEC/D3 cell; Rgp
• ISSN: 0197-0186; 1872-9754
• DOI: 10.1016/j.neuint.2022.105282

Despite a clear correlation between the infiltration of periodontal pathogens in the brain and cognitive decline in Alzheimer's disease (AD), the precise mechanism underlying bacteria crossing the blood-brain barrier (BBB) remains unclear. The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteases termed gingipains. Gingipains appear to be key virulence factors that exacerbate sporadic AD. We herein report that gingipains are involved in increasing permeability of hCMEC/D3 cell monolayer, human cerebral microvascular endothelial cell lines, through degradation of tight junction proteins including Zonula occludens-1 (ZO-1) and occludin. There was a significant decrease in the mean protein levels of ZO-1 and occludin after infection of hCMEC/D3 cells with wild-type (WT) P. gingivalis. However, infection of these cells with a gingipain-deficient P. gingivalis strain showed significantly lower reduction of the mean protein levels of either ZO-1 and occludin, compared to the WT strain. Similar results were obtained after treatment with culture supernatant from WT and gingipain-deficient P. gingivalis strains. In vitro digestion of human recombinant ZO-1 and occludin by WT P. gingivalis culture supernatant in the absence or presence of gingipain inhibitors indicated that gingipains directly degraded these tight junction proteins. A close immunohistochemical examination using anti-gingipain antibody further revealed that gingipains localized in the cytosol and nuclei of hCMEC/D3 cells after infection with WT P. gingivalis and treatment with its culture supernatant. Furthermore, intracellular localization of outer membrane vesicles (OMVs) bound gingipains from WT P. gingivalis and OMV-induced degradation of ZO-1 and occludin were also observed in hCMEC/D3 cells. Thus, the delivery of gingipains into the cerebral microvascular endothelial cells, probably through OMV, may be responsible for the BBB damage through intracellular degradation of ZO-1 and occludin. •Gingipains increased permeability of human cerebral endothelial cell monolayer.•Gingipains directly degraded tight junction proteins, ZO-1 and occludin.•PAR2 activation was not associated with the degradation of ZO-1 and occludin.•Intracellular localization of gingipains was found after P. gingivalis infection.•P. gingivalis OMVs deliver gingipains into cerebral endothelial cells.