Porphyromonas gingivalis entry into gingival epithelial cells modulated by Fusobacterium nucleatum is dependent on lipid rafts

Abstract

Host cell invasion by a major periodontal pathogen, Porphyromonas gingivalis, has been proposed as an important mechanism involved in host–pathogen interactions in periodontal and cardiovascular diseases. The present study sought to gain insight into the underlying mechanism(s) involved in previously demonstrated fusobacterial modulation of host cell invasion by P. gingivalis. An immortalized human gingival cell line Ca9-22 was dually infected with P. gingivalis ATCC 33277 and Fusobacterium nucleatum TDC 100, and intracellular invasion was assessed by scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). SEM observation showed that P. gingivalis and F. nucleatum formed consortia and were in the process of penetrating into Ca9-22 by 30–60 min after infection. In CSLM, Ca9-22 cells that contained both P. gingivalis and F. nucleatum were frequently observed after 2 h, although cells that contained exclusively P. gingivalis were also found. Infection by P. gingivalis and/or F. nucleatum revealed evident colocalization with a lipid raft marker, GM1-containing membrane microdomains. In an antibiotic protection assay, depletion of epithelial plasma membrane cholesterol resulted in a significant reduction of recovered P. gingivalis or F. nucleatum (~33% of untreated control; p < 0.001). This inhibition was also confirmed by CSLM. Sequential infection experiments showed that timing of infection by each species could critically influence the invasion profile. Co-infection with F. nucleatum significantly enhanced host cell invasion by P. gingivalis 33277, its serine phophatase SerB mutant and complemented strains, suggesting that the SerB does not play a major role in this fusobacterial enhancement of P. gingivalis invasion. Thus, the interaction between F. nucleatum and host cells may be important in the fusobacterial enhancement of P. gingivalis invasion. Collectively, these results suggest that lipid raft-mediated process is at least one of the potential mechanisms involved in fusobacterium-modulated host cell invasion by P. gingivalis.

Porphyromonas gingivalis Capsule‐Mediated Coaggregation as a Virulence Factor in Mixed Infection With Fusobacterium nucleatum

“…Capsulated Pg in mixed infection displayed a higher capacity for keratinocyte invasion than the non‐capsulated form, whereas elimination of coaggregation reduced capsulated Pg invasion. Also Saito et al 19 found mixed infection by Pg and Fn led to greater invasion of human keratinocytes by Pg (using the non‐capsulated ATCC 33277 strain). These changes may explain both the augmented virulence of mixed infection with capsulated Pg and also its attenuation once coaggregation is eliminated.…”

“…These changes may explain both the augmented virulence of mixed infection with capsulated Pg and also its attenuation once coaggregation is eliminated. Saito et al 19 also reported that Pg invasion in mixed infection was not dependent on SerB, which is crucial for Pg invasion in the monoinfection mode. This highlights the finding that bacterial invasion in mixed infection relies on a mechanism specific to the mixed infection, and that unique interactions (such as coaggregation) may play a critical role in the virulence of mixed infection.…”

Polymicrobial synergy and dysbiosis in inflammatory disease

“…Intracellular bacteria disrupt host cell signaling pathways and gene expression patterns which can have profound effects on immune activity and cellular physiology [37]. Co-operative invasion among periodontal bacteria has been documented, for example consortia of P. gingivalis and F. nucleatum invade GECs in higher numbers than either organism alone [38]. Filifactor alocis and P. gingivalis also exhibit synergistic infection of epithelial cells and dual species invasion elicits a distinct pattern of host cell response [39].…”

Attenuation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway by Porphyromonas gingivalis Gingipains RgpA, RgpB, and Kgp

“…gingivalis-induced Akt Inactivation Is Independent of P. gingivalis Invasion-P. gingivalis is capable of invading gingival epithelial cells and fibroblasts (13,42,43), and the virulence factors produced by P. gingivalis including outer membrane vesicles, which enter into host cells (44), and SerB, which is a serine phosphatase and contributes to P. gingivalis invasion (45), also interacts with host proteins in gingival epithelial cells. Lipid rafts are also involved in P. gingivalis invasion (42,46). To investigate whether Akt dephosphorylation is caused by invading P. gingivalis or endocytosed virulence factors, we employed CytD and MCD.…”