Invasion of Aortic and Heart Endothelial Cells by Porphyromonas gingivalis

Abstract

Invasion of host cells is believed to be an important strategy utilized by a number of pathogens, which affords them protection from the host immune system. The connective tissues of the periodontium are extremely well vascularized, which allows invading microorganisms, such as the periodontal pathogen Porphyromonas gingivalis, to readily enter the bloodstream. However, the ability of P. gingivalis to actively invade endothelial cells has not been previously examined. In this study, we demonstrate that P. gingivalis can invade bovine and human endothelial cells as assessed by an antibiotic protection assay and by transmission and scanning electron microscopy. P. gingivalis A7436 was demonstrated to adhere to and to invade fetal bovine heart endothelial cells (FBHEC), bovine aortic endothelial cells (BAEC), and human umbilical vein endothelial cells (HUVEC). Invasion efficiencies of 0.1, 0.2, and 0.3% were obtained with BAEC, HUVEC, and FBHEC, respectively. Invasion of FBHEC and BAEC by P. gingivalis A7436 assessed by electron microscopy revealed the formation of microvillus-like extensions around adherent bacteria followed by the engulfment of the pathogen within vacuoles. Invasion of BAEC by P. gingivalisA7436 was inhibited by cytochalasin D, nocodazole, staurosporine, protease inhibitors, and sodium azide, indicating that cytoskeletal rearrangements, protein phosphorylation, energy metabolism, andP. gingivalis proteases are essential for invasion. In contrast, addition of rifampin, nalidixic acid, and chloramphenicol had little effect on invasion, indicating that bacterial RNA, DNA, and de novo protein synthesis are not required for P. gingivalisinvasion of endothelial cells. Likewise de novo protein synthesis by endothelial cells was not required for invasion by P. gingivalis. P. gingivalis 381 was demonstrated to adhere to and to invade BAEC (0.11 and 0.1% efficiency, respectively). However, adherence and invasion of the corresponding fimA mutant DPG3, which lacks the major fimbriae, was not detected. These results indicate thatP. gingivalis can actively invade endothelial cells and that fimbriae are required for this process. P. gingivalisinvasion of endothelial cells may represent another strategy utilized by this pathogen to thwart the host immune response.

Blockade of protease-activated receptors on T cells correlates with altered proteolysis of CD27 by gingipains ofPorphyromonas gingivalis

“…In this study, we reported that PAR-4 is strongly expressed on resting T cells; therefore, cleavage and activation of PAR-4 by RgpA could be an important early event. Findings from the present study support this contention: (1) PAR-1, -2 and -4 expression on the cell surface was up-regulated by RgpA; (2) up-regulation of CD69 on T cells by gingipain alone was inhibited by blocking peptide against PAR-2. These results indicating the capacity of RgpA to activate PAR-2 on T cells are in agreement with previous reports [25,26] that PAR-2 plays a crucial role in the local inflammatory reaction within the pathological periodontal pocket.…”

Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells

“…The ability of microbial pathogens, such as P. gingivalis and C. pneumoniae, to potentiate atherosclerosis by establishment of an inflammatory state within the endothelium, is receiving increased attention. Previous reports have established that bacterial infection of endothelial cells stimulates inflammatory markers such as tissue factor, IL-6, chemokines and CAMs (Fryer et al, 1997;Deshpande et al, 1998;Molestina et al, 1998;Dechend et al, 1999;Khlgatian et al, 2002). In this study, we have utilized a series of P. gingivalis fimbria mutants to demonstrate that the expression of 41 kDa (major) fimbria is required for efficient invasion of HAEC.…”

“…Invasion of HAEC cells by P. gingivalis strains was quantified by determining the number of cfu following infection at a multiplicity of infection (moi) of 100. HAEC cells were incubated with P. gingivalis strains for 1, 2 and 6 h followed by treatment with metronidazole for killing of adherent external bacteria as previously described (Deshpande et al, 1998). For 'enhanced rate of infection' assays only, bacteria were centrifuged onto HAEC monolayers (350 g for 5 min) as described (Walter et al, 2004).…”

Lipid raft-dependent uptake, signalling and intracellular fate ofPorphyromonas gingivalisin mouse macrophages

“…On the other hand, the inhibitory effects of MCD could not be attributed to its ability to suppress P. gingivalis phagocytosis. Indeed, pretreatment with cytochalasin D, which prevents phagocytosis by blocking actin polymerization (Deshpande et al, 1998), had no significant influence on P. gingivalis-induced cell activation (Fig. 5).…”

“…Quantification of the degree of colocalization under the different experimental conditions examined revealed that P. gingivalis was about five times less likely to colocalize with lipid rafts in cholesterol-depleted cells than in untreated or cholesterol-reconstituted cells (Table 1). Although cytochalasin D is known to inhibit phagocytosis (Deshpande et al, 1998), it had no effect on the ability of P. gingivalis to colocalize with lipid rafts ( Fig. 2D and Table 1).…”