Role for Gingipains inPorphyromonas gingivalisTraffic to Phagolysosomes and Survival in Human Aortic Endothelial Cells

Abstract

Gingipains are cysteine proteinases that are responsible for the virulence of Porphyromonas gingivalis. Recent studies have shown that P. gingivalis is trapped within autophagic compartments of infected cells, where it promotes survival. In this study we investigated the role of gingipains in the intracellular trafficking and survival of this bacterium in human aortic endothelial cells and any possible involvement of these enzymes in the autophagic pathway. Although autophagic events were enhanced by infection with either wild-type (WT) P. gingivalis strains (ATCC 33277, 381, and W83) or an ATCC 33277 mutant lacking gingipains (KDP136), we have found that more than 90% of intracellular WT and KDP136 colocalized with cathepsin B, a lysosome marker, and only a few of the internalized cells colocalized with LC3, an autophagosome marker, during the 0.5-to 4-h postinfection period. This was further substantiated by immunogold electron microscopic analyses, thus implying that P. gingivalis evades the autophagic pathway and instead directly traffics to the endocytic pathway to lysosomes. At the late stages after infection, WT strains in phagolysosomes retained their double-membrane structures. KDP136 in these compartments, however, lost its double-membrane structures, representing a characteristic feature of its vulnerability to rupture. Together with the ultrastructural observations, we found that the number of intracellular viable WT cells decreased more slowly than that of KDP136 cells, thus suggesting that gingipains contribute to bacterial survival, but not to trafficking, within the infected cells.

Cell entry and exit by periodontal pathogen via recycling pathway

“…7 On the other hand, no bacteria were found in the cytoplasmic spaces of endothelial cells. 11,12 We showed that P. gingivalis is internalized in early endosomes and about half of the intracellular bacteria were found to be sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes. 6 Intracellular trafficking of P. gingivalis may depend on the phenotypic and cells in a mechanism of cell-to-cell spreading (Fig.…”

“…Intracellular P. gingivalis reportedly localizes in various cellular compartments, such as the cytoplasm, 7-10 endosomes, [8][9][10][11][12] and autophagosomes, [11][12][13] which are distinctive double-membraned vacuoles that are part of the autophagic pathway, with autophagosome localization noted to occur in endothelial and smooth muscle cells, 11 but not in gingival epithelial cells. 7 On the other hand, no bacteria were found in the cytoplasmic spaces of endothelial cells.…”

Transglutaminase 2 is essential for adherence of Porphyromonas gingivalis to host cells

“…The mouse NIH/3T3 fibroblast cell line was purchased from the American Type Culture Collection (ATCC) and cultivated in DMEM supplemented with 10% FCS. P. gingivalis strain ATCC 33277 is fimbriated and has high gingipain activity; both virulence factors are required for successful attachment and internalization (47)(48)(49). P. gingivalis was cultured as described previously (7).…”

Potential Value of a Rice Protein Extract, Containing Proteinaceous Inhibitors against Cysteine Proteinases fromPorphyromonas gingivalis, for Managing Periodontal Diseases

“…Gingipains consist of two molecular species that show differing substrate specificity: Arg-specific (Rgp) and Lys-specific (Kgp) enzymes. 14) These enzymes are essential for the growth and survival of the bacterium in vitro and in vivo, [15][16][17][18] and play critical roles in the degradation of host proteins [19][20][21] and cell invasion by the bacterium. 22,23) Gingipains are the major determinants in the pathogenicity of P. gingivalis, so that inhibitory agents towards these enzymes can be expected to be effective in preventing and improving P. gingivalis-associated diseases.…”