Nuclear Targeting ofPorphyromonas gingivalisW50 Protease in Epithelial Cells

Abstract

Porphyromonas gingivalis is an important pathogen associated with destructive periodontal disease and is able to invade the epithelial cell barrier. Its cysteine proteases are recognized as major virulence factors, and in this study, we examined the interaction of the arginine-specific protease with epithelial cells in culture. Three cell lines (KB, HeLa, and SCC4) were incubated with strain W50 culture supernatant; stained with monoclonal antibody 1A1, which recognizes an epitope on the adhesin (␤) component of the cysteine protease-adhesin (␣/␤) heterodimer; and viewed using immunofluorescence microscopy. Within 1 h, the protease traversed the plasma membrane and was localized around the nucleus before becoming concentrated in the cytoplasm after 24 to 48 h. In contrast, the purified arginine-specific heterodimeric protease (HRgpA) rapidly entered the nucleus within 15 to 30 min. This nuclear targeting (i) was seen with active and N␣-p-tosyl-L-lysine chloromethyl ketone (TLCK)-inactivated HRgpA, indicating it was independent of the proteolytic activity; (ii) occurred at both 4 and 37°C; and (iii) failed to occur with the monomeric protease (RgpA cat ), indicating the importance of the adhesin chain of the HRgpA protease to this process. Rapid cell entry was also observed with recombinant catalytic (␣) and adhesin (␤) chains, with the latter again targeting the nuclear area. After 48 h of incubation with HRgpA, significant dose-dependent stimulation of metabolic activity was observed (measured by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), and a doubling of mitotic activity combined with the presence of apoptotic cells indicated that HRgpA may interfere with cell cycle control mechanisms. These effects were seen with both active and TLCK-inactivated protease, confirming that they were not dependent on proteolytic activity, and thus provide new insights into the functioning of this P. gingivalis protease.Periodontal diseases are chronic inflammatory conditions resulting from the host response to microbial challenge and are characterized by the formation of deepening periodontal pockets and the destruction of both hard and soft tissues supporting the teeth. An essential step in initiating any infection involves adherence to, and/or invasion of, host cells by pathogens or their products. As epithelial cells form the first barrier to invasion by bacteria which colonize the gingival crevice, the nature of their interaction with potential pathogens is likely to have a major influence on the progression of disease.Porphyromonas gingivalis is an anaerobic gram-negative bacterium important in destructive periodontal disease whose main ecological niche is the subgingival crevice adjacent to the tooth surface and the epithelial attachment apparatus of the soft tissues. The organism can invade gingival tissue in advanced periodontitis (18) and can be taken up by gingival and pocket epithelial cells, oral epithelial cell lines, and endothelial cells in vitro (13,23,33,40). The nature of the ...

Functional Implication of the Hydrolysis of Platelet Endothelial Cell Adhesion Molecule 1 (CD31) by Gingipains ofPorphyromonas gingivalisfor the Pathology of Periodontal Disease

“…A recent study with chimeric constructs of human PECAM-1 revealed that homophilic binding of PECAM-1 on adjacent cells and positively charged sequences ( 599 RKAKA K 604 ) in the juxtamembrane region of the transmembrane domain of PECAM-1 are important for its localization to cell-cell borders (62). In this context, the hemagglutinin domains have been reported to regulate intracellular dispersion of gingipains taken up by epithelial cells (57), and the proteinases have specificity for arginine and lysine residues (54), with the 599 RK AKAK 604 sequence a potential target. The major proteinase inhibitors present in human serum are alpha-1-antitrypsin, alpha-1-antichymotrypsin, and alpha-2-macroglobulin (␣ 2 M).…”

Gingipain-dependent interactions with the host are important for survival of Porphyromonas gingivalis

“…Moreover, we have observed that a high concentration of Kgp can cause BCAEC detachment and apoptosis (our unpublished observations) suggesting that there is a threshold level of gingipain activity below which a cell can remain viable, but at gingipain activity above this level the cell cannot overcome gingipain-induced effects and becomes apoptotic. This threshold phenomenon has been demonstrated in epithelial cells treated with HRgpA (175).…”

“…HRgpA localizes to the nucleus (175) and may be able to cleave lamin B and subsequently gain access to the nucleus to cleave Topo I and possibly modulate other nuclear proteins, producing the nuclear apoptotic morphology. Kgp may function similarly to and work with HRgpA, since its nuclear targeting ability was not dependent on proteolytic activity (175). Figure 5 is a comparison of potential caspaseindependent apoptosis pathway members induced by gingipain activity.…”

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

“…Based on karyotyping, the HEp-2 cell line has been reclassified as HeLa (44). Both cell lines have been used extensively to study host-bacterial interactions, including those with P. gingivalis (5,9,45,46), and, in the present study, we used HEp-2 cells to maintain consistency with previous work (7,17). The mouse NIH/3T3 fibroblast cell line was purchased from the American Type Culture Collection (ATCC) and cultivated in DMEM supplemented with 10% FCS.…”

“…gingivalis binds to several human cell types and is internalized upon attachment; adherence and entry are mediated by bacterial surface structures such as fimbriae (5,6) and gingipain cysteine proteinases (7)(8)(9). A number of surface components of eukaryotic cells have been suggested to serve as receptors.…”