Two Small Molecules Block Oral Epithelial Cell Invasion by Porphyromons gingivalis

Abstract

Porphyromonas gingivalis is a keystone pathogen of periodontitis. One of its bacterial characteristics is the ability to invade various host cells, including nonphagocytic epithelial cells and fibroblasts, which is known to facilitate P. gingivalis adaptation and survival in the gingival environment. In this study, we investigated two small compounds, Alop1 and dynasore, for their role in inhibition of P. gingivalis invasion. Using confocal microscopy, we showed that these two compounds significantly reduced invasion of P. gingivalis and its outer membrane vesicles into human oral keratinocytes in a dose-dependent manner. The inhibitory effects of dynasore, a dynamin inhibitor, on the bacterial entry is consistent with the notion that P. gingivalis invasion is mediated by a clathrin-mediated endocytic machinery. We also observed that microtubule arrangement, but not actin, was altered in the host cells treated with Alop1 or dynasore, suggesting an involvement of microtubule in this inhibitory activity. This work provides an opportunity to develop compounds against P. gingivalis infection.

Fig 1. Invasive activity of P. gingivalis vesicles into HOKs in the presence of different doses of Alop1 and dynasore for 2 h.

(A) P. gingivalis vesicles were stained with anti-33277 serum and a secondary antibody conjugated with Alex Fluor 546 (red), nuclei were stained with DAPI (blue), and HOKs were visualized with confocal microscopy. Scale bar, 20 μm. (B) After treatment with Alop1 or dynasore, the number of HOKs carrying intercellular P. gingivalis vesicles (infection rate) was determined by counting the infected HOKs in 30 random areas. Each bar represents the percentage of HOKs with intercellular vesicles. The SEs are indicated (n = 3). An asterisk indicates the statistical significance of invasive rates between P. gingivalis vesicles in the presence or absence of compounds (P < 0.05; t test).

Fig 2. Comparison of inhibitory activities of compounds in internalization of P. gingivalis vesicles.

(A) HOKs treated with 30 μM of different compounds including DMSO, Alop1, dynasore, cytochalasin D, and nocodazole for 2 h. P. gingivalis vesicles were stained with anti-33277 serum and a secondary antibody conjugated with Alex Fluor 546 (red), nuclei were stained with DAPI (blue), and HOKs were visualized with confocal microscopy. Scale bar, 20 μm. (B) Infection rate and level (average of fluorescence intensity in each cell) of HOKs treated with different compounds are presented and compared with a DMSO control. Means and SDs are indicated (n = 3). An asterisk indicates the statistical significance of invasive rates and levels between P. gingivalis vesicles (P < 0.05; t test).

Fig 3. Inhibition of P. gingivalis invasion by different compounds.

HOK nuclei were stained with DAPI (blue), and internalized P. gingivalis cells were stained with primary anti-33277 serum and a secondary antibody conjugated with Alex Fluor 546 (red) and visualized with confocal microscopy. Scale bars, 20 μm. (B) Each bar represents relative P. gingivalis infection rate or level of HOKs treated with compounds compared to that of untreated HOKs. An asterisk indicates the statistical significance between invasive rates or levels between P. gingivalis cells in the presence or absence of compounds (P < 0.05; t test).

Fig 4. Microtubule rearrangement in HOKs induced by Alop1 and dynasore.

After treated with Alop1 (30 μM) or dynasore (30 μM) for 0, 10, 30 min, or 2 h as well as recovery from treatment, HOKs were stained with anti-α-tubulin, anti-IgG with Alex Fluor 546 (red) and DAPI (blue) and visualized under a confocal microscope. Scale bar, 20 μm.

Fig 5. Actin arrangement in HOKs treated with compounds.

After treated with Alop1 or dynasore for 0, 10, 30 min, or 2 h, HOKs were stained with anti-actin antibodies, anti-IgG with Alex Fluor 546 (red) and DAPI (blue) and visualized under a confocal microscope. Scale bar, 20 μm.

Fig 6. Exit of intracellular P. gingivalis cells from HOKs.

(A) HOKs were cultured in the presence of antibiotics (gentamicin and metronidazole) as well as Alop1 and dynasore (30 μM) for 20 h after infection with P. gingivalis 33277. P. gingivalis cells were stained with anti-33277 serum and a secondary antibody conjugated with Alex Fluor 546 (red), nuclei were stained with DAPI (blue), and HOKs were visualized with confocal microscopy. Scale bar, 20 μm. (B) Each bar represents average of fluorescence intensity (red) in 100 infected cells cultured with Alop11 or dynasore relative to that without compounds.

Fig 7. Effects of Alop1 and dynasore on bacterial growth and gene expression in P. gingivalis.

(A) Comparison of the growth curves of P. gingivalis 33277 in the presence of compounds. Cells were grown in TSB media in the presence or absence of Alop1 or dynasore (30 μM). Shown in the curves are means of four samples, with error bars representing SEM. One ml aliquots were taken and the OD600 was measured over a period of 44 hr. (B) Gene expression in P. gingivalis in the presence or absence of Alop1 or dynasore (30 μM) was determined using qRT-PCR analysis. Expression levels were normalized with 16s rRNA. Representative data are shown as means with standard deviation of three biological replicates and relative to expression level of the housekeeping gene glk.