Role of Porphyromonas gingivalis SerB in gingival epithelial cell cytoskeletal remodeling and cytokine production

Abstract

The SerB protein of Porphyromonas gingivalis is a HAD family serine phosphatase that plays a critical role in entry and survival of the organism in gingival epithelial cells. SerB is secreted by P. gingivalis upon contact with epithelial cells. Here it is shown by microarray analysis that SerB impacts the transcriptional profile of gingival epithelial cells, with pathways involving the actin cytoskeleton and cytokine production among those significantly overpopulated with differentially regulated genes. Consistent with the transcriptional profile, a SerB mutant of P. gingivalis exhibited defective remodeling of actin in epithelial cells. Interaction between gingival epithelial cells and isolated SerB protein resulted in actin rearrangement and an increase in the F/G actin ratio. SerB protein was also required for P. gingivalis to antagonize interleukin-8 accumulation following stimulation of epithelial cells with Fusobacterium nucleatum. SerB is thus capable of modulating host cell signal transduction that impacts the actin cytoskeleton and cytokine production.

FIG. 1.

Comparison of transcriptional profiles of HIGK cells following coculture with wild-type P. gingivalis or SerB mutant strain. The expression pattern of the cRNAs analyzed by microarray is represented as a supervised analysis of the variance-normalized data set of differentially expressed genes with the algorithm Cluster and displayed with TreeView. Each row represents an individual DNA element spotted on the array, and each column represents the expression states of cRNAs for the challenge condition indicated. Each expression data point represents the ratio of the fluorescence intensity of the cRNA from P. gingivalis 33277-infected cells (Pg columns, replicates [R] 1 to 4) to the fluorescence intensity of the cRNA from SerB mutant-infected cells (SerB columns, R 1 to 4). The distance matrix used to show the relatedness of samples through gene expression space was the Pearson's correlation coefficient. The cluster is subdivided into three groups consisting of genes that were repressed (green), genes that were induced (red), and genes whose expression did not change (black). The variation in expression for a given gene is expressed as the distance from the mean observation for that gene according to the color scale presented below the heat map.

FIG. 2.

Summary of epithelial cell actin and MAP kinase-related genes differentially regulated (P < 0.005) following infection with the P. gingivalis SerB mutant compared to the parental strain. The pathway was adapted from Pathway Express and utilizes the Kegg nomenclature. Red represents upregulation, green represent downregulation, and gray represents no change in expression. +P indicates phosphorylation; −P indicates dephosphorylation. Pointed arrows indicate molecular interactions resulting in activation; flat arrows indicate molecular interactions resulting in inhibition. Solid lines show a direct interaction, and dotted lines are interactions involving intermediates.

FIG. 3.

SerB modulates the actin cytoskeleton in gingival epithelial cells. (A) HIGK cells were infected with P. gingivalis 33277 (WT) or SerB mutant (SerB) for 10 min or 6 h. Control (Ctrl) cells were uninfected. The actin cytoskeleton was visualized with rhodamine-phalloidin (red), and bacteria were stained with P. gingivalis antibody and fluorescein-labeled secondary antibody (green). The image is a confocal 0.2-μm optical x-y projection magnified 40×. Arrows indicate areas of actin redistribution around P. gingivalis. (B) Boxed areas from panel A are shown, along with x-z and y-z projections. (C) Quantitative image analysis of actin fluorescence in the stack of z-projections representing whole cells shown in panel A. Data are expressed as the ratio of actin in infected versus control HIGK cells. *, P < 0.01 (t test) at 6 h compared to 10 min.

FIG. 4.

Extracellular SerB regulates F actin in gingival epithelial cells. HIGK cells were reacted with rSerB or rPG1170. The control (Ctrl) was prepared in the absence of phosphatase. (A) Actin was visualized with rhodamine-phalloidin (red), and bacterial phosphatases were visualized with SerB antibody and fluorescein-labeled secondary antibody (green). Confocal x-y projections of a z-stack (×40) are shown, along with x-z and y-z projections for the merged images (×60) with enzyme-reacted cells. (B) F/G actin ratio in HIGKs reacted with SerB. Error bars represent standard deviations. Values are statistically different at P < 0.01 (t test).

FIG. 5.

Enzyme-linked immunosorbent assay of IL-8 accumulation in HIGK supernatants following stimulation with F. nucleatum (Fn) alone and with P. gingivalis 33277 (WT), SerB mutant (SerB-), or the complemented SerB mutant (cSerB) for the times indicated. Error bars represent standard deviations (n = 3).