Subcellular localization and cytotoxic activity of the GroEL-like protein isolated from Actinobacillus actinomycetemcomitans

Abstract

The subcellular locations, ultrastructure, and cytotoxic activity of the GroEL-like protein from Actinobacillus actinomycetemcomitans were investigated. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that synthesis of the GroEL-like protein is substantially increased after a thermal shock. Analysis of the purified native GroEL-like protein by transmission electron microscopy revealed the typical 14-mer cylindrical molecule, which had a diameter of about 12 nm. A. actinomycetemcomitans cells grown at 35 degreesC and heat shocked at 43 degreesC were fractionated, and fractions were separated by SDS-PAGE and analyzed by Western immunoblotting using antibodies to GroEL- and DnaK-like proteins. The GroEL-like protein was found in both the soluble and membrane fractions, whereas the DnaK-like protein was mostly found in the cytoplasm. An increase in specific proteins, including the GroEL- and DnaK-like proteins, was found in heat-shocked cells. The subcellular localization of the GroEL-like protein was examined by immunoelectron microscopy of whole cells. More GroEL-like protein was detected in stressed cells than in unstressed cells, and most of it was found not directly associated with outer membranes but rather in extracellular material. The native GroEL-like protein was assessed for cytotoxic activities. The GroEL-like protein increased the proliferation of periodontal ligament epithelial cells at concentrations between 0.4 and 1.0 microgram/ml. The number of cells in the culture decreased significantly at higher concentrations. A cell viability assay using HaCaT epithelial cells indicated that the GroEL-like protein was strongly toxic for the cells. These studies suggest the extracellular nature of the GroEL-like protein and its putative role in disease initiation.

FIG. 1

Two-dimensional gel electrophoresis of proteins from unstressed (A) and stressed (B) cells. The isoelectric focusing gel is oriented with the acidic side to the left and high-molecular-mass proteins at the top. The GroEL-like protein (64 kDa) and the DnaK-like protein (74 kDa) are indicated by arrows in panel B. Circled dots in panel A are examples of proteins whose synthesis was lowered by the heat shock.

FIG. 2

Ultrastructure of the purified native GroEL-like protein stained with uranyl acetate. The sevenfold symmetry can be seen and easily recognized in the enlargement (upper left corner). Bar = 20 nm.

FIG. 3

Presence of the GroEL-like protein in various cell fractions. Cell fractionation was performed according to the method described in the text. Each lane contains 10 μg of proteins. Lanes 1, unstressed A. actinomycetemcomitans cells; lanes 2, stressed A. actinomycetemcomitans cells; lanes 3, cytoplasm; lanes 4, periplasm; lanes 5, outer membrane; lanes 6, cytoplasmic membrane; lanes 7, extracellular vesicles. The protein profile of each fraction was analyzed by SDS-PAGE and staining with Coomassie brilliant blue R-250 (A) and by Western immunoblot analysis with an anti-GroEL-like antibody (B) or with an anti-DnaK antibody (C).

FIG. 4

Electron micrographs showing immunogold detection of GroEL-like protein in ultrathin sections of A. actinomycetemcomitans at 35°C (A) or 43°C (B). Sections were probed with an anti-GroEL-like protein followed by a 10-nm gold–anti-rabbit IgG conjugate. Stressed cells (B) exhibited many more gold particles both inside and outside the cells. The background level of immunogold labeling was evaluated with a rabbit anti-pig IgG (C) or without any first antibody (D), and little or no labeling was found. Immunogold detection of DnaK-like protein in ultrathin sections of A. actinomycetemcomitans at 35°C (E) or 43°C (F) indicated that stressed cells (F) showed more internal labeling than unstressed cells, but little or no labeling was seen outside the cells. In this case, sections were probed with an anti-DnaK followed by a 10-nm gold–anti-rabbit IgG conjugate. Bar = 100 nm.

FIG. 5

Effects of native GroEL-like protein on numbers of cells in cultures of periodontal ligament epithelial cells. Cells were first cultured for 48 h, and then culture was continued in the presence of different concentrations of the GroEL-like protein for 7 days. The numbers of cells in the cultures were measured by staining with crystal violet and measuring the OD₅₇₀ of the dissolved stain. Values are means plus standard deviations for four samples. Differences between the control and the GroEL-like protein at 0.4 to 4.0 μg/ml were statistically significant (P < 0.05 by Sheffe’s F test).