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°C and heat shocked at 43°C 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 μg/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.

Localization of heat shock proteins in clinical Actinobacillus actinomycetemcomitans strains and their effects on epithelial cell proliferation

“…Epithelial proliferation leads to an increase in pocket depth and, thus, to a larger area for bacteria to grow. Therefore, GroEL-like Hsp may be important for bacterial survival in the periodontal pocket and play a role in the pathogenesis of periodontitis as has been suggested earlier by our study [15] and the study of Kirby and coworkers on A. actinomycetemcomitans laboratory strains [16]. Both proliferation and death of epithelial cells occur during the formation of periodontal pockets and are important mechanisms in periodontal disease progression.…”

“…The membrane fraction of A. actinomycetemcomitans is of special interest because the Hsp localization on the bacterial cell surface enables rapid Hsp expression and makes the quick contact of Hsp with human cells possible. Membrane blebs se- creted by the bacteria also carry GroEL-like Hsp [15]. Epithelial proliferation leads to an increase in pocket depth and, thus, to a larger area for bacteria to grow.…”

“…GroEL-like Hsp from A. actinomycetemcomitans may induce osteolysis [16]. We have observed that GroEL-like protein promotes epithelial cell proliferation at lower concentrations, but has toxic e¡ects at higher concentrations [15]. In this study we localized the GroEL-like and DnaKlike heat shock proteins in the subcellular fractions of 12 A. actinomycetemcomitans strains of various clinical origin and of di¡erent serotypes.…”

“…Fractionation was performed as previously described [15]. Brie£y, bacterial cells were exposed to osmotic shock with ice-cold distilled water for 5 min, the suspension was centrifuged, and the supernatant containing the periplasmic material was lyophilized.…”

“…SDS-PAGE was performed according to Laemmli [17] using 10% slab gels (Mini-Protean II, Bio-Rad Laboratories). Western immunoblotting was performed as described previously [15,18] using rabbit antisera against the puri¢ed A. actinomycetemcomitans GroEL-like protein (1:3000 dilution) and a commercial antibody against Hsp70 protein (1:1000 dilution) (Dako Corporation, Carpinteria, CA, USA). Incubation with the secondary antibody, goat anti-rabbit IgG conjugated to alkaline phosphatase (1:3000 dilution) (Bio-Rad Laboratories), was for 1 h. The reactions were visualized by alkaline phosphatase staining.…”