SUBCELLULAR-LOCALIZATION AND CYTOTOXIC ACTIVITY OF THE GROEL-LIKE PROTEIN ISOLATED FROM ACTINOBACILLUS-ACTINOMYCETEMCOMITANS

The subcellular locations, ultrastructure, and cytotoxic activity of t he GroEL-like protein from Actinobacillus actinomycetemcomitans were i nvestigated. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that synthesis of the Gr oEL-like protein is substantially increased after a thermal shock. Ana lysis of the purified native GroEL-like protein by transmission electr on microscopy revealed the typical 14-mer cylindrical molecule, which had a diameter of about 12 nm. A. actinomycetemcomitans cells grown at 35 degrees C and heat shocked at 43 degrees C were fractionated, and fractions were separated by SDS PAGE and analyzed by Western immunoblo tting using antibodies to GroEL- and DnaK-like proteins. The GroEL-lik e protein was found in both the soluble and membrane fractions, wherea s the DnaK-like protein was mostly found in the cytoplasm. An increase in specific proteins, including the GroEL- and DnaK-like proteins, ma s found in heat-shocked cells. The subcellular localization of the Gro EL-like protein was examined by immunoelectron microscopy of whole cel ls. More GroEL-like protein was detected in stressed cells than in uns tressed cells, and most of it was found not directly associated with o uter membranes but rather in extracellular material. The native GroEL- like protein was assessed for cytotoxic activities. The GroEL-like pro tein increased the proliferation of periodontal ligament epithelial ce lls at concentrations between 0.4 and 1.0 mu g/ml. The number of cells in the culture decreased significantly at higher concentrations. A ce ll viability assay using HaCaT epithelial cells indicated that the Gro EL-like protein was strongly toxic for the cells. These studies sugges t the extracellular nature of the GroEL-like protein and its putative role in disease initiation.