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Infection and Immunity, November 1998, p. 5307-5313, Vol. 66, No. 11
Groupe de Recherche en Écologie
Buccale,
Received 6 April 1998/Returned for modification 12 June
1998/Accepted 20 August 1998
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.
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Subcellular Localization and Cytotoxic Activity of
the GroEL-Like Protein Isolated from Actinobacillus
actinomycetemcomitans
*
Corresponding author. Mailing address: GREB,
Faculté de Médecine Dentaire, Université Laval,
Cité Universitaire, Québec, Canada, G1K 7P4. Phone: (418)
656-5669. Fax: (418) 656-2861. E-mail: denis.mayrand{at}bcm.ulaval.ca.
Infection and Immunity, November 1998, p. 5307-5313, Vol. 66, No. 11
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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