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Infection and Immunity, February 2002, p. 535-543, Vol. 70, No. 2
0019-9567/01/$04.00+0     DOI: 10.1128/IAI.70.2.535-543.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Surface Accessibility of the 70-Kilodalton Chlamydia trachomatis Heat Shock Protein following Reduction of Outer Membrane Protein Disulfide Bonds

Departments of Microbiology,¹ Departments of Pathology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614-0579,² Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7290³

Received 2 July 2001/ Returned for modification 6 September 2001/ Accepted 24 October 2001

Numerous investigations have shown that 70-kDa heat shock protein (Hsp70) homologs interact tightly with hydrophobic proteins and functionally assist proteins in membranous organelles and environments. One such protein is the Chlamydia trachomatis Hsp70 that is associated with isolated outer membrane complexes of infectious elementary bodies (EB). Previous observations have indicated that chlamydial Hsp70 plays a role in EB attachment to, or entry into, endometrial epithelial cells. In this study, immunofluorescence microscopy and transmission electron microscopy observations showed that chlamydial Hsp70 is not a surface-displayed ligand on purified EB. However, brief exposure of EB to the thiol reducing agent dithiothreitol (DTT) led to surface accessibility of the Hsp70 substrate-binding domain. Reduction of the highly disulfide-cross-linked EB outer membrane proteins with DTT resulted in a decrease in EB attachment and infectivity. Interestingly, exposure of EB to the membrane-impermeable thiol-alkylating reagent 5,5'-dithiobis(2-nitrobenzoic acid) enhanced attachment but compromised infectivity, suggesting that EB outer membrane proteins must be reduced for entry and productive infection. Together, our data suggest that (i) the structural integrity of the EB outer membrane, maintained by protein disulfide bonds, is important during the initial stages of attachment; (ii) reduction occurs within the localized microenvironment of host cell surfaces once intimate contact is established between EB and host cells; and (iii) subsequent conformational changes in EB ultrastructure allow productive infection in host cells. The accessibility of the Hsp70 substrate-binding domain may support the hypothesis that this protein plays a role in events following the initial stage of attachment instead of serving as a primary, surface-displayed adhesin.

Editor: D. L. Burns

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