Surface Accessibility of the 70-KilodaltonChlamydia trachomatisHeat Shock Protein following Reduction of Outer Membrane Protein Disulfide Bonds

Abstract

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.A distinguishing feature of the chlamydiae is their transition between infectious elementary bodies (EB) that bind to and enter host cells and noninfectious reticulate bodies that replicate intracellularly within a membrane-bound inclusion. EB are small (diameter, 300 nm) particles with an unusually rigid ultrastructure due to cysteine-rich membrane proteins that exhibit intra-and intermolecular disulfide cross-linking in the envelope (22,36). Several chlamydial molecules, including the cysteine-rich proteins, have been examined to determine their roles in EB attachment to eukaryotic cells (25,26,38,43,46,48,49,50,51). There is evidence that both the 60-kDa cysteinerich membrane protein and the major outer membrane protein (MOMP) serve as receptors for sulfated glycosaminoglycans in a tethering event between EB and host cell surfaces (46,48,49). Adherence mediated by the 40-kDa MOMP appears to be initiated by charge-charge interactions involving surface-exposed domains (50) and a high-mannose oligomannose oligosaccharide (26). It is likely that additional, unidentified surface components also participate in establishing contact between EB and the host cell surface.Although many d...

Proteic boost enhances humoral response induced by DNA vaccination with the dnaK gene of Chlamydophila abortus but fails to protect pregnant mice against a virulence challenge

“…This absence of a neutralizing effect could be due to the inaccessibility of the antigen on the cell surface. Indeed, a recent report suggests that Hsp70 is not a surface displayed ligand on purified elementary bodies and needs a partial reduction of the cell wall to be exposed [11]. This lack of a neutralizing effect was confirmed by the evaluation of the protection induced by pcDNA3.1::DnaK/DnaK boost vaccination after a chlamydial challenge.…”

Disulfide Bonding within Components of the Chlamydia Type III Secretion Apparatus Correlates with Development

“…Thus far, disulfide bonding among MOMP, OmcA, and OmcB are thought to constitute a highly crosslinked supramolecular envelope complex in EBs (14,19). Conversion of EBs to RBs is accompanied by reduction of these disulfide bonds (17,32) and appears to occur as early as invasion, since evidence suggests that EB proteins must be reduced for productive infection (2,11,26,35). Later in development, during RB to EB differentiation, disulfide bonds are re-formed (4,17,20,21,32,33,36).…”

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…RB cell walls lack the latticework of cross-linked proteins found in EB cell walls (45,46) and have low and constricted levels of peptidoglycan (47), potentially facilitating DNA uptake. RBs also undergo cell division and express DNA repair enzymes that mediate the chromosomal integration of DNA by homologous recombination during division, and thus RBs are likely to be naturally competent for transformation.…”