Protein Disulfide Isomerase, a Component of the Estrogen Receptor Complex, Is Associated with Chlamydia trachomatis Serovar E Attached to Human Endometrial Epithelial Cells

Chlamydia trachomatis serovars D to K are the predominant cause of bacterial sexually transmitted diseases and sequelae in the United States and worldwide (25). These bacteria are thought to be luminal pathogens, entering and exiting the apical surfaces of target columnar epithelial cells lining the genital mucosae. They ascend canalicularly in the female genital tract from the endocervix to the endometrium and, subsequently, to the fallopian tubes. In contrast, the Lymphogranuloma venereum (LGV) serovars L1 to L3 of C. trachomatis are disseminating pathogens, exiting the basal surfaces of epithelial cells lining the endocervix or the urethra. The chlamydiae then spread through the submucosa to regional lymph nodes; an inguinal lymphadenopathy is a common clinical manifestation of this sexually transmitted disease syndrome (25). Since these bacteria are obligate intracellular pathogens, they must initially attach to and enter the apical surfaces of epithelial host cells. While a few chlamydial envelope components have been proposed as adhesins (2, 6, 12, 19, 21, 27, 29, 32) and even fewer epithelial plasma membrane components have been proposed as receptors (14, 31, 33, 36), the leading candidate adhesin-receptor combination seems to be heparan sulfate, at least in in vitro studies. In a series of studies by Stephens and colleagues (reviewed in reference 27), a chlamydia biosynthetically directed heparan sulfate was proposed as the adhesin for the LGV L2 serovar (18). These data have recently been confirmed, although there may still be some controversy as to the origin of the heparan sulfate, i.e., is it prokaryote or eukaryote derived (33)? While the chlamydial genomes have no open reading frames encoding genes for heparan sulfate, it has been suggested that some of the chlamydial open reading frames coding for the enzymatic machinery may yet be unassigned, or alternatively, the chlamydiae may exploit substrate intermediates supplied by the host (18). Su et al. (31) suggested that heparan sulfate also serves as the receptor for the C. trachomatis mouse pneumonitis serovar by expressing in Escherichia coli a recombinant maltose binding protein-major outer membrane protein (MBP-MOMP) fusion protein which, in turn, binds specifically to heparan sulfate receptors on HeLa cells. However, this same group also found that heparan sulfate had no competitive inhibitory effect on establishment of chlamydial genital infection in mice (30). Perhaps the extended molecules of the high-mannose oligomannose oligosaccharide glycosylated to MOMP (12) serve as an initial adhesin to bring the infectious form of chlamydia—the elementary body (EB)—closer to the epithelial cell for MOMP-heparan sulfate interaction, leading to entry, or alternatively, they use separately mannose-binding receptors. Little information is available on the receptor for the nondisseminating, non-LGV serovars of C. trachomatis (14). The EB of these D to K serovars are less susceptible to interference of attachment to HeLa cells by heparan sulfate (4, 33). Further, Hayashi et al. (9) demonstrated by immunocytochemistry at both the light- and electron-microscopic levels that heparan sulfate was localized only to the basal surfaces of genital columnar epithelial cells of mouse tissues in vivo. Such basal laminal or interstitial matrix proteins may be redistributed differently, i.e., circumferentially, in nonpolarized epithelial cells cultured in vitro (37). Our laboratory previously examined the endometrial epithelial cell line HEC-1B, grown on coverslips, for the presence of integrins (39). These endometrial cells were moderately to strongly positive for 13 out of 24 major integrins, mostly α, αv, and β. Monoclonal antibodies recognizing seven of these integrins were added to HEC-1B cells, but this maneuver had no effect on blocking serovar E EB attachment, whereas there was markedly decreased attachment of the positive control Yersinia pseudotuberculosis, which binds RGD sequences. Recent chlamydial genomic analyses of the multigene family encoding polymorphic outer membrane proteins (POMPs) suggest the POMPs may have autotransporter function (10). Indeed, the passenger domain of POMPs from several Chlamydia species, including C. trachomatis, contains RDG motifs. While the authors point out that RGD motifs are often associated with proteins predicted to be adhesins, these functional motifs are common and also are seen in proteins that do not require cell adhesion properties. This study presents a different approach designed to identify receptor components on HEC-1B cells involved in attachment of C. trachomatis serovar E EB. The key strategy was to isolate and identify by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) chemiluminescence-labeled apical membrane proteins associated with EB during attachment, subsequently immunoprecipitated with EB, and resolved by isoelectric focusing. Our report focuses on one member of the estrogen receptor complex (13) which carries out thiol-disulfide exchange reactions at cell surfaces—a prerequisite for EB infection.