Opa binding to cellular CD66 receptors mediates the transcellular traversal of Neisseria gonorrhoeae across polarized T84 epithelial cell monolayers

Abstract

SummaryWe have analysed the capacity of the 11 phase-variable, opacity-associated (Opa) proteins encoded by Neisseria gonorrhoeae MS11 to mediate traversal across polarized monolayers of the human colonic carcinoma T84 cell line. Gonococci expressing either the heparan sulphate proteoglycan (HSPG) binding Opa protein (Opa 50 ) or no Opa protein (Opa ¹ ) did not interact with the apical pole of T84 monolayers, whereas the 10 variant Opa proteins previously shown to bind CD66 receptors were found to mediate efficient gonococcal adherence and transepithelial traversal. Consistent with this, T84 cells were shown by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting to co-express CD66a (BGP), CD66c (NCA) and CD66e (CEA). The recruitment of CD66 receptors by Opa-expressing gonococci indicates their involvement in mediating adherence to the surface of T84 cells, and these bacterial interactions could be inhibited completely using polyclonal antibodies cross-reacting with all of the CD66 proteins co-expressed on T84 cells. Consistent results were obtained when Opa proteins were expressed in Escherichia coli, suggesting that the Opa-CD66 interaction is sufficient to mediate bacterial traversal. Transcytosis of Opa-expressing N. gonorrhoeae or E. coli did not disrupt the barrier function of infected monolayers, as indicated by a sustained transepithelial electrical resistance (TEER) throughout the course of infection, and confocal laser scanning and electron microscopy both suggest a transcellular rather than a paracellular route of traversal across the monolayers. Parallels between the results seen here and previous work done with organ cultures confirm that T84 monolayers provide a valid model for studying neisserial interactions with the mucosal surface, and suggest that CD66 receptors contribute to this process in vivo.

Distinct Roles of Secreted HtrA Proteases from Gram-negative Pathogens in Cleaving the Junctional Protein and Tumor Suppressor E-cadherin

“…Strikingly, a single Q 3 A mutation was sufficient to convert NgHtrA into an E-cadherin-cleaving protease. Obviously N. gonorrhoeae does not require E-cadherin processing because it crosses the epithelium through transcytosis (17), whereas other pathogens may benefit from HtrA-mediated E-cadherin shedding.…”

Selection for a CEACAM Receptor-Specific Binding Phenotype during Neisseria gonorrhoeae Infection of the Human Genital Tract

“…Pilus facilitates initial bacterial attachment to the urogenital mucosa (4), where other adhesins can then confer a tighter secondary binding to epithelium-expressed receptors (3,18,58,59). Of these, the Opa proteins mediate tight adherence and transcytosis to the subepithelial space (25,26). The phase variability of the genes encoding Opa proteins and pilus allows N. gonorrhoeae to change the patterns of expression of these adhesins randomly.…”

“…CEACAM1, CEACAM3, CEACAM5 (carcino-embryonic antigen [CEA]), and CEACAM6 (but apparently not other CEACAMs) are each capable of mediating neisserial adherence to and engulfment by the various tissues on which they are differentially expressed (16-20, 23, 24). Epithelial CEACAMs (CEACAM1, CEACAM5, and CEACAM6) are all presumed to facilitate bacterial colonization (25,26). In the female genital tract, squamous epithelia express CEACAM5, whereas CEACAM1 is expressed on columnar epithelia of the endocervix and uterus (27), allowing each to be accessible for direct docking by the gonococci.…”

CEACAM1 Dynamics during Neisseria gonorrhoeae Suppression of CD4+ T Lymphocyte Activation

“…More intimate attachment is then established by one of a variety of adhesins (6). Of these, the colony opacity-associated (Opa) 3 proteins have been shown to allow bacterial transcellular transcytosis through polarized epithelial monolayers (7) so that they ultimately emerge in the subepithelial tissues (7,8). Each gonococcal strain encodes up to 11 related but antigenically distinct Opa variants (9).…”