Amino-terminal Igv-like domains of CEACAM1 from human (hCEA1), mouse

(mCEA1), dog (cCEA1), or cattle (isoform a, bCEA1a; isoform b, bCEA1b) were expressed in human cells as soluble GFP-fusion proteins. Binding of Neisseria gonorrhoeae to the amino-terminal domain of CEACAM1 is human specific Bucladesine in vitro The soluble GFP-tagged amino-terminal domains of CEACAM1 orthologues were incubated with isogenic strains of the human pathogen N. gonorrhoeae. The bacterial strains used either expressed a specific Opa protein, which is known to bind human CEACAM1 and other human CEACAMs (Ngo OpaCEA), or they did not express any Opa protein (Ngo Opa-). Opa expression by the gonococci was confirmed by Western blotting with a monoclonal antibody against neisserial Opa proteins Fulvestrant mouse (Fig. 2A). Following incubation with the amino-terminal CEACAM1 domains from different mammalian species, the samples were washed, and the bacteria-associated fluorescence was measured by flow cytometry. Clearly, the non-opaque Entinostat bacteria (Ngo Opa-) did not reveal a positive signal in the

GFP channel for any tested protein, confirming that Opa proteins are the sole neisserial factor necessary for CEACAM recognition (Fig. 2B). In contrast to the non-opaque gonococci, the OpaCEA-expressing bacteria clearly associated with the isolated amino-terminal Igv-like domain of human CEACAM1 (Fig. 2B). Most importantly, OpaCEA-positive gonococci did not associate with the Igv-like domains of murine, canine or bovine origin (Fig. 2B). These results demonstrate that the association of Neisseria gonorrhoeae with CEACAM1 is limited to the human orthologue of this protein and suggests that CEACAM1 recognition is species-specific. Figure 2 Opa CEA protein expressing Neisseria gonorrhoeae selectively binds to human CEACAM1. (A) Neisseria gonorrhoeae MS11 strains lacking Opa protein expression (Ngo Opa-) or expressing a CEACAM-binding Opa protein

(Ngo OpaCEA) were lysed and the Opa protein expression was determined by Western blotting with a monoclonal anti-Opa antibody (clone 4B12/C11). (B) Expression of the soluble GFP-fusion proteins of CEACAM1 Igv-like domains was determined C-X-C chemokine receptor type 7 (CXCR-7) by Western blotting of culture supernatants with polyclonal anti-GFP antibody. Culture supernatants from cells transfected with a vector encoding cytoplasmically expressed GFP served as control. (C) Culture supernatants containing soluble GFP-tagged amino-terminal domains of the indicated mammalian CEACAMs or a control culture supernatant from GFP-transfected cells (neg. control) were incubated with OpaCEA protein-expressing N. gonorrhoeae (Ngo OpaCEA) or the non-opaque strain (Ngo Opa-). After washing, bacteria were analysed by flow cytometry and the bacteria-associated GFP-fluorescence was determined. Only human CEACAM1 (hCEA1) binds to Ngo OpaCEA.