CEACAM1 recognition by bacterial pathogens is species-specific

Abstract

Background: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an immunoglobulin (Ig)-related glycoprotein, serves as cellular receptor for a variety of Gram-negative bacterial pathogens associated with the human mucosa. In particular, Neisseria gonorrhoeae, N. meningitidis, Moraxella catarrhalis, and Haemophilus influenzae possess well-characterized CEACAM1-binding adhesins. CEACAM1 is typically involved in cell-cell attachment, epithelial differentiation, neovascularisation and regulation of T-cell proliferation, and is one of the few CEACAM family members with homologues in different mammalian lineages. However, it is unknown whether bacterial adhesins of human pathogens can recognize CEACAM1 orthologues from other mammals.

Results: Sequence comparisons of the amino-terminal Ig-variable-like domain of CEACAM1 reveal that the highest sequence divergence between human, murine, canine and bovine orthologues is found in the beta-strands comprising the bacteria-binding CC'FG-face of the Ig-fold. Using GFP-tagged, soluble amino-terminal domains of CEACAM1, we demonstrate that bacterial pathogens selectively associate with human, but not other mammalian CEACAM1 orthologues. Whereas full-length human CEACAM1 can mediate internalization of Neisseria gonorrhoeae in transfected cells, murine CEACAM1 fails to support bacterial internalization, demonstrating that the sequence divergence of CEACAM1 orthologues has functional consequences with regard to bacterial recognition and cellular invasion.

Conclusions: Our results establish the selective interaction of several human-restricted bacterial pathogens with human CEACAM1 and suggest that co-evolution of microbial adhesins with their corresponding receptors on mammalian cells contributes to the limited host range of these highly adapted infectious agents.

Figure 1

Amino acid sequence alignment and expression of soluble CEACAM1 proteins of different mammals. (A) Amino acid sequence alignment of the N-terminal domains of human, murine, bovine and canine CEACAM1 proteins. The following sequences were used: human CEACAM1 (hCEA1, NM_001712), murine CEACAM1a (mCEA1, BC016891), canine CEACAM1 (cCEA1, NM_001097557.1), bovine CEACAM1 (bCEA1, AY345129), bovine CEACAM1 isoform b (bCEA1b, AY487418). Amino acids identical to the human CEACAM1 sequence are indicated by dots. The characteristic beta-strands of the Ig variable-like domain are marked by blue lines and letters above the human sequence. (B) Amino acid identity between different mammalian CEACAM1 orthologues. Percent identity compared to the human sequence is given for amino acid residues comprising the beta strands of either the AA'BDE-face or the CC'C"FG-face of the immunoglobulin fold. (C) Schematic illustration of the proteins used in this study. Human CEACAM1-4L isoform containing a long cytoplasmic domain (hCEA1-4L), the human CEACAM1 isoform containing a short cytoplasmic domain (hCEA1-4S), and the corresponding murine isoform (mCEA1-4S) were expressed as GFP or cerulean fusion proteins. 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.

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 Opa_CEA) 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 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 Opa_CEA protein-expressing N. gonorrhoeae (Ngo Opa_CEA) 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 Opa_CEA.

Figure 3

Binding of Neisseria meningitidis and Moraxella catarrhalis to CEACAM1 orthologues. Culture supernatants containing soluble GFP-tagged amnio-terminal domains of the indicated mammalian CEACAMs or a control culture supernatant from GFP-transfected cells (neg. control) were incubated with Opa_CEA protein-expressing N. meningitidis or UspA1-expressing M. catarrhalis. After washing, bacteria were analysed by flow cytometry and the bacteria-associated GFP-fluorescence was determined. Only human CEACAM1 (hCEA1) binds to the pathogenic bacteria.

Figure 4

Internalization of Opa_CEA-expressing Neisseria gonorrhoeae is only mediated by human CEACAM1. (A) 293 cells were transfected with constructs encoding the indicated human or murine CEACAM1 isoforms fused to GFP or Cerulean. Cells transfected with a GFP-encoding vector served as control. After 48 h, cells were lysed and the expression was determined by Western blotting with a polyclonal anti-GFP antibody. (B) Cells transfected as in A) were infected with Opa-negative (Ngo Opa-) or Opa_CEA-expressing N. gonorrhoeae (Ngo Opa_CEA) at an MOI of 30 for 2 h. The number of viable intracellular bacteria was determined by gentamicin protection assay. Bars represent mean values ± SEM of three independent experiments done in triplicate. For statistical analysis, samples were compared against control transfected cells by one-tailed Mann-Whitney U-test; *, p < 0.001. (C) 293 cells were transfected with constructs encoding human CEACAM1 isoform containing a short cytoplasmic domain (hCEA1), the corresponding murine isoform (mCEA1) or an empty control vector. Cells were infected with fluorescein-labelled Opa-negative (Ngo Opa-) or Opa_CEA-expressing N. gonorrhoeae (Ngo Opa_CEA) at an MOI of 30 for 2 h. The uptake index was determined by flow cytometry as described in Material and Methods. Bars represent mean values ± SEM of three independent experiments.

Figure 5

Microscopic verification of N. gonorrhoeae uptake via human CEACAM1. 293 cells were transfected with constructs encoding GFP, human CEACAM1-4S-GFP, or murine CEACAM1-4S-GFP as indicated. Cells were infected for 2 h with biotin- and rhodamine-labelled non-opaque (Ngo Opa-) or Opa_CEA-expressing N. gonorrhoeae (Ngo Opa_CEA). Infected cells were fixed, but not permeabilized, and samples were stained with AlexaFluor647-streptavidin to label extracellular bacteria (Extr. bacteria). Intracellular bacteria (small arrow) are marked by their selective rhodamine labelling, whereas extracellular bacteria (arrowheads) are stained with both rhodamine and AlexaFluor647. Bars represent 5 μm.