CEACAM1 recognition by bacterial pathogens is species-specific
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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.
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 β-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.
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.
KeywordsNeisseria Gonorrhoeae Intracellular Bacterium Moraxella Catarrhalis Mammalian Lineage Extracellular Bacterium
The immunoglobulin (Ig) superfamily contains a large number of receptors that serve as cell adhesion molecules (CAMs) mediating homotypic cell-cell-adhesion in multicellular animals. One group of mammalian IgCAMs is named according to the carcinoembryonic antigen (CEA), a tumor marker used in the surveillance of colon cancer . Interestingly, CEA is only known from primates, where it is expressed by mucosal epithelial cells. Similar to CEA, most other CEA-related CAMs (CEACAMs) are restricted to specific mammalian lineages, and only a few CEACAMs, such as CEACAM1 or CEACAM16-20, have orthologues in distantly related mammals [2, 3, 4]. Accordingly, sequence comparisons based on published genome data have provided evidence that CEACAMs have independently diversified in each mammalian order [3, 5].
In humans, CEACAM1 is the target of several Gram-negative commensal and pathogenic bacteria that inhabit the nasopharyngeal, intestinal, or urogenital mucosa. In particular, Neisseria gonorrhoeae, N. lactamica, N. meningitidis, N. subflava, Haemophilus influenzae, Moraxella catarrhalis, and Escherichia coli strains have been found to associate with the protein core or carbohydrate structures of this glycoprotein [6, 7, 8, 9, 10, 11]. These bacterial species utilize distinct surface proteins (adhesins) to engage CEACAMs. For example, the neisserial colony opacity associated (Opa) proteins allow gonococci and meningococci to bind several CEACAM family members including CEACAM1, CEA, and CEACAM6, which are expressed on the apical surface of mucosal epithelial cells. Opa proteins are integral outer membrane proteins with 8 transmembrane β-strands and 4 small extracellular loops, with the central loops participating in CEACAM recognition . Opa-like proteins with a similar β-barrel structure are also found in commensal Neisseria species and can mediate the association with CEACAM1 . In addition, several typeable and non-typeable strains of Haemophilus influenzae, a species that shares the mucosal habitat and lifestyle of Neisseria, can engage CEACAM1 via their outer membrane protein P5 . Another inhabitant of the human oro-pharyngeal mucosa, Moraxella catarrhalis, can bind via the UspA1 surface protein to the N-terminal domain of CEACAMs . UspA1 belongs to the family of trimeric autotransporter or oligomeric coiled-coil adhesin (Oca) family. The prototype of the Oca family is the adhesin YadA of enteropathogenic Yersiniae that has a lollipop structure with a head group, an extended coiled-coil stalk region and a membrane anchor domain . The mature trimeric UspA1 with a size of about 250 - 300 kDa protrudes up to 60 nm from the bacterial surface and is therefore completely distinct from membrane-embedded neisserial Opa proteins or the Haemophilus protein P5 . Surprisingly, CEACAM recognition by the Moraxella UspA1 is mediated by a short sequence within the stalk region requiring a bend conformation of the UspA1 extracellular domain to accommodate CEACAM1 binding . Moraxella strains lacking this peptide sequence within their stalk region fail to bind to CEACAMs .
The striking convergent evolution of structurally distinct adhesive proteins to engage CEACAM1 suggests that this binding is important during the life cycle of these bacteria. As all CEACAM-binding bacteria greatly differ in their pathogenic potential, but share the same ecological niche, it is highly likely that CEACAM-binding promotes colonization of the mucosa. Indeed, in vitro experiments have suggested that CEACAM-binding is not only a means to firmly attach to the host cell surface, but also suppresses the detachment of infected epithelial cells . CEACAM-targeting bacterial adhesins might therefore represent colonization factors that promote the ability of bacteria to establish a firm foothold in their ecological niche. Whether this specialization is also a determinant of the host range of these bacterial pathogens is not known. Though bacterial species expressing CEACAM-binding adhesive proteins are in most cases human-specific, and have no other natural host organism, it has not been experimentally tested whether their adhesins selectively recognize human CEACAMs or can also bind to orthologues from other mammalian species.
In the present study, we analysed the binding of CEACAM1 orthologues from several mammals to bacterial pathogens with distinct adhesive proteins. In particular, we tested Opa protein-expressing N. gonorrhoeae and N. meningitidis as well as UspA1-expressing M. catarrhalis for their ability to recognize CEACAM1 homologues of human, murine, canine or bovine origin. Biochemical binding studies clearly demonstrate that these bacteria selectively interact with human CEACAM1. Furthermore, analyses of bacterial internalization show that the observed amino acid changes in the amino-terminal domain of mammalian CEACAM1 orthologues have clear-cut functional consequences. Accordingly, our data not only demonstrate that bacterial adhesins have co-evolved with the receptor molecules of their mammalian host, but also support the view that the diversification of CEACAMs in mammalian lineages is a pathogen-driven process.
Amino acid sequence alignment
For the amino acid sequence alignment of the N-terminal domains of CEACAM1 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). The alignment was performed using CLUSTALW.
Cell culture and transfection
The human embryonic kidney cell line 293T (293 cells) was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum at 37°C in 5% CO2 and subcultured every second to third day. 293T cells were transfected by calcium-phosphate coprecipitation using 5 - 8 μg of plasmid DNA for each 10 cm culture dish.
Bacteria and infection
Opa52-expressing (OpaCEA), non-piliated N. gonorrhoeae MS11-B2.1 (strain N309), and non-piliated, non-opaque gonococci MS11-B2.1 (strain N302) were kindly provided by T.F. Meyer (Max-Planck Institut für Infektionsbiologie, Berlin, Germany) and were cultured as described previously . Opa-expressing, non-encapsulated N. meningitidis (SiaD mutant of strain MC58) was obtained from Matthias Frosch (Institut für Hygiene und Mikrobiologie, Universität Würzburg, Germany). M. catarrhalis strain ATCC 25238 was obtained from DSMZ (Braunschweig, Germany). Both Moraxella and Neisseriae were grown on GC agar plates (Difco BRL, Paisley, UK) supplemented with vitamins at 37°C, 5% CO2 and subcultured daily. For infection, bacteria were suspended in DMEM and the optical density of the suspension was used to estimate the number of the microorganisms according to a standard curve generated for each strain.
Recombinant plasmid constructs
Mammalian expression plasmids encoding GFP-tagged human CEACAM1-4L (hCEACAM1-4L), human CEACAM1-4S, and the amino-terminal domain of human CEACAM1 (hCEA1-N) were described previously [18, 19]. Murine CEACAM1-4S was constructed by amplifying the full-length cDNA of murine CEACAM1-4S (clone BF584691; ImaGenes, Berlin, Germany) with primers mCEACAM1-sense 5'-GAAGTTATCAGTCGACATGGAGCTGGCCTCAGCAC-3' and mCEACAM1-anti 5'-ATGGTCTAGAAAGCTTCCGCCAGACTTCCTGG-3'. The amino-terminal domain of murine CEACAM1 was amplified with primers mCEACAM1-sense and mCEACAM1-N-anti 5'-ATGGTCTAGAAAGCTTGGGTGTACATGAAATCGC-3'. The N-terminal domains of bovine CEACAM1 isoforms a and b as well as canine CEACAM1 were amplified from full-length cDNA using primers bovine CEACAM1abN for 5'-GAAGTTATCAGTCGACATGGGGACCCCCTCAG-3', bovine CEACAM1aN rev 5'-ATGGGTCTAGAAAGCTTGGGAGTATGTGGAGGTGTCCAG-3', bovine CEACAM1bN rev 5'-ATGGTCTAGAAAGCTTTGGAGTACGTGGAGGTGTCC-3', canine CEACAM1N for 5'-GAAGTTATCAGTCGACATGGAGCCCCCCTCG-3' and canine CEACAM1N rev 5'-ATGGTCTAGAAAGCTTGGGAATACTTGGAGCTGTCC-3'. All the resulting PCR fragments were cloned into pDNR-Dual using the In-Fusion PCR Cloning Kit (Clontech, Mountain View, CA) and transferred by Cre-mediated recombination into pLPS-3'EGFP (Clontech) resulting in GFP fused to the carboxy-terminus of the expressed proteins. Full-length human CEACAM1-4S and murine CEACAM1-4S were also transferred from pDNR-Dual into pLPS3'mCerulean resulting in mCerulean fused to the carboxy-terminus of the expressed proteins. pLPS3'mCerulean was generated by replacing the GFP coding sequence in pLPS3'EGFP with the cDNA encoding mCerulean  generously provided by D.W. Piston (Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA).
Cell lysis and Western blotting
Cell lysis and Western blotting were performed as described  using a rabbit polyclonal antibody against His-tagged GFP (produced at the animal core facility; University of Konstanz) or a monoclonal antibody against Opa proteins (clone 4B2/C11; generous gift of Marc Achtman, MPI für Infektionsbiologie, Berlin, Germany). Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA).
Binding studies of the different pathogens
Binding studies of the different pathogens with the soluble N-terminal domains of human, murine, bovine and canine CEACAM1 were performed as described . Briefly, 4 × 107 bacteria were added to CEACAM1-N-domain-containing cell culture supernatants in a total volume of 1 ml and incubated for 30 min. After four washing steps, the samples were analysed on a LSR II flow cytometer (BD Bioscience, Heidelberg, Germany) by gating on the bacteria (based on forward and sideward scatter) and measuring bacteria-associated GFP fluorescence. In each case, 10 000 events per sample were obtained.
Gentamicin protection assay
Gentamicin protection assays were conducted as described . Briefly, 5 × 105 293 cells were seeded in 24-well plates coated with 10 μg/ml poly-L-lysine. Cells were infected with 30 bacteria/cell (MOI 30) for two hours. Then, the medium was replaced with DMEM containing 50 μg/ml gentamicin. After 45 min of incubation in gentamicin-containing medium, cells were lysed by the addition of 1% saponin in PBS for 10 min. Suitable dilutions were plated in triplicates on GC agar to determine the number of recovered viable bacteria.
Flow cytometry invasion assay
Bacterial uptake by transfected 293 cells was analysed by flow cytometry as described . Prior to infection, bacteria were labelled with 0.2 μg/ml 5-(6)-carboxyfluorescein-succinylester (fluorescein; Invitrogen-Molecular Probes, Karlsruhe, Germany) in PBS at 37°C for 30 min. Cells were infected with labelled bacteria at an MOI of 30 for 2 h. After infection, cells were washed with PBS and the samples were analysed on a LSR II flow cytometer (BD Bioscience) by gating on the cells based on forward and sideward scatter. Cell-associated fluorescein fluorescence was measured in the presence of 2 mg/ml trypan blue to quench fluorescence of extracellular bacteria and to selectively detect the fluorescence derived from intracellular bacteria. The percentage of fluorescein-positive cells was multiplied by the mean fluorescence intensity of the sample to obtain an estimate of the total number of internalized bacteria (uptake index). In each sample 10,000 cells were counted.
293 cells transfected with the indicated constructs were seeded onto poly-L-lysine- and fibronectin-coated (10 μg/ml and 4 μg/ml, respectively, in PBS) coverslips in 24-well plates. Cells were infected for 2 h with 5-(and-6)-carboxytetramethylrhodamine-succinimidyl- and biotin-labelled OpaCEA-expressing N. gonorrhoeae at an MOI of 20 essentially as described . To discriminate between extracellular and intracellular bacteria, infected samples were fixed with 4% paraformaldehyde in PBS and washed three times with PBS, prior to incubation in blocking buffer (PBS, 10% FCS) for 15 min. Extracellular bacteria were stained with AlexaFluor647-streptavidin (Invitrogen, Karlsruhe, Germany) diluted 1:100 in blocking buffer for 1 h. Following three washes, samples were embedded in mounting medium (Dako, Glastrup, DK).
Samples were viewed with a TCS SP5 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) using a 63×, 1.3 NA Plan Neofluar oil-immersion objective. Fluorescence signals of triple-labelled specimens were serially recorded to avoid bleed-through. Images were digitally processed with NIH ImageJ and merged to yield pseudo-coloured pictures.
Mammalian CEACAM1 orthologues show conserved as well as divergent regions in their amino-terminal domains
Binding of Neisseria gonorrhoeae to the amino-terminal domain of CEACAM1 is human specific
Binding of Moraxella catarrhalis and Neisseria meningitidis to the amino-terminal domain of CEACAM1 is species-specific
Human, but not murine CEACAM1 mediates internalization of Neisseria gonorrhoeae
CEACAM engagement by OpaCEA-expressing N. gonorrhoeae was evaluated through functional analysis of bacterial uptake by the transfected cells. In a first set of experiments, we used an antibiotic protection assay that is based on recovery of viable intracellular bacteria after treatment of the infected cells with gentamicin, an antibiotic that kills extracellular bacteria. In the case of non-opaque gonococci, only very low numbers of bacteria were recovered from murine or human CEACAM1-4S expressing cells similar to the numbers isolated from control transfected cells (Fig. 4B). In contrast, upon infection with OpaCEA-expressing N. gonorrhoeae, 50 - 100 times more bacteria were recovered from cells expressing human CEACAM1 (Fig. 4B). Similar to what has been observed before , both the short and the long isoform of human CEACAM1-4 were able to mediate efficient uptake of the pathogens (Fig. 4B). Importantly, murine CEACAM1-4S was not able to mediate internalization of OpaCEA-expressing N. gonorrhoeae consistent with the lack of bacterial binding to the Igv-like amino-terminal domain of murine CEACAM1 (Fig. 4B).
To further confirm that full length murine CEACAM1-4S does not mediate bacterial internalization, we analysed transfected cells upon infection with fluorescein-labeled bacteria by an established flow cytometry method . Addition of trypan blue quenches the fluorescence emitted by extracellular bacteria, resulting in cell-associated fluorescence signals derived exclusively from intracellular bacteria. In line with the results of the antibiotic protection assay, non-opaque N. gonorrhoeae was not internalized, whereas OpaCEA-expressing bacteria were taken up by cells transfected with human CEACAM1-4S (Fig. 4C). Moreover, cells expressing murine CEACAM1-4S did not harbor intracellular bacteria, further corroborating the notion that OpaCEA proteins of N. gonorrhoeae do not functionally engage CEACAM1 orthologues of other mammalian species (Fig. 4C).
Microscopic determination of Neisseria gonorrhoeae internalization via CEACAM1
Members of the CEACAM family serve as receptors for a variety of Gram-negative bacteria that live on mucosal surfaces of the human body. In an example of convergent evolution these microbes have evolved distinct CEACAM-binding adhesins that seem to promote the colonization of the mucosa. Here we provide evidence that CEACAM-binding adhesins from pathogenic Neisseriae and Moraxella catarrhalis display a high selectivity for human CEACAMs and do not associate with orthologues from non-primate mammalian species. Accordingly, the amino acid sequence divergence, that is particularly high in the CC'C"FG-face of the amino-terminal Igv-like domain of mammalian CEACAM1 orthologues, results in functional differences with regard to bacterial binding. CEACAM-binding bacterial species, which specifically colonize and infect humans, only recognize human CEACAM1 suggesting that the microbial adhesive proteins have co-evolved with their host receptor.
It has been observed earlier, that CEACAM1 orthologues from different mammalian species display high sequence diversity [4, 5]. Starting from a primordial CEACAM1-like gene, CEACAMs seem to have undergone independent duplication and diversification events in different mammalian lineages resulting in an expanded family of closely related surface molecules [2, 26]. Therefore, even within a mammalian order such as the primates it is difficult to assign orthologues genes except for CEACAM1 . As several members of the CEACAM family are exploited by viral and bacterial pathogens, it has been suggested that the driving force behind the rapid diversification of CEACAMs in different mammalian lineages might be the selective pressure by pathogens [3, 28].
An additional example of CEACAM1 recognition by pathogens is found in rodents, where the mouse hepatitis virus strain A59 (MHV-A59), belonging to the coronavirus complex, binds via its spike protein to murine CEACAM1 [29, 30]. Of the two CEACAM1 alleles present in the mouse population, MHV-A59 selectively recognizes CEACAM1a and only marginally binds to the CEACAM1b allele . Therefore, inbred mouse lines that carry the CEACAM1a allele are susceptible, whereas lines carrying the CEACAM1b allele or CEACAM1-deficient mice are resistant to MHV-A59 . However, despite this selectivity for the murine CEACAM1a allele, it has been shown that several MHV strains, including A59 and MHV-2, can utilize human CEACAM1 as well as CEA to infect eukaryotic cells in vitro .
In contrast to this promiscuity of host receptor utilization, our results highlight the specificity of bacterial adhesins for human CEACAMs. Consistent with the strict selectivity of these pathogens for humans as natural host organisms, they only associate with human CEACAM1. Accordingly, the bacteria can efficiently invade only cells that express the human orthologue of CEACAM1, but not the murine orthologue. It is interesting to note, that additional pathogenicity factors of these bacteria show a similar exquisite specialisation for human molecules. For example, the neisserial IgA1 protease  only cleaves human IgA1 molecules, but not IgA molecules from other mammalian species. Similarly, the transferrin-binding protein, that is critical for iron acquisition in the human host, can utilize only transferrin from human sources or from closely related apes such as chimpanzee [35, 36]. Gonococci are also able to escape from host complement attack by recruiting complement component 4b-binding protein (C4bp) . This ability is again specific for human C4bp and even chimpanzee C4bp only provides protection from complement for some, but not all gonococcal strains .
Besides immune escape and nutrient acquisition, our results reveal another area, where these Gram-negative pathogens employ species-specific pathogenicity factors. Clearly, adhesion to the mucosal surface epithelium is the initial step in the colonization by CEACAM-binding bacteria, and the possession of adhesive proteins specifically targeting human CEACAMs might promote this step. However, at the same time this specialization could contribute to the limited host spectrum not only of pathogenic Neisseriae, but also of M. catarrhalis and Haemophilus influenzae.
Recognition of host surface structures is critical for many bacterial pathogens to establish a first foothold in their target organism. Whereas a high degree of specificity might allow intimate binding of the microorganisms to eukaryotic cells, it might at the same time limit the host range of the pathogen. Here we reveal a selective interaction between bacteria and the human form of the cell surface receptor CEACAM1 that correlates with the human-restricted pathogenicity of these microbes. Our analysis not only points to an ongoing pathogen-host co-evolution at the level of receptor-adhesin interaction, but further strengthens the idea that the OpaCEA protein-mediated interaction with human CEACAMs might provide an access point for preventing or limiting infection.
We thank M. Frosch (Universität Würzburg, Germany) and T.F. Meyer (Max-Planck-Institute für Infektionsbiologie, Berlin, Germany) for the bacterial strains used in this study. We thank D.W. Piston (Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN) for Cerulean cDNA, S. Feindler-Boeckh and R. Hohenberger-Bregger for expert technical assistance. MV and CRH acknowledge the support by the Konstanz Research School-Chemical Biology. This study was supported by funds from the DFG (Ha2856/6-1) to C.R.H.
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