Carcinoembryonic antigens are targeted by diverse strains of typable and non-typable Haemophilus influenzae

Haemophilus influenzae (Hi), a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We show that distinct strains belonging to typable (THi) and non-typable (NTHi) H. influenzae target human carcinoembryonic antigens (the membrane associated CEA family of cell adhesion molecules, are now termed CEACAMs). All strains of H. influenzae biogroup aegyptius (Hi-aeg) and more than 70% of THi and NTHi strains tested specifically recognize CEACAMI-Fc soluble constructs. Furthermore, transfection of Chinese hamster ovary cells with human CEACAM1 cDNA alone was sufficient for promoting Hi interactions with the transfected cells. The majority of the Hi-aeg strains tested interacted with soluble constructs containing only the N-terminal domain. In contrast, several THi and NTHi strains reacted with soluble constructs only when additional extracellular A and B domains of the receptor were present. The use of monoclonal antibodies confirmed that THi and NTHi strains also interact primarily at the N-domain. We used site-directed mutants of CEACAM1 that contained substitutions at surface exposed amino acids and a molecular model of the N-domain to identify the residues involved in interactions with Hi ligands. The studies show that a common region exposed at the CFG face of the molecule is targeted by diverse Hi strains. However, mutation at distinct sites within this area affected the interactions of distinct strains signifying the potential for tissue tropism via this receptor. Analyses of the molecular basis of interaction with human cell lines and purified CEA show that Hi strains, especially those belonging to Hi-aeg, interact with multiple CEACAMs. Because Neisseria meningitidis (Nm) strains are also known to bind at the CFG face of the receptor, we used Nm and Hi strains in co-infection experiments and demonstrate competition between these mucosal pathogens in colonization of target cells via CEACAMs.     

Critical determinants of host receptor targeting by Neisseria meningitidis and Neisseria gonorrhoeae: identification of Opa adhesiotopes on the N-domain of CD66 molecules

The human pathogens Neisseria meningitidis and Neisseria gonorrhoeae express a family of variable outer membrane opacity-associated (Opa) proteins that recognize multiple human cell surface receptors. Most Opa proteins target the highly conserved N-terminal domain of the CD66 family of adhesion molecules, although a few also interact with heparan sulphate proteoglycans. In this study, we observed that at least two Opa proteins of a N. meningitidis strain C751 have the dual capacity to interact with both receptors. In addition, all three Opa proteins of C751 bind equally well to HeLa cells transfected with cDNA encoding the carcinoembryonic antigen [CEA (CD66e)] subgroup of the CD66 family, but show distinct tropism for CGM1- (CD66d) and NCA (CD66c)-expressing cells. Because the C751 Opa proteins make up distinct structures via the surface-exposed hypervariable domains (HV-1 and HV-2), these combinations appear to be involved in tropism for the distinct CD66 subgroups. To define the determinants of receptor recognition, we used mutant proteins of biliary glycoprotein [BGP (CD66a)] carrying substitutions at several predicted exposed sites in the N-domain and compared their interactions with several Opa proteins of both N. meningitidis and N. gonorrhoeae. The observations applied to the molecular model of the BGP N-domain that we constructed show that the binding of all Opa proteins tested occurs at the non-glycosylated (CFG) face of the molecule and, in general, appears to require Tyr-34 and Ile-91. Further, efficient interaction of distinct Opa proteins depends on different non-adjacent amino acids. In the three-dimensional model, these residues lie in close proximity to Tyr-34 and Ile-91 at the CFG face, making continuous binding domains (adhesiotopes). The epitope of the monoclonal antibody YTH71.3 that inhibits Opa/CD66 interactions was also identified within the Opa adhesiotopes on the N-domain. These studies define the molecular basis that directs the Opa specificity for the CD66 family and the rationale for tropism of the Opa proteins for the CD66 subgroups.     

Synthetic peptides from the N-domains of CEACAMs activate neutrophils.

Four members of the carcinoembryonic antigen family, CEACAM1, CEACAM8, CEACAM6 and CEACAM3, recognized by CD66a, CD66b, CD66c and CD66d monoclonal antibodies (mAb), respectively, are expressed on human neutrophils. CD66a, CD66b, CD66c and CD66d mAb binding to neutrophils triggers an activation signal that regulates the adhesive activity of CD11/CD18, resulting in an increase in neutrophil adhesion to human umbilical vein endothelial cells. Molecular modeling of CEACAM1 using IgG and CD4 as models has been performed, and three peptides from the N-terminal domain were found to increase neutrophil adhesion to human umbilical vein endothelial cell monolayers. The peptides were 14 amino acids in length and were predicted to be present at loops and turns between beta-sheets. To better understand the amino acid sequences critical for this biological activity, in the present study we examined the other neutrophil CEACAMs and the highly homologous CEACAM, CEA. Molecular modeling of the N-terminal domains of human CEACAM8, -6, -3 and CEA was performed. Twenty peptides, each 14 amino acids in length, that were homologous to the previously reported peptides from the N-domains of CEACAM1, were synthesized and tested for their ability to alter neutrophil adhesion. Only one new peptide, from the N-domain of CEA, was found to increase neutrophil adhesion, and this peptide differed from the corresponding CEACAM1 peptide by only a single conservative amino acid substitution. Importantly, minor amino acid differences between active and inactive homologous peptides suggest regions of these peptides that are critical for biological activity. The data suggest that the regions SMPF of peptide CD66a-1, QLFG of peptide CD66a-2 and NRQIV of peptide CD66a-3 are critical for the activities of these peptides, and for the native CEACAMs.     

Expression of pathogen-like Opa adhesins in commensal Neisseria: genetic and functional analysis

Several species of commensal Neisseriae (Cn) may colonize the human nasopharynx, but little is known about their adhesion mechanisms. We have investigated structural and functional similarities between adhesins of Cn and of Neisseria meningitidis (Nm), also a frequent colonizer of the nasopharynx. In this study, we demonstrate the expression of Opa-like proteins in nine strains of Cn. Phylogenetic analysis segregated the majority of the Cn Opa in a cluster separated from the pathogenic cluster with a few exceptions. One Opa, which located within the pathogenic cluster, was strikingly similar (74%) to an Opa of a Neisseria gonorrhoeae (Ng) strain and, like Ng, it lacked the extra Y¹¹ or the ¹³⁶DKF¹³⁸ triplet insert, which are conserved among many N. meningitidis Opa proteins. Most importantly, the majority of the Cn Opa proteins were able to interact with human CEACAM1 (CD66a) molecules, previously identified as receptors for pathogenic Opa proteins. By the use of CEACAM1 N-domain mutants, we demonstrate that Cn Opa target the same region of the N-domain of the receptor as that used by Nm. Furthermore, Cn strains bound to cell-expressed human CEACAM1. In competition assays, adherent Cn strain C450, exhibiting high affinity for CEACAM1, was not displaced by a Nm isolate and vice versa . But in simultaneous incubation, Nm out-competed the Cn strain. This is the first study to demonstrate the expression of adhesins in Cn that are structurally and functionally closely related to pathogenic adhesins. The studies imply that some Cn have the potential to occupy and thus compete with the pathogens for receptors on human mucosa, their common and exclusive niche.     

A subfamily of Dr adhesins of Escherichia coli bind independently to decay-accelerating factor and the N-domain of carcinoembryonic antigen

Escherichia coli expressing the Dr family of adhesins adheres to epithelial cells by binding to decay-accelerating factor (DAF) and carcinoembryonic antigen (CEA)-related cell surface proteins. The attachment of bacteria expressing Dr adhesins to DAF induces clustering of DAF around bacterial cells and also recruitment of CEA-related cell adhesion molecules. CEA, CEACAM1, and CEACAM6 have been shown to serve as receptors for some Dr adhesins (AfaE-I, AfaE-III, DraE, and DaaE). We demonstrate that AfaE-I, AfaE-V, DraE, and DaaE adhesins bind to the N-domain of CEA. To identify the residues involved in the N-CEA/DraE interaction, we performed SPR binding analyses of naturally occurring variants and a number of randomly generated mutants in DraE and N-CEA. Additionally, we used chemical shift mapping by NMR to determine the surface of DraE involved in N-CEA binding. These results show a distinct CEA binding site located primarily in the A, B, E, and D strands of the Dr adhesin. Interestingly, this site is located opposite to the beta-sheet encompassing the previously determined binding site for DAF, which implies that the adhesin can bind simultaneously to both receptors on the epithelial cell surface. The recognition of CEACAMs from a highly diverse DrCEA subfamily of Dr adhesins indicates that interaction with these receptors plays an important role in niche adaptation of E. coli strains expressing Dr adhesins.     

The specific innate immune receptor CEACAM3 triggers neutrophil bactericidal activities via a Syk kinase-dependent pathway

The human-restricted pathogens Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae and Moraxella catarrhalis colonize host tissues via carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs). One such receptor, CEACAM3, acts in a host-protective manner by orchestrating the capture and engulfment of invasive bacteria by human neutrophils. Herein, we show that bacterial binding to CEACAM3 causes recruitment of the cytoplasmic tyrosine kinase Syk, resulting in the phosphorylation of both CEACAM3 and Syk. This interaction is specific for the immunoreceptor tyrosine-based activation motif (ITAM) in the CEACAM3 cytoplasmic domain. While dispensable for the phagocytic uptake of single bacteria by CEACAM3, Syk is necessary for internalization when cargo size increases or when the density of CEACAM-binding ligand on the cargo surface is below a critical threshold. Moreover, Syk engagement is required for an effective bacterial killing response, including the neutrophil oxidative burst and degranulation functions in response to N. gonorrhoeae. These data reveal CEACAM3 as a specific innate immune receptor that mediates the opsonin-independent clearance of CEACAM-binding bacteria via Syk, a molecular trigger for functional immunoreceptor responses of both the adaptive (TCR, BCR, FcR) and innate (Dectin-1, CEACAM3) immune systems.     

Selection for functional diversity drives accumulation of point mutations in Dr adhesins of Escherichia coli

Immune escape is considered to be the driving force behind structural variability of major antigens on the surface of bacterial pathogens, such as fimbriae. In the Dr family of Escherichia coli adhesins, structural and adhesive functions are carried out by the same subunit. Dr adhesins have been shown to bind decay-accelerating factor (DAF), collagen IV, and carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). We show that genes encoding Dr adhesins from 100 E. coli strains form eight structural groups with a high level of amino acid sequence diversity between them. However, genes comprising each group differ from each other by only a small number of point mutations. Out of 66 polymorphisms identified within the groups, only three were synonymous mutations, indicating strong positive selection for amino acid replacements. Functional analysis of intragroup variants comprising the Dr haemagglutinin (DraE) group revealed that the point mutations result in distinctly different binding phenotypes, with a tendency of increased affinity to DAF, decreased sensitivity of DAF binding to inhibition by chloramphenicol, and loss of binding capability to collagen, CEACAM3 and CEACAM6. Thus, variability by point mutation of major antigenic proteins on the bacterial surface can be a signature of selection for functional modification.     

Profiling of bacterial adhesin--host receptor recognition by soluble immunoglobulin superfamily domains

Several gram-negative human pathogens recognize members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Pathogenic Neisseriae employ distinct isoforms of the colony opacity-associated proteins (Opa(CEA) proteins) to bind to the amino-terminal domains of CEACAMs. Here we present a novel approach to rapidly determine the CEACAM-binding properties of single bacteria. Expression of the isolated amino-terminal domains of various CEACAMs in eukaryotic cells yields soluble probes that selectively recognize Opa(CEA)-expressing bacteria in a pull-down assay format. Furthermore, by expressing soluble CEACAMs as fusions to green-fluorescent protein (CEACAM-N-GFP), CEACAM-binding bacteria can be decorated with a fluorescent label and analysed by flow cytometry allowing the specific detection of receptor binding events on the level of single bacteria. Besides its potential for rapid and quantitative analysis of pathogen-receptor interactions, this novel approach allows the detection of receptor recognition in heterogeneous bacterial populations and might represent a valuable tool for profiling the host binding capabilities of various microorganisms.     

Opa proteins and CEACAMs: pathways of immune engagement for pathogenic Neisseria

Neisseria meningitidis and Neisseria gonorrhoeae are globally important pathogens, which in part owe their success to their ability to successfully evade human immune responses over long periods. The phase-variable opacity-associated (Opa) adhesin proteins are a major surface component of these organisms, and are responsible for bacterial adherence and entry into host cells and interactions with the immune system. Most immune interactions are mediated via binding to members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family. These Opa variants are able to bind to different receptors of the CEACAM family on epithelial cells, neutrophils, and T and B lymphocytes, influencing the innate and adaptive immune responses. Increased epithelial cell adhesion creates the potential for prolonged infection, invasion and dissemination. Furthermore, Opa proteins may inhibit T-lymphocyte activation and proliferation, B-cell antibody production, and innate inflammatory responses by infected epithelia, in addition to conferring increased resistance to antibody-dependent, complement-mediated killing. While vaccines containing Opa proteins could induce adhesion-blocking and bactericidal antibodies, the consequence of CEACAM binding by a candidate Opa-containing vaccine requires further investigation. This review summarizes current knowledge of the immunological consequences of the interaction between meningococcal and gonococcal Opa proteins and human CEACAMs, considering the implications for pathogenesis and vaccine development.     

Outer membrane protein P1 is the CEACAM‐binding adhesin of Haemophilus influenzae

Haemophilus influenzae is a Gram‐negative pathogen colonizing the upper respiratory tract mucosa. H. influenzae is one of several human‐restricted bacteria, which bind to carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs) on the epithelium leading to bacterial uptake by the eukaryotic cells. Adhesion to CEACAMs is thought to be mediated by the H. influenzae outer membrane protein (OMP) P5. However, CEACAMs still bound to H. influenzae lacking OMP P5 expression, and soluble CEACAM receptor ectodomains failed to bind to OMP P5, when heterologously expressed in Escherichia coli. Screening of a panel of H. influenzae OMP mutants revealed that lack of OMP P1 completely abrogated CEACAM binding and supressed CEACAM‐mediated engulfment of H. influenzae by epithelial cells. Moreover, ectopic expression of OMP P1 in E. coli was sufficient to induce CEACAM binding and to promote attachment to and internalization into CEACAM‐expressing cells. Interestingly, OMP P1 selectively recognizes human CEACAMs, but not homologs from other mammals and this binding preference is preserved upon expression in E. coli. Together, our data identify OMP P1 as the bona fide CEACAM‐binding invasin of H. influenzae. This is the first report providing evidence for an involvement of the major OMP P1 of H. influenzae in pathogenesis.     

Extracellular N-domain alone can mediate specific heterophilic adhesion between members of the carcinoembryonic antigen family, CEACAM6 and CEACAM8

The domain(s) responsible for the specific heterophilic adhesion between two members of the carcinoembryonic antigen (CEA) family, CEACAM6 and CEACAM8, both of which with three extracellular domains, were investigated using Chinese hamster ovary (CHO) transfectants expressing chimeric antigens. Using a chimeric antigen in which the N-domain, a sole extracellular domain, of CEACAM3 was substituted with that of CEACAM6, it was shown that the N-domain of CEACAM6 alone was able to mediate specific adhesion to CEACAM8. Furthermore, the chimeric antigen was shown to bind significantly to chimeric CEA whose N-domain was substituted with that of CEACAM8, but not to unsubstituted CEA. These results demonstrate that the N-domain alone is sufficient and other domains of CEACAM6 or CEACAM8 are not required for this specific binding. We therefore propose a model of heterophilic interaction between the N-domains, which is distinct from that of CEA-CEA homophilic binding.     

The CEACAM1 transmembrane domain, but not the cytoplasmic domain, directs internalization of human pathogens via membrane microdomains

Several bacterial pathogens exploit carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to promote attachment and uptake into eukaryotic host cells. The widely expressed isoform CEACAM1 is involved in cell–cell adhesion, regulation of cell proliferation, insulin homeostasis, and neo-angiogenesis, processes that depend on the cytoplasmic domain of CEACAM1. By analysing the molecular requirements for CEACAM1-mediated internalization of bacteria, we surprisingly find that the CEACAM1 cytoplasmic domain is completely obsolete for bacterial uptake. Accordingly, CEACAM1-4L as well as a CEACAM1 mutant with a complete deletion of the cytoplasmic domain (CEACAM1 ΔCT) promote equivalent internalization of several human pathogens. CEACAM1-4L- and CEACAM1 ΔCT-mediated uptake proceeds in the presence of inhibitors of actin microfilament dynamics, which is in contrast to CEACAM3-mediated internalization. Bacteria-engaged CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3, localize to a gangliosid GM1- and GPI-anchored protein-containing portion of the plasma membrane. In addition, interference with cholesterol-rich membrane microdomains severely blocks bacterial uptake via CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3. Similar to GPI-anchored CEACAM6, both CEACAM1-4L as well as CEACAM1 ΔCT partition into a low-density, Triton-insoluble membrane fraction upon receptor clustering, whereas CEACAM3 is not detected in this fraction. Bacterial uptake by truncated CEACAM1 or chimeric CEACAM1/CEACAM3 molecules reveals that the transmembrane domain of CEACAM1 is responsible for its association with membrane microdomains. Together, these data argue for a functional role of lipid rafts in CEACAM1-mediated endocytosis that is promoted by the transmembrane domain of the receptor and that might be relevant for CEACAM1 function in physiologic settings.     

Binding of Dr adhesins of Escherichia coli to carcinoembryonic antigen triggers receptor dissociation

Carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) are host receptors for the Dr family of adhesins of Escherichia coli. To define the mechanism for binding of Dr adhesins to CEACAM receptors, we carried out structural studies on the N-terminal domain of CEA and its complex with the Dr adhesin. The crystal structure of CEA reveals a dimer similar to other dimers formed by receptors with IgV-like domains. The structure of the CEA/Dr adhesin complex is proposed based on NMR spectroscopy and mutagenesis data in combination with biochemical characterization. The Dr adhesin/CEA interface overlaps appreciably with the region responsible for CEA dimerization. Binding kinetics, mutational analysis and spectroscopic examination of CEA dimers suggest that Dr adhesins can dissociate CEA dimers prior to the binding of monomeric forms. Our conclusions include a plausible mechanism for how E. coli, and perhaps other bacterial and viral pathogens, exploit CEACAMs. The present structure of the complex provides a powerful tool for the design of novel inhibitory strategies to treat E. coli infections.     

The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs to facilitate translocation of the oncoprotein CagA

Helicobacter pylori infects half of the world's population, and strains that encode the cag type IV secretion system for injection of the oncoprotein CagA into host gastric epithelial cells are associated with elevated levels of cancer. CagA translocation into host cells is dependent on interactions between the H. pylori adhesin protein HopQ and human CEACAMs. Here, we present high‐resolution structures of several HopQ‐CEACAM complexes and CEACAMs in their monomeric and dimeric forms establishing that HopQ uses a coupled folding and binding mechanism to engage the canonical CEACAM dimerization interface for CEACAM recognition. By combining mutagenesis with biophysical and functional analyses, we show that the modes of CEACAM recognition by HopQ and CEACAMs themselves are starkly different. Our data describe precise molecular mechanisms by which microbes exploit host CEACAMs for infection and enable future development of novel oncoprotein translocation inhibitors and H. pylori‐specific antimicrobial agents.     

Mapping the binding domains on meningococcal Opa proteins for CEACAM1 and CEA receptors

The opacity (Opa) proteins of pathogenic Neisseria spp. are adhesins, which play an important role in adhesion and invasion of host cells. Most members of this highly variable family of outer membrane proteins can bind to the human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). Several studies have identified the Opa-binding region on the CEACAM receptors; however, not much is known about the binding sites on the Opa proteins for the corresponding CEACAM-receptors. The high degree of sequence variation in the surface-exposed loops of Opa proteins raises the question how the binding sites for the CEACAM receptors are conserved. Neisseria meningitidis strain H44/76 possesses four different Opa proteins, of which OpaA and OpaJ bind to CEACAM1, while OpaB and OpaD bind to CEACAM1 and CEA. A sequence motif involved in binding to CEACAM1 was identified by alanine scanning mutagenesis of those amino acid residues conserved within the hypervariable (HV) regions of all four Opa proteins. Hybrid Opa variants with different combinations of HV-1 and HV-2 derived from OpaB and OpaJ showed a reduced binding to CEACAM1 and CEA, indicating that particular combinations of HV-1 and HV-2 are required for the Opa binding capacity. Homologue scanning mutagenesis was used to generate more refined hybrids containing novel combinations of OpaB and OpaJ sequences within HV-1 and HV-2. They could be used to identify residues determining the specificity for CEA binding. The combined results obtained with mutants and hybrids strongly suggest the existence of a conserved binding site for CEACAM receptors by the interaction of HV-1 and HV-2 regions.     

H-NMR study of rabbit skeletal muscle troponin C: Ca(2+)-dependent interaction with mastoparan.

1H-NMR spectroscopy is employed to study the interaction between rabbit skeletal muscle troponin (C (TnC) and wasp venom tetradecapeptide mastoparan. We monitored the spectral change of the following species of TnC as a function of mastoparan concentration: apoTnC, Ca(2+)-saturated TnC (Ca4TnC) and Ca(2+)-half loaded TnC (Ca2TnC). When apo-TnC is titrated with mastoparan, line-broadening is observed for the ring-current shifted resonance of Phe-23, Ile-34, Val-62 and Phe-72 and the downfield-shifted CH alpha-resonances of Asp-33, Thr-69 and Asp-71; these residues are located in the N-domain. When Ca4TnC is titrated with mastoparan, chemical shift change is observed for the ring-current shifted resonances of Phe-99, Ile-110 and Phe-148 and the downfield-shifted CH alpha-resonances of Asn-105, Ala-106, Ile-110 and Ile-146 and aromatic resonance of Tyr-109 and His-125; these residues are located in the C-domain. The resonance of Phe-23, Asp-33, Asp-71, Phe-72, Phe-99, Tyr-109, Ile-146, His-125 and Phe-148 in both N- and C-domains changes when Ca2TnC is titrated with mastoparan. These results suggest that mastoparan binds to the N-domain of apo-TnC, the C-domain of Ca4TnC and the N- and C-domains of Ca2TnC; the hydrophobic cluster in each domain is involved in binding. As mastoparan binds to TnC, the above resonances shift to their normal chemical shift positions. The stability of the cluster and the beta-sheet is reduced by mastoparan-binding. These results suggest that the conformation of the hydrophobic cluster and the neighboring beta-sheet change to a loose form. The stability of the N-domain of Ca2TnC and Ca4TnC increases when these species bind 1 mol of mastoparan at the C-domain. These results suggest a mastoparan-induced interaction between the N- and C-domains of TnC.     

The critical role of residues 43R and 44Q of carcinoembryonic antigen cell adhesion molecules-1 in the protection from killing by human NK cells

The multifunctional carcinoembryonic Ag cell adhesion molecule (CEACAM)1 protein has recently become the focus of intense immunological research. We have previously shown that the CEACAM1 homophilic interactions inhibit the killing activity of NK cells. This novel inhibitory mechanism plays a key role in melanoma immune evasion, inhibition of decidual immune response, and controlling NK autoreactivity in TAP2-deficient patients. These roles are mediated mainly by homophilic interactions, which are mediated through the N-domain of the CEACAM1. The N-domain of the various members of the CEACAM family shares a high degree of similarity. However, it is still unclear which of the CEACAM family members is able to interact with CEACAM1 and what are the amino acid residues that control this interaction. In this study we demonstrate that CEACAM1 interacts with CEACAM5, but not with CEACAM6. Importantly, we provide the molecular basis for CEACAM1 recognition of various CEACAM family members. Sequence alignment reveals a dichotomy among the CEACAM family members: both CEACAM1 and CEACAM5 contain the R and Q residues in positions 43 and 44, respectively, whereas CEACAM3 and CEACAM6 contain the S and L residues, respectively. Mutational analysis revealed that both 43R and 44Q residues are necessary for CEACAM1 interactions. Implications for differential expression of CEACAM family members in tumors are discussed.     

Quaternary structure of coronavirus spikes in complex with carcinoembryonic antigen-related cell adhesion molecule cellular receptors

Oligomeric spike (S) glycoproteins extend from coronavirus membranes. These integral membrane proteins assemble within the endoplasmic reticulum of infected cells and are subsequently endoproteolyzed in the Golgi, generating noncovalently associated S1 and S2 fragments. Once on the surface of infected cells and virions, peripheral S1 fragments bind carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors, and this triggers membrane fusion reactions mediated by integral membrane S2 fragments. We focused on the quaternary structure of S and its interaction with CEACAMs. We discovered that soluble S1 fragments were dimers and that CEACAM binding was entirely dependent on this quaternary structure. However, two differentially tagged CEACAMs could not co-precipitate with the S dimers, suggesting that binding sites were closely juxtaposed in the dimer (steric hindrance) or that a single CEACAM generated global conformational changes that precluded additional interactions (negative cooperativity). CEACAM binding did indeed alter S1 conformations, generating alternative disulfide linkages that were revealed on SDS gels. CEACAM binding also induced separation of S1 and S2. Differentially tagged S2 fragments that were free of S1 dimers were not co-precipitated, suggesting that S1 harbored the primary oligomerization determinants. We discuss the distinctions between the S.CEACAM interaction and other virus-receptor complexes involved in receptor-triggered entry.     

CEACAM1 dynamics during neisseria gonorrhoeae suppression of CD4+ T lymphocyte activation

Neisseria gonorrhoeae colony opacity-associated (Opa) proteins bind to human carcinoembryonic antigen cellular adhesion molecules (CEACAM) found on host cells including T lymphocytes. Opa binding to CEACAM1 suppresses the activation of CD4(+) T cells in response to a variety of stimuli. In this study, we use primary human CD4(+) T cells isolated from peripheral blood to define the molecular events occurring subsequent to Opa-CEACAM1 binding. We establish that, in contrast to other cell types, T cells do not engulf N. gonorrhoeae upon CEACAM1 binding. Instead, the bacteria recruit CEACAM1 from intracellular stores and maintain it on the T cell surface. Upon TCR ligation, the co-engaged CEACAM1 becomes phosphorylated on tyrosine residues within the ITIMs apparent in the cytoplasmic domain. This allows the recruitment and subsequent activation of the src homology domain 2-containing tyrosine phosphatases SHP-1 and SHP-2 at the site of bacterial attachment, which prevents the normal tyrosine phosphorylation of the CD3zeta-chain and ZAP-70 kinase in response to TCR engagement. Combined, this dynamic response allows the bacteria to effectively harness the coinhibitory function of CEACAM1 to suppress the adaptive immune response at its earliest step.