Neisseria meningitidis Has Two Independent Modes of Recognizing Its Human Receptor CEACAM1

BACKGROUND: Several human-restricted gram-negative bacteria exploit carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) for host colonization. For example, Neisseria meningitidis engages these human receptors via outer membrane proteins of the colony opacity-associated (Opa) protein family triggering internalization into non-phagocytic cells. PRINCIPAL FINDINGS: We report that a non-opaque strain of N. meningitidis selectively interacts with CEACAM1, but not other CEACAM family members. Using functional assays of bacterial adhesion and internalisation, microscopic analysis, and a panel of CEACAM1 deletion mutants we demonstrate that the engagement of CEACAM1 by non-opaque meningococci occurs in a manner distinct from Opa protein-mediated association. In particular, the amino-terminal domain of CEACAM1 is necessary, but not sufficient for Opa protein-independent binding, which requires multiple extracellular domains of the human receptor in a cellular context. Knock-down of CEACAM1 interferes with binding to lung epithelial cells, whereas chemical or pharmacological disruption of host protein glycosylation does not abrogate CEACAM1 recognition by non-opaque meningococci. The previously characterized meningococcal invasins NadA or Opc do not operate in a CEACAM1-dependent manner. CONCLUSIONS: The results demonstrate a mechanistically distinct, Opa protein-independent interaction between N. meningitidis and human CEACAM1. Our functional investigations suggest the presence of a second CEACAM1-binding invasin on the meningococcal surface that associates with the protein backbone and not the carbohydrate structures of CEACAM1. The redundancy in meningococcal CEACAM1-binding factors further highlights the important role of CEACAM recognition in the biology of this human-adapted pathogen

CEACAMs : their role in physiology and pathophysiology

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) belong to a group of mammalian immunoglobulin-related glycoproteins. They are involved in cell cell recognition and modulate cellular processes that range from the shaping of tissue architecture and neovascularization to the regulation of insulin homeostasis and T-cell proliferation. CEACAMs have also been identified as receptors for host-specific viruses and bacteria in mice and humans, respectively, making these proteins an interesting example of pathogen host co-evolution. Forward and reverse genetics in the mouse now provide powerful novel models to elucidate the action of CEACAM family members in vivo

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 Opac[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

Known meningococcal invasins do not contribute to CEACAM1-mediated internalization of non-opaque meningococci.

<p>(<b>A</b>) 293 cells were transfected with pcDNA CEACAM1-4L-HA or with the empty control vector (pcDNA). Two days after transfection, cells were infected for 2 h with <i>E. coli</i> strains recombinantly expressing NadA, Opc, or the parent strain. The number of internalised bacteria was determined by gentamicin protection assays. Results represent mean ± standard deviation of three independent experiments done in triplicate and show the percentage of recovered bacteria compared to CEACAM1. (<b>B</b>) Cells as in (A) were infected with Opa_CEA-expressing gonococci (Ngo Opa_CEA) and the number of internalised bacteria was determined as in (A). (<b>C</b>) Expression of recombinant NadA and Opc in <i>E. coli</i>. Expression of Opc was analysed in bacterial lysates by Western Blot using monoclonal anti-Opc antibody. Expression of recombinant NadA in <i>E. coli</i> was analysed by SDS-PAGE after induction with IPTG. The multimeric, functional form of NadA is indicated (arrow). As a negative control, non-induced <i>E. coli</i> or <i>E. coli</i> not transformed with recombinant proteins were used.</p

CEACAM1 – but not other members of the CEACAM family - mediates uptake of Opa protein-negative meningococci.

<p>293 cells were transfected with constructs encoding CEACAM1, CEACAM3, CEA, CEACAM6, CEACAM7, CEACAM8 or an empty control plasmid (pcDNA). Two days after transfection cells were infected for 2 h with (<b>A</b>) Opa protein-negative meningococci (Nm Opa-) or (<b>B</b>) Opa protein-positive meningococci (Nm Opa_CEA) and the number of internalised bacteria was determined by gentamicin protection assays. Results represent mean ± standard deviation of three independent experiments done in triplicate. Shown is the percentage of recovered bacteria compared to CEACAM1-expressing cells.</p

Opa protein-negative meningococci associate with and invade into CEACAM1-expressing cells.

<p>(<b>A</b>) Lysates of Opa protein-positive and Opa protein-negative <i>N. meningitidis</i> MC 58 (Nm) and <i>N. gonorrhoeae</i> MS11 (Ngo) were analysed for Opa protein expression using a monoclonal anti Opa protein-antibody. (<b>B</b>) 293 cells were transfected with an empty control vector (pcDNA) or a CEACAM1-encoding vector. Whole cell lysates (WCLs) of the transfected cells were analysed by Western blotting with a CEACAM-specific monoclonal antibody. (<b>C</b>) 293 cells as in (B) were infected for 2 h with Opa protein-negative (Nm Opa-) or Opa protein-positive meningococci (Nm Opa_CEA) or the corresponding gonococcal strains (Ngo Opa- or Ngo Opa_CEA, respectively). The number of internalised bacteria was determined by gentamicin protection assays. Results represent mean ± standard deviation of three independent experiments done in triplicate. Shown is the percentage of recovered bacteria compared to CEACAM1-expressing cells (left axis) as well as the absolute number of internalized colony forming units (right axis). (<b>D</b>) 293 cells as in (B) were infected for 2 h with Opa protein-negative meningococci (Nm Opa-). The number of cell-associated bacteria was determined by an adherence assay. Results represent mean ± standard deviation of one representative experiment done in triplicate. Shown is the percentage of cell-associated bacteria compared to CEACAM1-expressing cells (left axis) as well as the absolute number of cell-associated colony forming units (right axis). (<b>E</b>) The four <i>opa-</i>genes encoded in the genome of the Opa protein-negative meningococcal strain were cloned in <i>E. coli</i>. Expression of recombinant Opa1, Opa2, Opa3, or Opa4 was induced or not by IPTG and bacterial lysates were analysed for Opa protein-expression as in (A).</p

CEACAMs: their role in physiology and pathophysiology

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) belong to a group of mammalian immunoglobulinrelated glycoproteins. They are involved in cell-cell recognition and modulate cellular processes that range from the shaping of tissue architecture and neovascularization to the regulation of insulin homeostasis and T-cell proliferation. CEACAMs have also been identified as receptors for host-specific viruses and bacteria in mice and humans, respectively, making these proteins an interesting example of pathogen-host co-evolution. Forward and reverse genetics in the mouse now provide powerful novel models to elucidate the action of CEACAM family members in vivo. Introduction In multicellular organisms, cell-cell adhesion is vital to guide cells to their proper location during embryonic development and to mediate the integration of single cells into functional tissues and organs. Members of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs) constitute a large group of cell surface glycoproteins with ancient roots in the animal kingdom that specialize in cell-cell adhesion. All IgCAMs posses at least one immunoglobulin (Ig)-like domain, a compact structure $85-110 amino acids long characterized by two b-sheets packed against each other Another prominent member of the IgCAM superfamily is the carcinoembryonic antigen (CEA), which is involved in homotypic and heterotypic interactions with other closely related IgCAMs and which constitutes a clinically relevant diagnostic marker in the surveillance of colon tumors. Together with its paralogues, CEA has been grouped in the CEA-related cell adhesion molecule (CEACAM) family, a subdivision of IgCAMs so far only known from mammals CEA In the mid-1960s, CEA was identified as a prominent tumor-associated antigen in human colon cancer Current Opinion in Cell Biology CEACAM1 in angiogenesis A number of previous studies have implicated CEA-CAM1 in angiogenesis. For example, soluble CEACAM1 exhibits pro-angiogenic effects by stimulating the proliferation, chemotaxis and capillary-like tube formation of human microvascular endothelial cells in vitro, as well as increasing the vascularization of the chorioallantoic membrane of chicken embryos in vivo Analyses with cDNA arrays revealed that CEACAM1 is part of the hypoxia-induced genetic program, as it is prominently induced on the microvessels of the left ventricle of chronically hypoxic rats as well as upon myocardial infarction of mice CEACAM1 helps to limit the action of insulin Besides its function in angiogenesis, CEACAM1 plays a role in the regulation of insulin action. Insulin binding to the insulin receptor (IR) promotes the phosphorylation of specific cellular target molecules, including members of the insulin receptor substrate (IRS) family, Shc, and CEACAM1. Indeed, the &apos;L&apos; isoform of CEACAM1 is a direct substrate of the IR, which phosphorylates CEA-CAM1 at Tyr-488, and this modification requires an intact Ser-503 residue in the cytoplasmic tail of CEA-CAM1 (reviewed in [31] To investigate the role of CEACAM1 on hepatic insulin metabolism in vivo, transgenic mice (L-SACC1) overexpressing a dominant-negative, phosphorylation-defective mutant of CEACAM1 (CEACAM1 S503A) in hepatocytes were generated CEACAM1 knock-out mice and the role of CEACAM1 in tumorigenesis Surprisingly, CEACAM1 knock-out mice (CEACAM

The granulocyte orphan receptor CEACAM4 is able to trigger phagocytosis of bacteria.

Human granulocytes express several glycoproteins of the CEACAM family. One family member, CEACAM3, operates as a single-chain phagocytic receptor, initiating the detection, internalization, and destruction of a limited set of gram-negative bacteria. In contrast, the function of CEACAM4, a closely related protein, is completely unknown. This is mainly a result of a lack of a specific ligand for CEACAM4. By generating chimeric proteins containing the extracellular bacteria-binding domain of CEACAM3 and the transmembrane and cytoplasmic part of CEACAM4 (CEACAM3/4) we demonstrate that this chimeric receptor can trigger efficient phagocytosis of attached particles. Uptake of CEACAM3/4-bound bacteria requires the intact ITAM of CEACAM4, and this motif is phosphorylated by Src family PTKs upon receptor clustering. Furthermore, SH2 domains derived from Src PTKs, PI3K, and the adapter molecule Nck are recruited and associate directly with the phosphorylated CEACAM4 ITAM. Deletion of this sequence motif or inhibition of Src PTKs blocks CEACAM4-mediated uptake. Together, our results suggest that this orphan receptor of the CEACAM family has phagocytic function and prompt efforts to identify CEACAM4 ligands