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

Fluorescence resonance energy transfer (FRET)-based subcellular visualization of pathogen-induced host receptor signaling

<p>Abstract</p> <p>Background</p> <p>Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET) to visualize pathogen-initiated signaling events in human cells.</p> <p>Results</p> <p>Live-cell microscopy revealed the transient recruitment of the Src family tyrosine kinase Hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3). In cells expressing a CEACAM3 variant lacking the cytoplasmic domain, the Src homology 2 (SH2) domain of Hck (Hck-SH2) was not recruited, even though bacteria still bound to the receptor. FRET measurements on the basis of whole cell lysates revealed intimate binding between Hck-SH2 (using enhanced yellow fluorescent protein (YPet)-Hck-SH2) and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type CEACAM3 (CEACAM3 WT-CyPet) and a flow cytometry-based FRET approach verified this association in intact cells. Using confocal microscopy and acceptor photobleaching, FRET between Hck-SH2 and CEACAM3 was localized to the sites of bacteria-host cell contact.</p> <p>Conclusion</p> <p>These data demonstrate not only the intimate binding of the SH2 domain of Hck to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 in intact cells, but furthermore, FRET measurements allow the subcellular localization of this process during bacterial infection. FRET-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell.</p

Signaling through focal adhesion kinase

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several di€erent signaling proteins such as Src-family PTKs, p130 Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAKmediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout ®broblasts (FAK � ) and how these cells exhibit de®cit

Extracellular IgC2 Constant Domains of CEACAMs Mediate PI3K Sensitivity during Uptake of Pathogens

Several pathogenic bacteria utilize receptors of the CEACAM family to attach to human cells. Binding to different members of this receptor family can result in uptake of the bacteria. Uptake of Neisseria gonorrhoeae, a gram-negative human pathogen, via CEACAMs found on epithelial cells, such as CEACAM1, CEA or CEACAM6, differs mechanistically from phagocytosis mediated by CEACAM3, a CEACAM family member expressed selectively by human granulocytes.We find that CEACAM1- as well as CEACAM3-mediated bacterial internalization are accompanied by a rapid increase in phosphatidylinositol-3,4,5 phosphate (PI(3,4,5)P) at the site of bacterial entry. However, pharmacological inhibition of phosphatidylinositol-3' kinase (PI3K) selectively affects CEACAM1-mediated uptake of Neisseria gonorrhoeae. Accordingly, overexpression of the PI(3,4,5)P phosphatase SHIP diminishes and expression of a constitutive active PI3K increases CEACAM1-mediated internalization of gonococci, without influencing uptake by CEACAM3. Furthermore, bacterial uptake by GPI-linked members of the CEACAM family (CEA and CEACAM6) and CEACAM1-mediated internalization of N. meningitidis by endothelial cells require PI3K activity. Sensitivity of CEACAM1-mediated uptake toward PI3K inhibition is independent of receptor localization in cholesterol-rich membrane microdomains and does not require the cytoplasmic or the transmembrane domain of CEACAM1. However, PI3K inhibitor sensitivity requires the Ig(C2)-like domains of CEACAM1, which are also present in CEA and CEACAM6, but which are absent from CEACAM3. Accordingly, overexpression of CEACAM1 Ig(C2) domains blocks CEACAM1-mediated internalization.Our results provide novel mechanistic insight into CEACAM1-mediated endocytosis and suggest that epithelial CEACAMs associate in cis with other membrane receptor(s) via their extracellular domains to trigger bacterial uptake in a PI3K-dependent manner

CEACAM1 recognition by bacterial pathogens is species-specific

<p>Abstract</p> <p>Background</p> <p>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, <it>Neisseria gonorrhoeae</it>, <it>N. meningitidis</it>, <it>Moraxella catarrhalis</it>, and <it>Haemophilus influenzae </it>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.</p> <p>Results</p> <p>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 <it>Neisseria gonorrhoeae </it>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.</p> <p>Conclusions</p> <p>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.</p

Granulocyte CEACAM3 Is a Phagocytic Receptor of the Innate Immune System that Mediates Recognition and Elimination of Human-specific Pathogens

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are used by several human pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that participates together with CEACAM1 and CEACAM6 in the recognition of CEACAM-binding microorganisms. Here we show that CEACAM3 can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella, and Haemophilus species. CEACAM3- but not CEACAM6-mediated uptake is blocked by dominant-negative versions of the small GTPase Rac. Moreover, CEACAM3 engagement triggers membrane recruitment and increased GTP loading of Rac that are not observed upon bacterial binding to CEACAM6. Internalization and Rac stimulation are also inhibited by compromising the integrity of an immunoreceptor tyrosine-based activation motif (ITAM)–like sequence in the cytoplasmic tail of CEACAM3 or by interference with Src family protein tyrosine kinases that phosphorylate CEACAM3. In contrast to interfering with CEACAM6, blockage of CEACAM3-mediated events reduces the ability of primary human granulocytes to internalize and eliminate CEACAM-binding bacteria, indicating an important role of CEACAM3 in the control of human-specific pathogens by the innate immune system

Differential regulation of cell motility and invasion by FAK

Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK−/− fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK−/− cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK−/− v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK–Src-p130Cas–Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways

The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria

Background: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment