Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is an immunoglobulin-related receptor expressed on human granulocytes. CEACAM3 functions as a single chain phagocytotic receptor recognizing Gram-negative bacteria such as Neisseria gonorrhoeae, which possess CEACAMbinding adhesins on their surface. The cytoplasmic domain of CEACAM3 contains an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is phosphorylated upon receptor engagement. Here we show that the SH2 domains of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K) bind to tyrosine residue 230 of CEACAM3 in a phosphorylationdependent manner. PI3K is rapidly recruited and directly associates with CEACAM3 upon bacterial binding as shown by FRET analysis. Although PI3K activity is not required for efficient uptake of the bacteria by CEACAM3-transfected cells or primary human granulocytes, it is critical for the stimulated production of reactive oxygen species by infected phagocytes and the intracellular degradation of CEACAM-binding bacteria. Together, our results highlight the ability of CEACAM3 to coordinate signaling events that not only mediate bacterial uptake, but also trigger the killing of internalized pathogens.The Gram-negative bacterium Neisseria gonorrhoeae (Ngo), the causative agent of gonorrhea, is a paradigm for antigenic and phase variation of surface components that allow escape from the acquired immune response of its sole natural host, the human (1, 2). One family of neisserial outer membrane proteins that is modulated by phase variation are the colony opacity associated (Opa) proteins (for review, see Ref.3). There are up to 12 opa genes encoded on the gonococcal chromosome that are constitutively transcribed, but the expression of each Opa protein is independently controlled by pentameric sequence repeats in the 5Ј coding region of each transcript. The number of these repetitive units determines if the full-length protein is translated or if, due to reading frameshifts and generation of a premature stop codon, a truncated, non-functional protein is made (4). Interestingly, most gonococcal Opa proteins characterized so far bind to various members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 3 family, a group of immunoglobulin-domain containing glycoproteins found on human cells (for review, see Ref. 5). CEACAMs harbor at least one characteristic immunoglobulin variable (Ig V )-like amino-terminal domain that is recognized by CEACAM-binding Opa proteins (Opa CEA proteins) (6, 7). Opa CEA protein binding to the host receptor is a direct protein-protein interaction that is species-specific in the sense that Opa CEA proteins only recognize human CEACAMs, but not orthologues from other mammals (8). Moreover, of the 12 CEACAM family members expressed in humans (9), only four have been found to associate with gonococcal Opa CEA proteins, namely CEACAM1, CEACAM3, CEA (the product of the CEACAM5 gene), and CEACAM6 (10 -14). The situation is further complicated by the f...

BackgroundCarcinoembryonic 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.ResultsSequence 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.ConclusionsOur 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.

Background 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. Principal Findings 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. Conclusions 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.

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