Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms

Abstract

Several pathogenic bacteria exploit human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) for adhesion to and invasion into their host cells. CEACAM isoforms have characteristic expression patterns on epithelial, endothelial, or hematopoietic cells, providing bacteria with distinct sets of receptors on particular tissues. For example, while CEACAM1 and CEACAM6 have a wide tissue distribution, CEACAM3, CEACAM4, and CEACAM8 are uniquely expressed on primary human granulocytes, whereas CEA and CEACAM7 are limited to epithelia. By reconstitution of a CEACAM-deficient cell line with individual CEACAMs, we have analyzed the requirements for CEACAM-mediated internalization of Neisseria gonorrhoeae. Our results point to two mechanistically different uptake pathways triggered by either epithelial CEACAMs (CEACAM1, CEA, and CEACAM6) or the granulocyte-specific CEACAM3. In particular, CEACAM3-mediated uptake critically depends on Src family protein tyrosine kinase (PTK) activity, and CEACAM3 associates with the SH2 domains of several Src PTKs. In contrast, epithelial CEACAMs require the integrity of cholesterol-rich membrane microdomains and are affected by cholesterol depletion, whereas CEACAM3-mediated uptake by transfected cells or the opsonin-independent phagocytosis by human granulocytes is not altered in the presence of cholesterol chelators. These results allow the subdivision of all human CEACAMs known to be utilized as pathogen receptors into functional groups and point to important consequences for bacterial engagement of distinct CEACAM isoforms.

CEACAM3: An innate immune receptor directed against human-restricted bacterial pathogens

“…In line with the critical role of Rac, actin cytoskeleton dynamics are essential for CEACAM3-initiated bacterial uptake, whereas other CEACAMs appear to be internalized via lipid raft-dependent processes (Muenzner et al, 2008;Schmitter et al, 2007b). Lipid raft association and, therefore, sensitivity to cholesterol depletion, discriminate between bacterial internalization via CEACAM1 or glycosylphosphatidyl-inositol (GPI)-anchored CEACAMs (CEA and CEACAM6) and CEACAM3-mediated uptake (Muenzner et al, 2008).…”

“…The tyrosines within the ITAM-like sequence serve as targets for protein tyrosine kinases (PTKs) of the Src family that are activated upon bacterial engagement of CEACAM3 . In fibroblasts deficient for the Src family PTKs c-Src, Fyn and cYes, CEACAM3 is not able to promote efficient uptake of Opa-expressing gonococci, whereas re-expression of c-Src in these cells re-constitutes bacterial internalization (Schmitter et al, 2007b). Upon receptor phosphorylation, the SH2 domain of c-Src is able to associate with CEACAM3 (Schmitter et al, 2007b).…”

“…In fibroblasts deficient for the Src family PTKs c-Src, Fyn and cYes, CEACAM3 is not able to promote efficient uptake of Opa-expressing gonococci, whereas re-expression of c-Src in these cells re-constitutes bacterial internalization (Schmitter et al, 2007b). Upon receptor phosphorylation, the SH2 domain of c-Src is able to associate with CEACAM3 (Schmitter et al, 2007b). Though c-Src is not strongly expressed in human granulocytes, the closely related PTK Hck is also able to bind to CEACAM3 in a phosphorylation-dependent manner via its SH2 domain, and Hck is found to be recruited and strongly activated in phagocytes upon encounter with Opa protein-expressing gonococci (Hauck et al, 1998;Schmitter et al, 2007b).…”

“…In particular, CEA-CAM3 has not been found to engage in cell-cell adhesion with other CEACAM family members, despite the high sequence similarity of the amino-terminal Ig Vlike domain of CEACAM3 with CEACAM1 (87% amino acid sequence identity), CEA (92% amino acid sequence identity), or CEACAM6 (89% amino acid sequence identity) (Kuroki et al, 1991). The finding that N. gonorrhoeae can be phagocytosed in an opsoninindependent, but CEACAM-dependent manner by human granulocytes (Chen and Gotschlich, 1996;GrayOwen et al, 1997a;Hauck et al, 1998;Virji et al, 1996), together with the demonstration that CEACAM3 is the main receptor responsible for this process (Schmitter et al, 2004(Schmitter et al, , 2007b, has led to the suggestion that CEACAM3 is not involved in cell-cell adhesion, but rather represents a germline-encoded innate immune receptor targeted against human-specific pathogens. In this review, we will discuss the current understanding of CEACAM3 function for phagocytosis of bacteria and speculate about the evolutionary origin of this particular CEACAM family member.…”

“…Upon receptor phosphorylation, the SH2 domain of c-Src is able to associate with CEACAM3 (Schmitter et al, 2007b). Though c-Src is not strongly expressed in human granulocytes, the closely related PTK Hck is also able to bind to CEACAM3 in a phosphorylation-dependent manner via its SH2 domain, and Hck is found to be recruited and strongly activated in phagocytes upon encounter with Opa protein-expressing gonococci (Hauck et al, 1998;Schmitter et al, 2007b). Interestingly, the cytoplasmic tyrosine kinase Syk, which is known to critically contribute to ITAMinitiated uptake in the case of the Fcg receptor, is not required to stimulate CEACAM3-initiated phagocytosis (Sarantis and Gray-Owen, 2007).…”