Internalization of pathogens in mammalian cells via CEACAM1

Abstract

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) belongs to the immunoglobulin superfamily and has several physiological functions in the body, in-cluding cell-cell adhesion, angiogenesis, insulin metabolism, and regulation of immune cell functions. Within the CEACAM family, CEACAM1 has the widest tissue distribution and is expressed on endothelial, epithelial, and hematopoietic cells. It also serves as receptor for human specific pathogens like Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Moraxella catarrhalis, and Escherichia coli together with CEACAM3, CEA and CEACAM6. These pathogens express distinct adhesins to associate with the CC´FG interface of the N-terminal domain of CEACAMs; in addition we could demonstrate the selective recognition of human CEACAMs, which presumably explains the absence of non-human hosts. Engagement of CEACAM1 by pathogens has several advantages, based on its physiological functions. Receptor binding leads also to internalization and transcytosis of the bacteria in mammalian cells, causing increased survival of the pathogens in the host. During invasion of the bacteria via the epithelial CEACAMs (CEACAM1, CEA and CEACAM6) the actin cytoskeleton plays only a minor role, but it is highly dependent on receptor relocalization into lipid rafts. Also, the cytoplasmic domain of CEACAM1 is not involved in uptake process. In contrast, bind-ing of the pathogens to the granulocyte specific CEACAM3 induces fast and highly efficient phagocytosis and killing of the bacteria, leading to the suggestion that CEACAM3 might function as immune receptor to get rid of these highly adapted pathogens. For CEACAM3-mediated uptake of pathogens, the uptake is regulated by phosphoryla-tion of two tyrosines in the ITAM-like motif of the cytoplasmic domain of CEACAM3 by Src kinases. The guanine nucleotide exchange factor Vav and the adaptor protein Nck are recruited to these phosphorylated tyrosines. Vav catalyzes the nucleotide exchange of the small GTPase Rac1, which activates the Nck-associated WAVE complex, inducing massive actin cytoskeleton rearrangements and efficient uptake of CEACAM3-bound pathogens. The activity of PI3Ks is not required during CEACAM3-mediated uptake of pathogens, which is surprising because of the involvement during Fcγ receptor mediated uptake in granulocytes. One of the major part of this dissertation was to examine the role of PI(3,4,5)P during pathogen uptake via the epithelial CEACAMs in comparison to CEACAM3. We could clearly demonstrate an important role of this phosphoinositide during the endocytotic process. Further investigations revealed the participation of the extracellular immunoglobulin constant type 2-like (IgC2-like) domains of the epithelial CEACAMs for PI3K dependent uptake of pathogens. This led us to the suggestion, that another membrane protein might be involved in the signaling process as co-receptor, responsible for signal transduction into the mammalian cell. Another important observation was the involvement of the small GTPase RhoG in endocytotic processes via epithelial CEACAMs in contrast to CEACAM3, regulating the uptake upstream of PI3K. Furthermore, we could identify Ephexin4 as responsible guanine nucleotide exchange factor for RhoG after CEACAM engagement by pathogens and we investigated the involvement of the known upstream regulator of Ephexin4, the receptor tyrosine kinase EphA2. Finally, we could clearly demonstrate the important role of EphA2 during CEACAM1-mediated endocytosis of pathogens, confirming our hypothesis of a possible co-receptor in this signaling process. To sum up, the data obtained from this study provide new mechanistic insight in the barley clarified pathogen uptake via epithelial CEACAMs and the involvement of another membrane protein together with CEACAM1 during uptake could be shown for the first time.