Internalization of pathogens in mammalian cells via CEACAM1

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2012
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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.

Zusammenfassung in einer weiteren Sprache

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) ist ein Mitglied der Immunglobulin-Superfamilie und spielt eine wichtige Rolle bei zahlreichen physiologischen Funktionen im Körper, wie Zell-Zell Adhäsion, Angiogenese, Insulin-Stoffwechsel und Regulation von Immunzell-Funktionen. CEACAM1 wird in vielen unterschiedlichen Geweben exprimiert, zum Beispiel in Endothel-, Epithel- und Hämatopoetische Zellen. Einige human-spezifische Pathogene wie Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Moraxella catarrhalis und Escherichia coli binden durch verschiedene Adhäsine an das CC´FG Motiv in der N-terminalen IgV-ähnlichen Domäne einiger Mitglieder der CEACAM Familie (CEACAM1, CEACAM3, CEA und CEACAM6). Diese Pathogene erkennen ausschließlich humane CEACAMs, was die strenge Wirtsspezifität der Pathogene erklären könnte. Die Bindung an CEACAM1 ist für die Pathogene von Vorteil, basierend auf den physiologischen Funktionen des Rezeptors. Die Bindung führt zur Internalisierung und Transzytose der Bakterien, um die Überlebensrate im Wirt zu erhöhen. Das Aktinzytoskelett spielt während der Endozytose der Bakterien über epitheliale CEACAMs (also CEACAM1, CEA und CEACAM6) eine untergeordnete Rolle, aber der Prozess ist stark abhängig von der veränderte Lokalisation der Rezeptoren in Membran Mikrodomänen (Lipid Rafts). Die zytoplasmatische Domäne von CEACAM1 ist nicht in die Bakterienaufnahme involviert. Im Gegensatz dazu induziert die Bindung der Pathogene an den Granulozyten-spezifischen Rezeptor CEACAM3 eine hocheffiziente Aufnahme und Abtötung der Bakterien, was vermuten lässt, dass CEACAM3 als Immunrezeptor zu verstehen ist, um die stark an den Wirt angepassten Bakterien zu bekämpfen. Die Aufnahme der Bakterien über CEACAM3 ist durch Src Kinasen reguliert, die zwei Tyrosinresten im ITAM-ähnlichen Motiv der zytoplasmatischen Domäne phosphorylieren. Dies führt zu einer Rekrutierung des Guanosin-Austausch Faktors Vav und des Adaptormoleküls Nck. Vav katalysiert den Nukleotid-Austausch der GTPase Rac1, die wiederrum den Nck-assoziierten WAVE Komplex aktiviert, um die massiven Aktinzytoskelett-Umlagerungen für effiziente Bakterienaufnahme zu induzieren. Die CEACAM3-vermittelte Aufnahme von Pathogenen benötigt nicht die Aktivität von PI3Ks. Ein großer Teil dieser Dissertation beinhaltet die Untersuchung der Rolle von PI(3,4,5)P während der Endozytose von Pathogenen durch epitheliale CEACAM Rezeptoren und wir konnten deutlich die wichtige Funktion dieses Phosphoinositids in diesem Prozess zeigen. Weitere Untersuchungen demonstrierten die Beteiligung der extrazellulären IgC2-ähnlichen Domänen der epithelialen CEACAM Rezeptoren bei der PI3K abhängigen Aufnahme der Pathogene, was auf eine Beteiligung eines weiteren Membranproteins als Ko-Rezeptor hinweist, der für die Signalweiterleitung in die Zelle verantwortlich ist. Außerdem konnten wir zeigen, dass die kleine GTPase RhoG an der Endozytose von Pathogenen durch epitheliale CEACAMs beteiligt ist, und zwar im Signalweg oberhalb der PI3K, reguliert durch den Guanosin-Austausch Faktor Ephexin4. Ephexin4 ist assoziiert mit der Rezeptor-Tyrosin-Kinase EphA2, deren wichtige Rolle in der Endozytose von Pathogenen über epitheliale CEACAMs ebenfalls demonstriert werden konnten. Somit wurde der beteiligte Ko-Rezeptor in der Endozytose von Pathogenen über die epithelialen CEACAMs identifiziert. Zusammenfassend kann man sagen, dass die Daten aus dieser Studie neue mechanistische Einblicke in die kaum aufgeklärte Pathogenaufnahme über epitheliale CEACAMs gibt, außerdem konnte zum ersten Mal die Beteiligung eines weiteren Rezeptors zusammen mit CEACAM1 während der Aufnahme gezeigt werden.

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570 Biowissenschaften, Biologie
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ISO 690VOGES, Maike, 2012. Internalization of pathogens in mammalian cells via CEACAM1 [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Voges2012Inter-20569,
  year={2012},
  title={Internalization of pathogens in mammalian cells via CEACAM1},
  author={Voges, Maike},
  address={Konstanz},
  school={Universität Konstanz}
}
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