Integrin-mediated signal transduction in brain endothelial cells during infection with N. meningitidis: mechanisms of crossing the blood-brain barrier
Zusammenfassung der Projektergebnisse
N. meningitidis is a common colonizing bacterium of the human nasopharynx and can be found in 8 to 20% of the healthy population. In rare cases, N. meningitidis overcomes the epithelial barrier and enters the bloodstream to cause severe septicaemia and or an acute purulent meningitis. An initial stage of the interaction of N. meningitidis to overcome the blood-brain barrier is the adherence and penetration of the brain endothelial cells forming this barrier (e. g. by uptake and transcytosis). Like many other bacterial pathogens N. meningitidis can activate host cell signal transduction pathways to establish adhesion and induce uptake. It is especially tyrosine phosphorylation of host proteins that occurs after binding of meningococci to eukaryotic cells, which results in cytoskeletal remodelling with subsequent engulfment of the pathogen. Tyrosine kinases are important players in processes involving receptor-mediated changes of the cytoskeleton as well as cell adhesion events. During this proposal we succeeded to determine the role of a key non receptor protein tyrosine kinase, the focal adhesion kinase (FAK) in the interaction of N. meningitidis with host cells. We could prove that FAK activity is required for meningococcal invasion and the autophosphorylation and kinase activity of FAK are essential for the uptake of N. meningitidis by the eukaryotic cell. In addition, we observed that meningococcal infection leads to cortactin phosphorylation. Mutation of critical cortactin amino acids either within the domain that interacts with dynamin or within the NTA domain that activates the Arp2/3 complex supported the hypothesis that both domains are critical for efficient bacterial uptake. In addition we determined the role of RTKs in the infection process. To obtain a comprehensive picture of activated RTKs in infected eukaryotic cells we used a commercial available phosphoarray that captures 39 RTKs and key signalling nodes from cell lysates. We detected several activated RTKs, including the ErbB family receptors. A detailed analysis revealed that pharmacological inhibition and genetic ablation of ErbB receptor tyrosine phosphorylation significantly interfered with bacterial uptake. Activation of EGFR and ErbB4 was shown to be mediated by transactivation of the common ligand HB-EGF. We furthermore demonstrated activation of three specific phosphorylation sites of EGFR, ErbB2 and ErB4 and revealed that phosphorylation of EGFR at Tyr845 and phosphorylation of ErbB4 at Tyr1284 are involved in mediating uptake of N. meningitidis by brain endothelial cells. The systematic approach to characterize kinase-dependent signaling cascades triggered by the interaction between N. meningitidis and the host cell revealed that several kinases are activated that have not been described before. Our analyses therefore served as a useful screen to identify new kinases that might be involved in the context of the interaction of N. meningitidis with host cells, which will be analyzed in a new follow-up grant (“Untersuchungen zum Einfluß der Meningokokkeninfektion auf die Aktivierung von Rzeptor-Tyrosinkinasen sowie auf den Zellzyklus”). Based on our preliminary data we aim to identify and functionally characterize activation of insulin receptor (InsR), tyrosine-related kinase B receptor (TrkB) and in particular ephrin receptors (EphA3 and EphB1), which have not been described before. Activation and cellular distribution will be monitored and their role in meningococcal attachment and invasion shall be determined. Taken together we could show how N. meningitidis has taken advantage to interfere with host protein kinase activation to achieve a dominating up-regulation of particular kinase activities to establish its uptake by the eukaryotic cell.
Projektbezogene Publikationen (Auswahl)
- (2011). Real-time impedance analysis of host cell response to meningococcal infection. J Microbiol Methods. Jan;84(1):101-8
Slanina H, König A, Claus H, Frosch M, Schubert-Unkmeir A
- Cell Invasion by Neisseria meningitidis requires a common interaction between the Focal Adhesion Kinase, Src and Cortactin. DGHM 2011
Slanina, H, Hebling, S, Hauck, CR, and Schubert-Unkmeir A
- (2012). Cell Invasion by Neisseria meningitidis Requires a Functional Interplay between the Focal Adhesion Kinase, Src and Cortactin. PLoS One. 7:e39613
Slanina, H, Hebling, S, Hauck, C.R., and Schubert-Unkmeir A.
(Siehe online unter https://doi.org/10.1371/journal.pone.0039613) - Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells. J Mol Microbiol Biotechnol. 2012;22(4):245-57
Slanina H, Schmutzler M, Christodoulides M, Kim KS, Schubert-Unkmeir A
(Siehe online unter https://doi.org/10.1159/000342909) - Transcriptome analyses in the interaction of Neisseria meningitidis with mammalian host cells. Methods Mol Biol. 799:267-93, 2012
Joseph B, Frosch M, Schoen C, Schubert-Unkmeir A
- Genome-based bacterial vaccines: current state and future outlook. BioDrugs. 2013 Oct; 27(5):419-30
Schubert-Unkmeir A, and Christodoulides M
- Role of epidermal growth factor receptor signaling in the interaction of Neisseria meningitidis with endothelial cells. Infect Immun.. 2014 Mar;82(3):1243-55
Slanina H, Mündlein S, Hebling S, Schubert-Unkmeir A
(Siehe online unter https://doi.org/10.1128/IAI.01346-13)
