Dokument: Molecular Characterization of Chlamydia – Host Interactions During the Early Stages of Infection
| Titel: | Molecular Characterization of Chlamydia – Host Interactions During the Early Stages of Infection | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=70603 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20250904-135615-2 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Kocher, Fabienne [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Hegemann, Johannes H. [Gutachter] Prof. Dr. Willbold, Dieter [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | Chlamydia trachomatis (Ctr) and Chlamydia pneumoniae (Cpn) are human pathogenic bacteria of the genus Chlamydia. They target epithelial cells, with Ctr causing infections of the eyes and the urogenital tract, and Cpn causing respiratory tract infections. Owing to their obligate intracellular lifestyle, adhesion to and internalization into host cells are crucial steps for establishing infection.
Chlamydial adhesion is mediated by several components, including polymorphic membrane proteins (Pmps), a protein family found in all chlamydial species. In Ctr, all nine Pmps (PmpA-PmpI) act as adhesins on epithelial cells, but for none of them have the host cell binding partners been identified. After adhesion, internalization is triggered through host endocytic processes, engulfing the bacteria in a membrane-enclosed vesicle, termed inclusion. To facilitate internalization, Chlamydia injects entry-related early effector proteins into the host cell cytosol via its Type III secretion system (T3SS), thereby manipulating the host endocytic machinery. After secretion, the Cpn effector SemD binds via its amphipathic helix to the inner leaflet of the plasma membrane (PM), underneath the attached chlamydial cell. There, it recruits key components of the host endocytic machinery, such as SNX9 and N-WASP via its proline-rich domain and its N-WASP binding domain, respectively. While SNX9 functions as a membrane bender and enhances scission of the matured vesicle from the PM, activated N-WASP is an actin modulator, mediating F-actin polymerization and branching via the Arp2/3 complex. Intriguingly, SemD recruiting N-WASP simultaneously leads to the activation of the latter in a yet unknown manner, independently of its endogenous activator Cdc42GTP. In the first part of this work, focusing on Ctr adhesion, two parallel approaches were used to identify a potential host cell binding partner for Ctr PmpD. In approach (I), a proteolytically processed PmpD fragment found during infection in vivo was recombinantly produced (rD72) and used as bait in pulldown assays on epithelial cells. Clusterin, a host cell protein with a secreted isoform (sCLU), was identified as a binding partner. sCLU is responsible for the clearance of misfolded proteins from the extracellular space by endocytosis and is an inhibitor of the terminal complement pathway. In vitro assays verified the direct interaction of rD72 and sCLU. Additionally, in vivo infection experiments showed significantly reduced Ctr infectivity in the absence of sCLU in the cell culture medium overlaying epithelial cells during the early infection stages, suggesting that sCLU facilitates Ctr infectivity by binding the Ctr adhesin PmpD. In approach (II), the adhesive domain of PmpD, shown to be chlamydial cell surface-localized, was genetically fused to APEX2, a peroxidase which biotinylates nearby proteins (1-10 nm). Transformed Ctr EBs, producing the fusion protein, were used for infecting epithelial cells. Biotinylated proteins were identified and showed significant enrichment of the host insulin-like growth factor 2 receptor (IGF2R) and future experiments are needed to confirm the direct interaction. In the second part of this work, Cpn internalization via SemD was investigated. Structural analysis of SemD demonstrated that by combining flexible and structured domains, steric hindrance between individual host cell binding sites of SemD is minimized, allowing the simultaneous interaction with the host cell PM, SNX9 and N WASP. Co-crystallization of SemD and N WASP revealed that SemD structurally mimics Cdc42GTP for N-WASP activation. In biochemical assays, it was further revealed that SemD binds N-WASP much more strongly than Cdc42GTP does, allowing SemD to displace Cdc42GTP from an already formed N-WASP – Cdc42GTP complex, making Cdc42GTP redundant during the early infection. Identification of PmpD host cell binding partners and an understanding of the activation mechanism of SemD on N-WASP may in future allow for the development of small inhibitors that block a chlamydial infection. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Funktionelle Genomforschung der Mikroorganismen | |||||||
| Dokument erstellt am: | 04.09.2025 | |||||||
| Dateien geändert am: | 04.09.2025 | |||||||
| Promotionsantrag am: | 06.10.2020 | |||||||
| Datum der Promotion: | 07.07.2025 |
