Publikationsserver der Universitätsbibliothek Marburg

Titel:Structural and functional studies on the homeostasis and type-III-secretion of flagellin
Autor:Altegoer, Florian
Weitere Beteiligte: Bange, Gert (Dr.)
Veröffentlicht:2017
URI:https://archiv.ub.uni-marburg.de/diss/z2017/0216
URN: urn:nbn:de:hebis:04-z2017-02167
DOI: https://doi.org/10.17192/z2017.0216
DDC:570 Biowissenschaften, Biologie
Titel(trans.):Strukturelle und funktionelle Analysen der Homöostase und Typ-III-Sekretion von flagellin
Publikationsdatum:2017-11-09
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Typ-III-Sekretion, flagellum, Post-transcriptional regulation, Post-transkriptionelle Regulation, type-III-secretion, Flagellum

Summary:
The ability to move towards favorable and avoid unfavorable conditions is key to the survival of many bacterial species. Bacterial movement relies on a sophisticated nanomachine, the flagellum. Despite being one of the tiniest motors in the biosphere, the flagellum exhibits a complex architecture and is composed of more than 30 different proteins in diverse stoichiometries. Flagellar architecture can be subdivided into a membrane-embedded basal body, a hook and a long helical filament. The process of flagellar assembly involves a plethora of accessory factors and is organized at different stages on the transcriptional, post-transcriptional and translational level. Furthermore, biogenesis of a flagellum is strictly sequential and requires the completion of a building phase prior to initiating the next one. The most abundant constituent within a flagellum is the protein flagellin that assembles into a helical filament with more than 20.000 monomers. The two proteins CsrA and FliW regulate flagellin homeostasis via a posttranscriptional mechanism only allowing flagellin translation when cytoplasmic levels are low, thereby ensuring that flagellin is directly secreted after production. A third protein, the intrinsic chaperone FliS is essential for the recognition and efficient secretion of flagellin. Together these proteins couple translation to secretion of flagellin and keep cytoplasmic flagellin concentrations around a low and narrow threshold. This work aims at unraveling the molecular mechanisms by which the above-named proteins regulate flagellin homeostasis. In enterobacteria CsrA activity is antagonized by small non-coding RNAs (sRNAs) that act as competitive inhibitors. Conversely, the FliW protein allosterically controls CsrA in a variety of flagellated bacteria, which seems to represent the ancestral state of CsrA regulation. This work furthermore demonstrates that interaction of FliW and flagellin seems to be cotranslational or strongly associated with translating ribosomes, therefore coupling homeostasis and secretion. Another level of regulation elucidated in this study is the influence of bactofilins on the process of flagellar biogenesis. This ubiquitous class of proteins is reminiscent of cytoskeleton factors but seems to rather provide a dynamic scaffold for diverse processes. In B. subtilis the bactofilins BacE and BacF are involved in flagellar assembly at the stage of hook-completion but BacE also directly interacts with FliW. Finally, this work includes a model explaining the coupling of homeostasis and secretion of flagellin at atomic resolution.

Zusammenfassung:
Die Fähigkeit günstige Lebensbedingungen zu finden und ungünstige zu vermeiden, ist ein zentraler Aspekt für die Überlebensfähigkeit vieler Bakterienarten. Das bakterielle Flagellum stellt einen der kleinsten Motoren in der Biosphäre dar, weist aber dennoch eine komplexe Architektur auf und besteht aus mehr als 30 unterschiedlichen Proteinen. Das Flagellum kann wie folgt untergliedert werden: Der zentrale Basalkörper ist in der Membran verankert und bietet das Grundgerüst für ein langes, helikales Filament, das über einen „Haken“ mit dem Basalkörper verbunden ist. Der flagellare Assemblierungsprozess umfasst eine Vielfalt an regulatorischen Faktoren und ist hierarchisch sowohl transkriptionell, als auch post-transkriptionell und translational organisiert. Darüber hinaus ist die Biogenese eines Flagellums streng sequentiell und erfordert so die Fertigstellung eines „Bauabschnittes“ bevor ein neuer begonnen werden kann. Das Filament ist aus mehr als 20.000 Kopien des Proteins Flagellin aufgebaut, dessen Moleküle in einem helikalen Muster angeordnet werden. Zwei Proteine, CsrA und FliW, regulieren die Flagellin Produktion über einen posttranskriptionellen Mechanismus. Dieser stellt sicher, dass nur dann Flagellin translatiert wird, wenn die zytoplasmatischen Level des Proteins niedrig sind, es also direkt sekretiert wird. Ein drittes Protein, das intrinsische Chaperon FliS, ist essentiell für die Erkennung und effiziente Sekretion von Flagellin. Zusammen kontrollieren diese Proteine die Produktion von Flagellin, so dass die zytoplasmatische Flagellin Konzentration um einen schmalen Schwellenwert oszilliert. Ziel dieser Arbeit ist es die molekularen Details der Flagellin Homöostase aufzuklären. In Enterobakterien wird der CsrA Aktivität durch kleine, nicht-kodierende RNAs (sRNAs), die als kompetetive Inhibitoren agieren, entgegengewirkt. Im Gegensatz dazu kontrolliert FliW das CsrA Protein allosterisch in einer Vielzahl flagellierter Bakterien. Diese Art der CsrA Regulation stellt vermutlich den evolutionär ursprünglichen Zustand in Eubakterien dar. Weiterhin wird in dieser Arbeit gezeigt, dass die Wechselwirkung von FliW und Flagellin entweder direkt co-translational zu sein scheint oder aber nah im ribosomalen Kontext stattfindet. Dies erlaubt die Kopplung von Flagellin Homöostase und Sekretion. Weiterführende Untersuchungen, die in dieser Studie durchgeführt wurden, zeigen den Einfluss von Bactofilinen auf den Prozess der Flagellen-Biosynthese. Diese ubiquitäre Klasse von Proteinen erinnert an Zytoskelettproteine, scheint aber eher ein dynamisches Gerüst für VIII verschiedene Prozesse zu bilden. In B. subtilis sind die Bactofiline BacE und BacF am Aufbau der Flagelle beteiligt, möglicherweise am Aufbau des „Hakens“. Weiterhin wird eine Interaktion von BacE mit FliW gezeigt und damit eine Verbindung zur Flagellin Homöostase. Abschließend liefert diese Arbeit ein Modell, dass die Kopplung von Homöostase und Sekretion von Flagellin auf struktureller Basis erklärt.

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