| Abstract |
The type III secretion injectisome is a complex nanomachine, that allows bacteria to deliver protein effectors across eukaryotic cellular membranes. In recent years, significant progress has been made in our understanding of its structure, assembly and mode of operation. In the present stage of knowledge, it consists of a basal structure that traverses the bacterial envelope and the peptidoglycan, surmounted by a needle and a filament. The whole structure can act as a hollow conduit, through which all the virulence proteins will be delivered to the cytosol of the host. A key element in the secretion system is a set of chaperones in the bacterial cytosol, the dedicated type III chaperones. Their exact role in the secretion process remains ainigmatic.
The main goal of this study is the investigation of the molecular role of the CesAB chaperone from the Enteropathogenic coli (EPEC) during secretion of its substrates, EspA and EspB. In parallel we are characterizing the EscN ATPase of the system, as a final acceptor of the soluble complexes between chaperones-substrates.
Firstly, we are characterizing structurally the chaperone alone or in complex with its substrate and we reveal a novel mechamism of recognition, the mutual folding during binding of a substrate to its chaperone.
In parallel with biochemical experiments, we are demonstrating that the CesAB chaperone consists of two distict, functionally and structurally, domains, the N-terminal domain (N-domain) and the C-terminal tail (C-tail). Through mutagenesis to the N-domain of the protein, we are clearly showing that this domain is responsible for the chaperone-like function of CesAB, only for EspA and not EspB substrate. Then we are demonstrating that the C-tail is absolutelly essential for the effective secretion of both substrates, not only because in the absence of the C-tail the substrates cannot be secreted, but also because they cannot be localized to the membrane fraction of the cell, their natural place before secretion. In addition, we are showing that the C-tail is involved in the formation of a super-molecular complex intracellularly in the secretion pathway.
The physiological significance of these two domains of the CesAB chaperone is tested using cell models, and their importance for the pathogeneceity is revealed. Finally we are managing to isolate the stable active form of the EscN ATPase of the system and meisure affinity for the C-tail of the CesAB chaperone. The results of this study reveal very important key points for the secretion process of the two translocalots, EspA and EspB, through the type III secretion system of EPEC.
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Ανάλυση του μοριακού μηχανισμού εκκρισης τύπου ΙΙΙ στο εντεροπαθογόνο E.coli
