| Abstract |
Type III secretion system (T3SS) is a widespread virulence system, in Gram negative bacteria, used to transport effector proteins directly into the eukaryotic host cell by spanning three membranes, two bacterial and one eukaryotic. The T3SS is a unique nanomachine that resembles a needle and can be divided in three basic compartments. First, the basal body, which forms a ring-like structure, is used to bridge the inner with the outer membrane of the bacterium. From the basal body a needle-filament construction is generated that will be attached to the host cell. In the cytosolic domain of the inner membrane the cytosolic ring and the ATPase complex is assembled in order to regulate and coordinate the secretion process. In order the system to become functional and active more than 50 proteins need to be synchronized so the assembly of the system and the protein secretion through it undergo sophisticated regulation. Although recent structural and biochemical studies provide information about the assembly of the system, the understanding of the molecular mechanism behind the translocation process remains elusive.
The main goal of this thesis was to shed more light to the molecular mechanism that proteins follow so to be secreted from T3SS. The aim was to investigate and map the pathway proteins follow from the bacterial cytosol towards the membrane at the base of the translocation pore. Determination of the protein-protein interactions that occur and elucidation of the mechanism that lies behind the membrane targeting of the secretory protein- chaperone complexes was our ultimate target.
Using Enteropathogenic E. coli as a model organism we combined structural, biochemical and biophysical approaches to achieve our goal.
Towards this end, first we performed a comprehensive analysis of the protein complexes formed in the bacterial cytosol during protein secretion through T3SS. For this analysis we combined Native Polyacrylamide gel electrophoresis and/or Size exclusion chromatography for protein separation in line with high resolution Mass spectrometry analysis. From this analysis, we identified many protein complexes related with T3SS. Moreover, we ended up with important information regarding protein complex formation in the cytosol as a function of time, hierarchy that is followed and regulation of the procedure, which were also correlated with real-time secretion analysis from the system. We established a general pipeline that could be followed for the protein identification and determination of protein complexes formed in different biological states.
Additionally, in a more targeted approach to map the interactions between the specialized chaperones of the system with the secretory proteins, and how these interactions are regulated, we used biochemical and biophysical methods in concert with in vivo experiments. As a model for secretory protein-chaperone complex, the CesAB chaperone in complex with its cognate substrate EspA was used.
First, we determined the mechanism that is followed in order for the chaperone to avoid aggregation and unspecific interactions in the cytoplasm. According to this model, the chaperone forms a homo-dimer when is on its own in solution that mimics the structure that it would occur if the secretory protein was bound. The main difference between these two similar overall structures, the homo- and hetero-dimers is that the homodimer retains a partially loose and unfolded structure, due to unfavored interactions between the two CesAB protomers.
Once the secretory protein EspA is added in solution, the homodimer of the chaperone dissociates and a stable heterodimer is generated with the chaperone CesAB and the secretory protein EspA. The formation of the 1:1 protein complex is essential so to keep the secretory protein in an unfolded and translocation-competent state. Conformational changes that occur on CesAB main body, upon EspA-binding, lead to a transition disorder-to-order state. These structural changes are important for the exposure of a targeting signal on the chaperone’s surface, which enables the interaction of the protein complex with the ATPase of the system.
Overall the results obtained, revealed, on one hand, a self-regulation mechanism that the chaperones follow so to prevent unspecific protein interactions. On the other hand, we established a substrate –activated conformational switch on the chaperone which encodes a targeting signal that could be the mechanism for protein targeting to the ATPase of the system.
Finally, in order to fulfill our goal for elucidating the membrane targeting procedure, we reconstituted the process in vitro. We established an in vitro assay where the binding of the cytoplasmic complexes at the T3SS can be monitored and quantified.
From the data derived from the in vitro analysis of the targeting process, we have clearly demonstrated that the carboxyl-terminal tail (hereafter C-tail) of the chaperone CesAB is essential for the targeting of the secretory protein-chaperone complex at the T3SS export apparatus. We have signified that when CesAB is bound on the secretory protein EspA, the protein complex can interact with the membrane, via the C-tail. This targeting process is independent of the existence of the ATPase, and other proteins that are located at the export apparatus of the system, like SepL, EscV and EscU are essential for the binding event to occur.
Collectively, a two-step targeting hypothesis is proposed. During the first step, once the chaperone-secretory complex has been formed it is targeted to the membrane of the T3SS, via the C-tail of the chaperone. Afterwards, the ATPase can bind to the main body of the CesAB, as a second docking event.
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