TOWARDS UNDERSTANDING THE FUNDAMENTAL ASPECTS OF SECRETION MECHANISMS IN E. COLI (Cytoplasmome/secretome divide in E. coli and in vitro reconstitution of the Type 3 Secretion in EPEC)

To function properly, proteins need to reach their final sub-cellular destinations and acquire their final folded state. Therefore, protein targeting and transport are essential cellular processes in all kingdoms of life. Understanding how proteins become transported and folded into a functional state reveals the fundamental aspects of cellular protein biology, like cell wall biogenesis, cell division, molecule-export/uptake/degradation etc. Moreover, this basic knowledge helps tackle diseases that are caused by folding malfunctions and microbial infections in which secreted toxins are commonly key players.

Cells have evolved specialized machineries to mediate protein export. Yet, the precise mechanisms of export for many of these different export systems remain poorly understood. This knowledge is required for the successful development of drugs that target these processes.

In this thesis, I focussed on two main aspects of protein export. First, I aimed to study the physicochemical properties of bacterial proteins that are secreted compared to those of cytoplasmic ones in order to define the structural features that accompany the secretion ability of polypeptides. Second, I studied a complex protein export system in pathogenic bacteria, called the Type III Secretion System (T3SS), in order to understand its export mechanism and how it can be inhibited.

To characterize the proteome and compare the different sub-cellular topology groups, we curated all the proteins in E. coli K-12 model organism and their physicochemical properties. By performing proteome-wide analysis we defined the properties of the cytoplasmic proteins compared to the secreted ones. We found that secreted proteins show enhanced flexibility, slow folding, looser structures and unique folds that differentiate them from the cytoplasmome. These adaptations protect the secretome from premature folding before reaching the final destination, optimize transport and have implications for the specific functions of each protein group. These findings have wide implications on the structural diversity in different cell compartments and the protein folding problem and suggest how the evolution of modern proteomes may have taken place. To my knowledge, this is the first extensive curation and analysis of the structural features of subcellular topology group properties of the model organism E. coli K-12. The collected data is publicly available on our online database STEPdb. To further strengthen our findings and hypotheses, experimental evidence is needed and is the future direction in our laboratory.

T3SS is a translocation pathway that delivers virulence proteins from the bacterial cytoplasm to the eukaryotic cytoplasm through the complicated membrane spanning machinery called the injectisome. To resolve the molecular mechanisms of the T3 secretion we have for the first time established in vitro assays. This approach allowed the dissection of the export system into well-defined mechanistic steps that can be studied separately. First we aimed to gain insight in the targeting step and revealed the major export apparatus component EscV/SctV to be the main receptor at the membrane. Additionally, the gatekeeper SepL/SctW bound to it increases the affinity of the middle substrates to the injectisome. After the secretion switch the gatekeeper leaves the injectisome via unknown mechanism, which increases the affinity of the late substrates for the export apparatus. Additionally, we have started to reconstitute consecutive steps of injectisome assembly. These efforts will be continued in the future. Better understanding of the T3SS opens a possibility of developing inhibitors of this process thus reducing virulence of pathogenic bacteria.

In summary, in this thesis I describe the research I conducted or have contributed to in order to understand the fundamental aspects of targeting and secretion in the Gram negative bacteria, E. coli and enteropathogenic E. coli. First, we have concluded that different topology groups like cytoplasmic and secreted proteins are significantly distinct, showing striking physicochemical and structural differences in E. coli K-12 strain. Additionally, we have studied how proteins are being targeted for the secretion across membranes in T3SS in pathogenic E. coli strain and the interplay of different injectisome components that support this process. We aim to use this knowledge and techniques in order to search and develop small molecule inhibitors to battle the bacterial infections.