| Abstract |
Enteropathogenic Escherichia coli (EPEC) is an important diarrheal pathogen and an endemic health threat in the developing world[1]. The virulence proteins responsible for causing diarrheal disease are translocated directly into the host intestinal epithelium through a type iii secretion system (T3S). The T3S is a nano-machine which consists of the basal body that penetrates the bacterial membrane, and expanded by a needle and a filament. Although a lot of research has been done, there are many questions that need to be answered about the function and regulation of this specific secretion system.
In this study, firstly, we tried to analyze the whole secretome of EPEC. In addition, in order to elucidate how proteins are being secreted from T3S, we analyzed the secretion profile of two translocator proteins, EspA and EspB and their chaperone, CesAB.
To achieve this goal, firstly, we determine the conditions where the secretion from T3S is being induced (pH, temperature). Afterwards, we analyzed all the proteins that are being secreted through T3S using mass spectrometry. Moreover, using deletion strains of EPEC we determine the hierarchy of secretion and how it is regulated by the absence of different factors.
Additionally, we tried to elucidate the interactions between effector proteins and their specific chaperones, using as a model CesAB chaperone and its substrates EspA and EspB. To clarify these interactions, we perform biochemical assays in vitro and ex vivo. In these experiments, we analyze the interactions between the chaperone and its substrates, and how these interactions are affected after generating mutations at chaperone’s gene.
Moreover, to illustrate the secretion pathway that EspA and EspB follow through secretion via T3S, we analyzed the complexes that are being extracted after hard and mild lysis of the EPEC. Furthermore, using mass spectrometry analysis, we tried to identify all the proteins that form the above complexes.
The results of this study reveal important elements to understand the molecular basis of T3S and compose a framework for future studies and analysis of this secretion system.
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Μοριακή ανάλυση του μηχανισμού έκκρισης τύπου ΙΙΙ στο εντεροπαθογόνο E.coli
