Aleanizy, Fadilah (2012) Investigating the molecular mechanisms of colicin import into Escherichia coli cells. PhD thesis, University of Nottingham.
Colicins are a family of bacterial toxins, which kill Escherichia coli cells and other closely related species. Their mode of action requires binding to an outer membrane receptor, translocation across the cell envelope leading to cytotoxicity at specific targets. The mechanism of colicin cytotoxicity includes a non-specific endonuclease activity or depolarizing of the cytoplasmic membrane by pore-forming activity. The cytotoxic activity can be inhibited by the high affinity binding of an immunity protein. For Group A colicins, translocation requires interaction between the N-terminal domain of the colicin and a series of membrane bound and periplasmic proteins called the Tol system (TolB, TolR, TolA, TolQ and Pal). In order to allow cytotoxicity, the immunity protein of enzymatic colicins must be lost after binding of the colicin to a target cell and the cytotoxic domain has to be translocated through both the outer and inner membranes.
This work has studied colicin translocation by using a periplasmic protection assay combined with an in vivo lux-reporter assay and a potassium release assay. Expressing the translocation domain of colicin A in the periplasm and challenging the cells with external colicins showed that the translocation of group A colicins is inhibited as it requires an interaction with the Tol system. Surprisingly, the TolA protein was found to play the major role during the translocation of both ColA and the endonuclease colicin E9 even though the latter colicin has no direct interaction with the TolA protein. This study also suggests that the interaction with TolB is important for both colicins A and E9. Moreover, a series of ColA constructs with a truncated T domain were made by site directed mutagenesis to define the important residues of the TolA and TolB boxes for their interaction with Tol proteins. The results showed that tyrosine 58 residue of the TolA box of colicin A is essential for TolA binding, whilst the glutamate 18 residue of the TolB box of ColA did not show an effect on TolB binding.
Colicin-producing cells are protected by the co-expressed immunity protein which prevents killing of the producing cells. The immunity protein must be released from the cytotoxic domain of endonuclease E colicins prior to reaching the E. coli cytoplasm to degrade DNA. Little is known about the mechanism of release of immunity protein from E colicin cytotoxic domains and the role of Tol proteins in the release of immunity protein. Finally in this work an attempt was made to develop a sensitive, real-time assay to study the mechanism by which immunity protein is released from colicin E9 and to study the physiological role of the Tol system in the release process. Further developments are required for improving this assay which would be of great value.
|Item Type:||Thesis (PhD)|
|Faculties/Schools:||UK Campuses > Faculty of Medicine and Health Sciences > School of Molecular Medical Sciences|
|Deposited By:||miss Fadilah Aleanizy|
|Deposited On:||21 Nov 2012 10:12|
|Last Modified:||21 Nov 2012 10:12|
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