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Analysis of the mechanisms controlling septum cleavage in Enterococcus faecalis and the impact of cell chain formation on pathogenesis

Salamaga, Bartlomiej (2017) Analysis of the mechanisms controlling septum cleavage in Enterococcus faecalis and the impact of cell chain formation on pathogenesis. PhD thesis, University of Sheffield.

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Bartlomiej Salamaga PhD Thesis.pdf
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Abstract

Bacterial cell growth and division rely on a vast number of proteins including peptidoglycan hydrolases (PGHs). Previous studies have shown that in the opportunistic pathogen Enterococcus faecalis, the N-acetylglucosaminidase AtlA is dedicated to septum cleavage at the end of the division cycle to release daughter cells. Deletion of the atlA gene leads to formation of long cell chains. AtlA is a modular enzyme consisting of (i) an N-terminal domain rich in threonine, proline and glutamic acid residues, (ii) a catalytic domain with an N-acetylglucosaminidase activity and (iii) a LysM domain responsible for non-covalent binding to peptidoglycan. In this study, we investigated the mechanisms controlling the septum cleavage activity of AtlA and explored the contribution of the cell separation process to pathogenesis. Using flow cytometry we showed that post-translational modifications of the N-terminal domain of AtlA modulate the activity of this enzyme. O-glycosylation inhibits the cells separation, whereas proteolytic cleavage of this domain promotes it. AtlA C-terminal domain, which consists of six LysM peptidoglycan binding modules, is required for an optimal septum cleavage. The truncation or replacement of AtlA LysM modules with modules from AtlB, another E. faecalis PGH, leads to formation of long cell chains. These changes in the LysM domain abolish AtlA surface display and targeting to the septum, resulting in an accumulation of the enzyme inside the cell. A protein called AdmA (AtlA display mutant A) required for AtlA surface display was identified. An in-frame deletion of admA in E. faecalis JH2-2 leads to the formation of long cell chains and abolishes the septal and polar targeting of AtlA. The impaired septum cleavage in the atlA mutant was shown to promote an increased uptake by phagocytes resulting in a reduced virulence in the zebrafish model of infection.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Science (Sheffield)
The University of Sheffield > Faculty of Science (Sheffield) > Molecular Biology and Biotechnology (Sheffield)
Depositing User: Mr Bartlomiej Salamaga
Date Deposited: 29 Jan 2018 09:25
Last Modified: 29 Jan 2018 09:25
URI: http://etheses.whiterose.ac.uk/id/eprint/19223

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