Author ORCID Identifier

0000-0001-6477-4687

Date of Graduation

5-2017

Document Type

Dissertation (PhD)

Program Affiliation

Microbiology and Molecular Genetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Barrett Harvey, Ph.D.

Committee Member

Jeffrey Actor, Ph.D.

Committee Member

Danielle Garsin, Ph.D.

Committee Member

Barbara Murray, M.D.

Committee Member

Ambro van Hoof, Ph.D.

Abstract

AtlA is the major peptidoglycan hydrolase of E. faecalis involved in cell separation of dividing cells. SalB is a secreted stress-induced protein regulated by the CroRS system. In addition, these two proteins also appear to be affected by the virulence factor, gelatinase (GelE). GelE is a secreted zinc metalloprotease known to impact various cellular functions by post- translational modification of protein substrates. The overall objective of this work was to understand how GelE cleavage of secreted proteins, specifically AtlA and SalB, changes their function. Herein, I discovered that GelE modifies both AtlA and SalB. As visualized by Western blot analysis and flow cytometry, when GelE is expressed, AtlA exists in a N-terminally truncated form. Furthermore, N-terminal-sequencing analysis identified the GelE-cleavage site within AtlA to occur near the catalytic region, Domain II. Thus, cleavage removes the majority of the N-terminal T/E rich region, Domain I. Truncation of AtlA at this site caused no significant difference in the peptidoglycan hydrolysis activity compared to the full-length protein. Nevertheless, the modification of AtlA was shown to be required for cell separation and the completion of cell division. Additionally, GelE-modified AtlA was shown to localize to the cell septum. Taken together, these results demonstrate that post-translational modification of AtlA by GelE regulates AtlA septum localization and successful cell separation. Similarly, in the presence of GelE, SalB was found in multiple fragments. Western blot and flow cytometry analysis demonstrated that SalB was found in the media supernatant, but not associated with the cell surface. Overall, this dissertation demonstrates that GelE post-translationally modifies these two secreted proteins, AtlA and SalB, impacting the function of AtlA in cell division. Future experiments will strengthen our knowledge of how these modifications impact E. faecalis virulence.

Keywords

GelE, gelatinase, AtlA, autolysin, SalB, septum localization, peptidoglycan hydrolase, Enterococcus faecalis, exoproteins

Available for download on Thursday, April 26, 2018

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