Download MSc in Biochemistry Dissertation Project – 2nd Cycle Student´s

MSc in Biochemistry
Dissertation Project – 2nd Cycle
Student´s Name:
Student email address:
No.
Supervisor(s): Rita Sobral and Inês Grilo
Supervisor(s) email address:[email protected] and [email protected]
Lab/Institution: Lab of Molecular Microbiology of Bacterial Pathogens (333), UCIBIO, UNL
TITLE:Presence of extracellular DNA and its regulation of the activity of the major autolysin of
Staphylococcus aureus Atl
BACKGROUND
Staphylococcus aureus is a remarkably versatile microorganism that is usually a commensal of the
human’s microbiota, but can also become invasive when the host’s defenses are breached,
becoming a very important and successful pathogen, with high morbidity and mortality rates across
the globe. In S. aureus, the major murein hydrolase is Atl, a bifunctional autolysin with amidase
(AM) and glucosaminidase (GL) catalytic domains, primarily involved in cell separation, as well as
in cell wall turnover and lysis. This protein has also been implicated in biofilm formation in S.
aureus, not only due to its role in autolysis and subsequent genomic DNA release, but also
because of its intrinsic adhesive properties. Both domains of the Atl protein are translated as a
single pro-peptide that is exported to the extracellular milieu, where the proteolytic processing into
the two mature AM and GL domains occurs, in a thus far uncharacterized manner.
We have previously reported the DNA-binding capacity of the GL domain of Atl, and we have
shown that Atl binds DNA at the cell surface, while we hypothesize that the presence of
extracellular DNA (eDNA) could induce the differential proteolysis and/or activity of Atl.
Furthermore, our results have shown that the GL and AM catalytic domains have distinct functions
in biofilm formation and are likely involved in other cellular processes differently.
OBJECTIVES
We aim to characterize the expression of the atl gene in several conditions (temperature, pH,
medium, presence of eDNA, etc) and to correlate it with the differential expression and activity
of the AM and GL domains, in various strains of S. aureus.
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MSc in Biochemistry
Dissertation Project – 2nd Cycle
PROJECT DESCRIPTION
The transcriptional expression of the atl gene will be assayed using a promoter-GFP reporter
system. Several strains with diverse genetic backgrounds will be used(Task 1), as well as
different conditions, including the presence of eDNA, mimicking different environments(Task
2). The expression of the AM and GL catalytic domains of Atl will be assessed by Western
blotting with specific antibodies(Task 3), while the activity of these proteins will be
characterized using hydrolysis assays in microtiter plates and zymography with different cell
wall substrates in various conditions(Task 4). The presence of extracellular DNA in particular
will be tested to assess if the presence of this molecule affects the expression and activity of
Atl(Task 5).
TIMELINE(use fill tool for the cells)
Month 1
Month 2
Month 3
Month 4
Month 5
Month 6
Month 7
Month 8
Month 9
Month 10
Task 1
Task 2
Task 3
Task 4
Task 5
Thesis
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