The Library
Interaction of translocase MraY with the antibacterial E protein from bacteriophage ΦX174
Tools
Rodolis, Maria T. (2013) Interaction of translocase MraY with the antibacterial E protein from bacteriophage ΦX174. PhD thesis, University of Warwick.
|
Text
WRAP_THESIS_Rodolis_2013.pdf - Submitted Version Download (11Mb) | Preview |
Official URL: http://webcat.warwick.ac.uk/record=b2693374~S1
Abstract
The widespread use of antibiotics has played a significant role in the emergence of
resistant bacteria. It is of great interest and need to develop novel, effective and safe
antimicrobial therapeutics. The biosynthesis of bacterial cell wall peptidoglycan is an
intricate process that has become a popular target for antibiotics. Lytic protein E of
Bacteriophage ΦX174 was found to inhibit peptidoglycan biosynthesis via an
unknown interaction with integral protein MraY. Genetic studies have revealed that
E-mediated lysis is dependent on the interaction between Phe288 of MraY and the
transmembrane segment of protein E.
We have constructed an α-helical model for the predicted transmembrane interactions
between protein E and MraY and shown that favourable interactions can be formed
between Phe288 and the RWXXW motif of protein E. In this thesis, analogues of the
RWXXW motif were synthesised in solution and via solid phase peptide synthesis
using 2-chlorotrityl chloride resin as the polymeric support. The inhibitory activity of
these analogues was determined on a continuous fluorescence assay against
membrane bound MraY. Inhibition studies on site-directed mutants of E. coli MraY
were also conducted. Testing the inhibitory activity of RWXXW analogues provided
compelling information on the importance of protein E residues for the inhibition of
MraY. Peptides which contained a tryptophan residue were especially good inhibitors
of MraY presumably due to their interaction with Phe288. Mutation of Phe288 caused
a dramatic decrease or complete loss to the inhibitory activity of peptides containing
an aromatic residue.
Some analogues also contained antibacterial activity across multiple strains of
bacteria including E. coli, B. subtilis and P. putida with MIC values as low as
8μg/mL. To confirm if MraY was the target enzyme, E. coli cells overexpressing
MraY were treated with RWXXW analogues. An increase in the MIC of RWXXW
analogues signified that the MraY was the lysis target.
In the course of the project, we noticed that members of the UPA class of natural
products contained some structural features that are also found in the RWXXW motif.
These natural products were tested for activity against site-directed mutants of E. coli
MraY. Results showed that Phe288 plays some role in the inhibition of MraY by
pacidamycin. This work identifies a promising target for the development of novel
antimicrobial agents that is located on the outer face of the cytoplasmic membrane.
Item Type: | Thesis (PhD) |
---|---|
Subjects: | Q Science > QP Physiology Q Science > QR Microbiology R Medicine > RM Therapeutics. Pharmacology |
Library of Congress Subject Headings (LCSH): | Bacteriophages, Anti-infective agents -- Research, Peptidoglycans -- Synthesis, Proteins -- Inhibitors -- Therapeutic use, Peptide antibiotics -- Research |
Official Date: | September 2013 |
Institution: | University of Warwick |
Theses Department: | Department of Chemistry |
Thesis Type: | PhD |
Publication Status: | Unpublished |
Supervisor(s)/Advisor: | Bugg, Tim |
Sponsors: | National Science Foundation (U.S.) (NSF) |
Extent: | xxvi, 294 leaves : illustrations. |
Language: | eng |
Request changes or add full text files to a record
Repository staff actions (login required)
View Item |
Downloads
Downloads per month over past year