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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:589892 |
| Date | January 2013 |
| Creators | Rodolis, Maria T. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/58786/ |
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