Date of Award

2019

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Prof. Aidan Coffey

Second Advisor

Dr. Mark Johan van Raaij

Third Advisor

Dr. Olivia McAuliffe

Abstract

Antibiotic resistance is becoming a serious public health concern. Infections that some decades ago could be treated with antibiotics now sometimes do not respond to traditional treatment, causing higher mortality and economic losses. An alternative to the use of antibiotics are bacteria's natural predators, bacteriophages (or phages), and specifically their lytic enzymes. These proteins are produced by phages to degrade bacterial peptidoglycan to inject their genetic material into the bacteria (virion-associated peptidoglycan hydrolases) or to release their progeny once the infection is finished (endolysins). They can be applied exogenously to lyse Gram-positive bacteria or be genetically engineered to lyse Gram-negative bacteria. Phages also code for proteins that allow them to reach their receptors (depolymerases) and recognize them (receptor binding proteins or fibres). These receptor-binding proteins can be used to detect and to identify pathogenic bacteria. More knowledge on the structure of these proteins is necessary to understand their mechanisms of action and to design chimeric or active-site mutant endolysins able to lyse different pathogenic bacteria. In this work, the structure of the N-acetylmuramidases from two bacteriophages (Pseudomonas bacteriophage vB_PaeM_KTN6 and Erwinia jumbo bacteriophage vB_EamM_Y3) have been determined by X-ray crystallography and catalytic mechanisms have been proposed. Although both proteins are inverting glycoside hydrolases with similar catalytic mechanisms, they present large morphological differences, illustrating the different ways phages solve similar biological problems. A third structure, the Cysteine-Histidine Aminopeptidase/Hydrolase (CHAP) domain from the endolysin of staphylococcal bacteriophage K, was determined in the presence of triglycine, an analogue of its natural substrate, revealing a potential secondary ligand binding site. Additional endolysins and fibres targeting both Gram-positive and Gram-negative bacteria have been purified and crystallized, increasing the knowledge on these proteins. In the cases where their structure could not be obtained, in-silico models were generated to explore the mechanism of these enzymes.

Access Level

info:eu-repo/semantics/openAccess

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