Testing the potential of mycobacteriophage endolysins fused to biodegradable nanobeads for controlling mycobacteria : a thesis presented in partial fulfilment of the requirements for the degree of Master of Natural Sciences at Massey University, Albany, New Zealand
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Date
2019
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Massey University
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Abstract
10.4 million people are diagnosed with tuberculosis, worldwide, every year, according to the World Health Organisation. Mycobacterium tuberculosis is a Gram-positive bacterial pathogen that can easily be transmitted to health care professionals and people supplying aid in these nations. Fortunately, over the last 100 years, the bacteriophage has gained traction as a suitable therapeutic, antibiotic-alternative against bacterial pathogens, such as M. tuberculosis. Herein I describe my work utilising endolysins the lytic protein that bacteriophages usually employ to burst out of the cell, but instead using these proteins to lyse “from without”. In order to develop a proof-of-principle product, we used the expansive M. smegmatis bacteriophage collection and chose eight endolysin candidates for testing against M. smegmatis. These endolysins were bioinformatically analysed for active domains before being synthesised and inserted into an expression vector to produce fused biodegradable nanobeads made of polyhydroxyalkanoate. These nanobeads were tested for activity against M. smegmatis, a safe mycobacterium closely related to pathogenic tuberculosis. Four distinct tests were carried out to test the efficiency of these beads in causing cell death in different situations (45 minutes and 5 hours, across concentrations of 10mg/ml, 20mg/ml and 80mg/ml. Ultimately the nanobead fusions of endolysin Inca (lysin B) caused the most cell death at 80mg/ml after exposure to M. smegmatis for 5 hours in a standing culture, at 78.87% cell death ± 5.21. When the nanobeads were applied to filter paper to mimic application to a hospital mask as a proof-of-concept approach before spraying with a bacterial aerosol, we saw that endolysin nanobead Jaws (lysin B) caused the most cell death with 75.54% ± 3.15 at 80mg/ml. These results are promising and present a unique opportunity to take advantage of an existing natural mechanism to use as a prophylactic defense against pathogenic bacteria in hospital settings.
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Figures 1, 2, 3B, 4 & 5A were removed for copyright reasons but may be accessed via their sources listed in the References.
Keywords
Bacteriophages, Bacteriolysis, Mycobacterium, Biopolymers