Investigating Mechanisms of Toxicity and Detoxification of BMAA in Escherichia coli

Italiano, Carly Jade

Investigating Mechanisms of Toxicity and Detoxification of BMAA in Escherichia coli

Italiano, Carly Jade

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Publication Type:: Thesis
Issue Date:: 2021

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Field	Value	Language
dc.contributor.author	Italiano, Carly Jade
dc.date.accessioned	2022-06-10T02:50:56Z
dc.date.available	2022-06-10T02:50:56Z
dc.date.issued	2021
dc.identifier.uri	http://hdl.handle.net/10453/158049
dc.description	University of Technology Sydney. Faculty of Science.	en_US.UTF-8
dc.description.abstract	Neurodegenerative diseases are becoming an increasingly common problem affecting our population. Research into environmental triggers contributing to development of these conditions is vital for understanding disease pathogenesis and prevention. Non-protein amino acids are a class of naturally occurring molecules that can interfere with metabolic processes in cells to cause toxicity. Exposure to the neurotoxic non-protein amino acid BMAA has been linked to the development of neurodegenerative disease. BMAA is known to be produced by cyanobacteria, and their wide distribution globally increases the likelihood of human contact with this toxin. Additionally, BMAA can bioaccumulate in ecosystems, compounding the risk of exposure. While there are many studies on the neurotoxic effects of BMAA in mammalian systems, less work has been conducted in prokaryotic species such as bacteria. Previous work has indicated that the bacterium Escherichia coli exhibits tolerance towards BMAA that contrasts with the toxicity seen in mammalian cells and animal models. However, there have been no studies into the mechanism behind this tolerance. In this project, screening of Escherichia coli mutants with disruptions to various amino acid biosynthesis pathways indicated that cysteine biosynthesis genes (cysE, cysK, and cysM) are important for BMAA tolerance. Disruption to any of these genes resulted in susceptibility to BMAA, which manifested as a delay in the onset of logarithmic growth. Severity of delay was dependent on BMAA concentration. However, following a period of BMAA-induced growth delay, a recovery in growth occurred. The possibility that additional genetic mutations spontaneously arise in cultures and suppress sensitivity to BMAA in Escherichia coli (cysE mutant) was also investigated. Results showed that genetic changes were not responsible for growth restoration following BMAA-induced inhibition. Analysis of gene expression changes in Escherichia coli exposed to BMAA showed a pattern indicative of an iron starvation response. There were also changes to sulfur homeostasis and amino acid metabolism (cysteine and threonine). O-acetylserine sulfhydrylase (cysM) from the cysteine biosynthetic pathway was tested for ability to enzymatically degrade BMAA, which would explain the requirement of cysteine biosynthetic genes for BMAA tolerance. While the enzyme did not degrade BMAA, the cofactor pyridoxal-5’phosphate which the enzyme uses to catalyse cysteine biosynthesis reactions did, resulting in the production of methylamine. Results from this project have explored different possibilities for Escherichia coli tolerance towards BMAA and provided insights into BMAA metabolism, opening many future directions of BMAA research including cysteine metabolism, enzymatic BMAA degradation, and iron chelating properties of BMAA.	en_US.UTF-8
dc.format	Thesis (PhD)
dc.language.iso	en_US	en_US.UTF-8
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/158049/2/02whole.pdf
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	au.edu.uts.lib/ppc
dc.rights	info:eu-repo/semantics/openAccess
dc.title	Investigating Mechanisms of Toxicity and Detoxification of BMAA in Escherichia coli	en_US.UTF-8
dc.type	Thesis
utslib.copyright.status	open_access	*

Abstract:

Neurodegenerative diseases are becoming an increasingly common problem affecting our population. Research into environmental triggers contributing to development of these conditions is vital for understanding disease pathogenesis and prevention. Non-protein amino acids are a class of naturally occurring molecules that can interfere with metabolic processes in cells to cause toxicity. Exposure to the neurotoxic non-protein amino acid BMAA has been linked to the development of neurodegenerative disease. BMAA is known to be produced by cyanobacteria, and their wide distribution globally increases the likelihood of human contact with this toxin. Additionally, BMAA can bioaccumulate in ecosystems, compounding the risk of exposure. While there are many studies on the neurotoxic effects of BMAA in mammalian systems, less work has been conducted in prokaryotic species such as bacteria. Previous work has indicated that the bacterium Escherichia coli exhibits tolerance towards BMAA that contrasts with the toxicity seen in mammalian cells and animal models. However, there have been no studies into the mechanism behind this tolerance. In this project, screening of Escherichia coli mutants with disruptions to various amino acid biosynthesis pathways indicated that cysteine biosynthesis genes (cysE, cysK, and cysM) are important for BMAA tolerance. Disruption to any of these genes resulted in susceptibility to BMAA, which manifested as a delay in the onset of logarithmic growth. Severity of delay was dependent on BMAA concentration. However, following a period of BMAA-induced growth delay, a recovery in growth occurred. The possibility that additional genetic mutations spontaneously arise in cultures and suppress sensitivity to BMAA in Escherichia coli (cysE mutant) was also investigated. Results showed that genetic changes were not responsible for growth restoration following BMAA-induced inhibition. Analysis of gene expression changes in Escherichia coli exposed to BMAA showed a pattern indicative of an iron starvation response. There were also changes to sulfur homeostasis and amino acid metabolism (cysteine and threonine). O-acetylserine sulfhydrylase (cysM) from the cysteine biosynthetic pathway was tested for ability to enzymatically degrade BMAA, which would explain the requirement of cysteine biosynthetic genes for BMAA tolerance. While the enzyme did not degrade BMAA, the cofactor pyridoxal-5’phosphate which the enzyme uses to catalyse cysteine biosynthesis reactions did, resulting in the production of methylamine. Results from this project have explored different possibilities for Escherichia coli tolerance towards BMAA and provided insights into BMAA metabolism, opening many future directions of BMAA research including cysteine metabolism, enzymatic BMAA degradation, and iron chelating properties of BMAA.

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