Investigating Mechanisms of Toxicity and Detoxification of BMAA in Escherichia coli

University of Technology Sydney. Faculty of Science.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