Kundu, Karishma (2024) Study of the Effects of Different Stressors on Cyanotoxin Production through Metabolomics and Metagenomics. [Tesi di dottorato]

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Abstract

Cyanobacteria are the most primitive group of organisms thriving on the earth’s surface for millions of years. During this course of time, they have adapted, modified and evolved regarding the environmental niches. They have turned out to be one of the most scientifically used organisms, whether it can be for pharmacological causes, ecological causes or anything non-ecological. Cyanobacteria are green, photosynthetic bacteria that produce most of their food and important metabolites themselves. This PhD Thesis focuses on growing Anabaena flos aqua, a marine cyanobacterium, under different stress conditions, mimicking natural stimuli and producing a diverse range of secondary metabolites; developing and optimizing a framework for producing more secondary metabolites at lab conditions and facilities; and finally testing the extract/fractions for cell viability in lethal pancreatic cancer model. Anabaena flos aquae were grown in variations in BG11 media, especially in various iron levels (as iron is one of the key factors in the production of secondary metabolites such as siderophore). The variation in iron concentration alone does produce novel varieties of siderophores, including Synechobactin C16:1, Synechobactin oxyC14, Desacetyl-synechobactin A, Desacetyl-Synechobactin C14, Deoxysynechobactin C14 and Deoxysynechobactin C14:1. Within this PhD Thesis workflow, we came up with establishing a framework called a 4-stage pipeline and validated it using the model organism, Anabaena flos aquae UTEX 1444, as a prototypical organism for increased siderophore expression/production. Our framework takes less time, is less expensive, and can be elaborated to any microorganism, making it a demanding strategy over the other approaches explained by researchers over the past few years, such as the OSMAC (One Strain Many Compound) framework. Studying the genetic orientation of the model organism (Anabaena variabilis), discovering the siderophore operons and analysing the promoters are all the initial stages of the pipeline. The reporter vectors were prepared, cloned with siderophore coding regions and inserted in E.coli to justify the gene expression under various environmental conditions such as temperature, pH, salinity, iron levels, citrate, sugars and amino acids such as lysine (all suggested to us after analysing the promoter region). The screening was achieved using as little as 50 µl sample, providing a quick and economical platform for testing media variations. Following this, the best culture conditions were used to optimize the culture conditions of Anabaena flos-aquae UTEX1444 for siderophore production in the laboratory. The produced siderophores were identified and quantified using mass spectroscopy (MS)-based molecular networking. Assuming that the structurally related compounds share similar fragmentation patterns, we discovered novel siderophores and in increased quantities. Schizokinen, Synechobactin A and Synechobactin C14 being the majority, especially in the culture with 3µM of ferric ammonium citrate (0-5 µM) confirming the bell-shaped connection between the amount of Fe3+ and siderophore production suggested by our in-vitro pipeline. In the end, we tested the fractions rich in siderophore (composition as stated in the above study) for cell viability in the PDAC (Pancreatic ductal adenocarcinoma) SUIT2 cell line. The three fractions, namely Butanol fraction, Methanol fraction, and Aqueous fraction, showed lower cell proliferation at the initial concentrations and noticeable cell death at high concentrations such as 25 µg/ml for Butanol Fraction, 25 µg/ml for Methanol Fraction and 250 µg/ml for Aqueous Fraction. To know more about the cell death pathway, we combined our extract and fractions with ferroptosis (a form of cell death regulated by the presence of iron) inducers and inhibitors. Erastin was used as a ferroptosis inducer (standard) and showed cell death, and its activity was successfully reversed by using four different anti-ferroptosis drugs (inhibitors). For various fractions, we got mixed results as the ferroptotic inhibitors failed to rescue the viability, hence suggesting a different pathway of ferroptosis, i.e. cell death. Future work includes testing the hallmarks for ferroptosis, such as GPX4 level, GSH activities, ROS levels and the amount of lipid peroxidation in the cells.

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