Elucidating Mechanisms of Action of Bioactive Substances by Chemical-Genetic Profiling

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  • The need for the development of antimicrobial agents has increased considerably in the last years due to the emergence of new infections, bioterrorism and antimicrobial resistance. This study aimed to investigate the modes of inhibitory activity of a variety of chemical species using a functional genomic approach followed by mechanistic studies to confirm the cellular/pathway targets elucidated by the large-scale phenotypic screens performed using non-essential gene deletion sets (Saccharomyces cerevisiae and Escherichia coli).The antifungal mode of action of chitosan was investigated using S. cerevisiae deletion mutant set (≈4600 mutants). Functional analysis showed that mutants with deletions of genes related to protein synthesis were sensitive to chitosan. Disruption of protein synthesis was confirmed by a β-galactosidase expression assay suggesting that this is a primary mode of antifungal action.The antifungal and cytotoxic modes of action of zinc oxide and silver nanoparticles (ZnONPs and AgNPs) were investigated using a S. cerevisiae deletion mutant set. The large-scale phenotypic screen for ZnONPs showed that mutants lacking genes involved in transmembrane and membrane transport, cellular ion homeostasis and cell wall organization or biogenesis exhibited high sensitivity to ZnONPs. Results from the same screen for Ag NPs indicated that mutants lacking genes involved in transcription and RNA processing, cellular respiration and, endocytosis and vesicular transport were highly sensitive to these NPs. Secondary assays confirmed the results obtained from the phenotypic screenings.The antibacterial mode of action of nisin was investigated using an E. coli mutant set (Keio) composed by ≈ 4000 mutants. Large-scale phenotypic screening indicated that nisin may interfere with the following processes: cell wall/membrane/envelope biogenesis, cell cycle and DNA replication, recombination and repair. A DNA cellular content reduction detected by flow cytometry suggested that nisin may interfere with DNA synthesis as a secondary mode of action.The antimicrobial mode of action of a fermented supernatant produced by Bifidobacterium breve was investigated using the same E. coli mutant set (Keio). Results from the large-scale phenotypic screen showed that the bifidobacterial supernatant may interfere with carbohydrate transport and metabolism, intracellular trafficking, secretion and vesicular transport, and energy production and conversion.

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  • Copyright © 2016 the author(s). Theses may be used for non-commercial research, educational, or related academic purposes only. Such uses include personal study, research, scholarship, and teaching. Theses may only be shared by linking to Carleton University Institutional Repository and no part may be used without proper attribution to the author. No part may be used for commercial purposes directly or indirectly via a for-profit platform; no adaptation or derivative works are permitted without consent from the copyright owner.
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  • 2016


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