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

Abstract

General AbstractThe need for the development of antimicrobial agents has increased considerably due to emerging pathogens, bioterrorism and antimicrobial resistance (Spellberg et al., 2003). As antimicrobial resistance (AMR) is a worldwide public health concern, identification of inhibitory substances that effectively overcome this problem is urgently needed. This study aimed to investigate the modes of inhibitory activity of a variety of chemical species, the biopolymer chitosan, zinc oxide and silver nanoparticles (ZnONPs and AgNPs), the bacteriocin nisin, and a crude bacterial extract with a putative, uncharacterized bacteriocin. I used large-scale phenotypic screens performed with Saccharomyces cerevisiae and/or Escherichia coli for these functional genomics studies, followed by mechanistic studies to confirm the cellular pathways elucidated by the 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 the S. cerevisiae deletion mutant set (≈4600 mutants). This functional analysis supported the standing hypothesis that chitosan perturbs membrane functions and revealed an additional novel mode of action: mutants with deletions in a set of genes related to protein synthesis were very 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 by chitosan.The antifungal and cytotoxic modes of action of ZnONPs and AgNPs were also investigated using the 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. Secondary assays confirmed that ZnONPs cause disruption and depolarization of cell membrane and, in addition, alter cell wall integrity in yeast. In contrast, mutants lacking genes involved in transcription and RNA processing, cellular respiration, and endocytosis and vesicular transport were highly sensitive to AgNPs. For AgNPs, reduction of transcription, disruption of the electron transport chain performance and interference with endocytosis were confirmed by secondary assays.The antibacterial mode of action of nisin was investigated using an E. coli mutant set (Keio) comprising ≈4000 mutants. Large-scale phenotypic screening indicated that nisin interferes with the following processes: cell wall/membrane/envelope biogenesis, cell cycle and DNA replication, recombination and repair. A DNA content assay based on flow cytometry suggested that nisin may interfere with DNA synthesis as a mode of action.Finally, the antimicrobial mode of action of a fermented supernatant produced by Bifidobacterium breve was investigated using the E. coli Keio mutant set. Results from the largescale phenotypic screen indicated that the bifidobacterial supernatant interferes wi...