Engineering novel bacterial biosensors for the characterization of membrane active natural products

• Main Author: Richard, Celine Sarah Marine
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: UiT The Arctic University of Norway 2024
• Subjects: Antibiotic resistance; bacterial biosensor; antimicrobial peptide; natural product; drug discovery; reporter gene; membrane; DOKTOR-002; Arctic; Moa
• Online Access: https://hdl.handle.net/10037/33610

Treatment of bacterial infections has become more challenging due to the expansion of antibiotic resistance. Especially, resistant Gram-negative pathogens are burdening healthcare systems worldwide. This increases the need for new antibiotics able to penetrate the outer-membrane (OM) of Gram-negatives. Natural products (NPs) from the marine environment e.g. antimicrobial peptides (AMPs) are interesting drug lead candidates as they often show potent activity against bacterial membranes and are still under-studied compared to NPs from the terrestrial environment. Mode of action (MoA) specific drug lead discovery requires new tools, which can be based on engineered bacterial cells as biosensors. To identify MoA of peptides in general, and the impact of AMPs on bacterial membranes specifically, bacterial whole-cell biosensors (BWCBs) based on different reporter gene constructs are one possible solution to facilitate effective discovery pipelines. The work conducted in this thesis aims to engineer novel BWCBs with relatively new reporter genes to facilitate a better understanding of the impact of marine AMPs on the bacterial membranes already during screening steps of drug discovery. In paper I, as part of the ongoing research for antimicrobial NPs, the BWCBs Escherichia coli (for Gram-negative) and Bacillus subtilis (for Gram-positive) carrying the bacterial luciferase lux operon or the eukaryotic click beetle luciferase lucGR were used to study the impact of compounds extracted from the arctic bryozoan Securiflustra securifrons , on the cell viability or membrane integrity, respectively. One of them, the Securamine H, was found to inhibit the viability of Gram-positive bacteria and reduce metabolic activity of B. subtilis but the MoA on this intracellular target still needs to be identified. In paper II, a recently discovered reporter gene, unaG , from the Japanese eel Anguilla japonicas , was used to engineer a novel MoA specific BWCB to investigate OM integrity of Gram-negative bacteria. We used the E. coli ...