Proteotoxic Stress Bioreporter Enables Mechanism-Informed Antibiotic Discovery

Area of Science:

• Microbiology
• Biochemistry
• Drug Discovery

Background:

• Antibiotic resistance is a critical global health threat, exacerbated by dwindling antibiotic discovery.
• Natural products are vital sources of antibiotics, but mechanistic insights are often limited.
• Current screening methods lack mechanistic information, necessitating advanced tools.

Purpose of the Study:

• To develop a novel bioreporter for detecting proteotoxic stress, a key indicator of antibacterial activity.
• To enable mechanism-informed antibiotic discovery and accelerate the identification of new antibacterial agents.
• To validate the bioreporter's utility in screening natural product extracts.

Main Methods:

• Development of a *Bacillus subtilis* bioreporter (P_clpE-lux) to signal heat-shock Clp-ATPase gene induction.
• Utilizing the bioreporter for high-throughput screening in both solid and liquid formats.
• Coupling the bioreporter with microfractionation-based metabolomics for metabolite identification.

Main Results:

• The P_clpE-lux bioreporter effectively monitors proteotoxic stress and identifies antibacterial agents.
• The bioreporter detected compounds not previously linked to proteotoxic stress.
• Integration with metabolomics accelerated the discovery of streptothricin derivatives, including novel analogues.

Conclusions:

• The P_clpE-lux bioreporter is a versatile tool for mechanism-informed antibiotic discovery.
• This approach enhances detection sensitivity and dereplication efficiency in natural product screening.
• The study provides a valuable method for identifying novel antibacterial compounds to combat resistance.