(337ah) Controlling Intracellular Mutagenesis: A Key to Targeted Therapeutic Strategies Against Antibiotic Resistance

• Understanding the genetic and molecular mechanisms of bacterial drug resistance/tolerance using single cell analysis techniques such as flow cytometry, fluorescence microscopy, molecular biology techniques such as PCR, molecular cloning, antibiotic tolerance/resistance assays, genetic perturbation techniques
• Identifying potential candidate drugs using high throughput screening of chemical library and in-vitro drug testing in bulk and in hollow fiber bioreactor for mimicking the continuous culture condition in-vivo
• Exploring novel strategies to combat antimicrobial resistance/tolerance such as drug combination therapy tested in highly pathogenic organisms such as Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae
• Redirecting intracellular mutagenesis for augmenting evolutionary rate using cyclic genetic perturbation treatment towards industrially relevant and environment-friendly processes such as Polyethylene Terephthalate (PET) degradation by Ideonella sakaiensis.

The emergence of antibiotic resistance represents a critical global health crisis that poses a substantial threat to the remarkable medical advancements achieved in the past century. Bacterial cells possess the ability to rapidly develop resistance to antibiotics, partly due to the error-prone DNA repair mechanisms involved in the SOS response, which becomes activated in response to DNA damage. Although the SOS response is a fundamental survival strategy that safeguards bacterial genomic integrity, its mutagenic processes can give rise to antibiotic-resistant cells. Hence, a comprehensive understanding of mutagenic mechanisms becomes imperative to devise effective clinical treatment strategies combating the worldwide health crisis of antibiotic resistance.

The primary objective of this project is to gain insights into these mechanisms by perturbing the SOS response using mutagenic agents such as UV radiation and conventional antibiotics. Employing an Escherichia coli promoter library, we established a high-throughput screening approach and identified several genes, namely recA, recN, rmuC, polB, and dinB, exhibiting heightened expression levels in response to DNA damage. Subsequent deletion of these genes either individually or in combination resulted in a significant reduction in intracellular mutagenesis. Notably, recA, the global regulatory gene of the SOS response, exhibited the highest upregulation during treatment, and further investigations revealed a positive correlation between the level of recA expression and the extent of mutant formation. Our findings were further substantiated through the evaluation of selected metabolic inhibitors that suppressed recA induction by impeding cellular transcription, translation of DNA repair genes, and/or ATP production, which is crucial for the energy-demanding SOS response pathway. Treatment with these inhibitors resulted in a considerable decrease in UV-induced mutagenesis. In an attempt to translate these findings into clinically relevant therapeutic measures against antibiotic resistance, we co-treated E. coli cells with a conventional fluoroquinolone (ciprofloxacin) and metabolic inhibitors including chlorpromazine, chloramphenicol, and arsenate. It was observed that the fluoroquinolone, known for specifically inducing the SOS response, failed to induce mutagenesis in the presence of these inhibitors, leading to a prominent reduction in recA expression levels in the co-treated E. coli cells.

Collectively, our study highlights the significance of understanding and regulating intracellular mutagenesis within the context of antibiotic resistance. Furthermore, it highlights the targeting of the SOS regulatory network as a potential therapeutic adjunct in combination therapies aimed at combating antibiotic resistance effectively.