MR/V027204/1 - Dissecting Gram-negative envelopes biogenesis using system approaches

The World Health Organization (WHO) has identified antibiotic-resistant pathogens as a major threat to global health, food security, and development, affecting people of all ages and nationalities. The increasing number of pathogens resistant to current antibiotics highlights the urgent need to act against these rapidly adapting bacteria. Even in advanced medical settings, untreatable infections lead to longer hospital stays and, in severe cases, death. The WHO warns of a looming post-antibiotic era where common infections and minor injuries could once again become deadly, urging greater investment in pathogen research to develop effective treatments. To guide this effort, the WHO has prioritized a list of critical pathogens, with Gram-negative bacteria at the top due to their near-untreatable status. This research proposal focuses on understanding how Gram-negative bacteria construct their protective cell envelope, aiming to develop strategies to overcome their resistance to antibiotics. The bacterial cell envelope, a multi-layered structure, shields cells from hostile environments, including antibiotics. Gram-negative bacterial envelopes are particularly significant because they serve as both a structural component and a permeability barrier. This barrier, created by an asymmetric lipid bilayer called the outer membrane, blocks toxic compounds, including many antibiotics, from entering the cell. Identifying the genes that maintain the envelope's structure and impermeability is crucial for understanding bacterial self-protection and finding new ways to breach this barrier for antibiotic delivery. However, genome-wide screens to assess envelope integrity in Gram-negative bacteria are currently lacking. This proposal addresses that gap by developing genome-wide, high-throughput assays to map the gene network involved in Gram-negative envelope biosynthesis. To study gene function, I will systematically delete individual genes from the genome and analyze the effects, such as changes in antibiotic response. Using a collection of single-gene deletion mutants, I will investigate envelope biogenesis in the Gram-negative pathogens Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. This approach will reveal how each gene deletion impacts responses to various antibiotics and environmental stresses. The resulting stress-response maps will provide insights into how these bacteria maintain envelope integrity under challenge. By analyzing these networks, I will identify genes critical to envelope biogenesis. Following the identification of key genes or pathways, I will further explore their cellular roles using my expertise in molecular biology. This will involve studying genetic interactions to determine if other genes depend on these key players. These findings will help pinpoint potential drug targets and strategies to overcome the envelope's molecular barrier, ultimately improving treatments for Gram-negative bacterial infections. By deepening our understanding of envelope biogenesis, this research will contribute to addressing the global challenge of antibiotic resistance, aligning with the WHO's call to tackle critical pathogens.