Rapid Stress Response in Bacterial Pathogens: the Role of Extant Porins in Antibiotic Tolerance

Project Summary Cells of most Gram-negative bacterial pathogens can develop tolerance to chemical stressors through rapid adaptations in the outer membrane that are poorly understood. This application focuses on uncovering the adaptation mechanisms responsible for stress-induced tolerance to chemical stressors, such as antibiotics, in major pathogenic bacterial species. A first-line defense in Gram-negative species is inhibiting porin-mediated uptake to minimize stressor accumulation within the cell. By preventing or delaying the intracellular buildup of bactericidal compounds, the cell may gain sufficient time to activate stress response pathways. The prevailing view suggests that phenotypically tolerant bacteria survive by inhibiting porin function through the downregulation of their synthesis. However, a critical gap exists in the current understanding of porin-mediated phenotypic tolerance: even if the expression of porins is downregulated, there are currently no known ways to eliminate existing porins in the outer membrane. Theoretically, the cell must divide multiple times under stressful conditions to gradually decrease porin abundance. This scenario is improbable because the stressful conditions might hinder cell division, or the cell could perish before dividing. New evidence from the PI's lab suggests that E. coli can adaptively restrict intracellular accumulation of drugs by inhibiting the functioning of extant porins within minutes of exposure to stress. The proposed work will test the central hypothesis that cells attempt to block mass transfer through their porins within seconds or minutes of exposure to a chemical stressor, with the purpose of limiting its intracellular accumulation. A combination of single-cell growth and CFU (colony-forming units) assays, fluorescent dye-uptake (fluorescence), and motility assays will be performed to probe physiological responses of cells under micro-confinement during stress. The experiments will be compared with mathematical models of drug transport to explain how heterogeneity in phenotypic tolerance mechanisms emerges in sensitive populations. In particular, the proposed work will explain how energetically active cells develop antibiotic tolerance while preserving ATP and the proton-motive force (PMF). The proposed work will define novel mechanisms of stress tolerance in pathogens, with wide implications for antibacterial therapies and human health.