A novel pathway altering OM permeability

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PROJECT SUMMARY The gram-negative outer membrane (OM) represents a strong permeability barrier that impedes the entry of many antibiotics. The majority of the species the US Centers for Disease Control and Prevention list as of “urgent” or “serious” concern for antibiotic resistance are gram-negative in part due to the impermeability of the OM. In recent years, it has become clear that the permeability of the OM can be altered by the physiological state of the cell. Specifically, clinically relevant stresses such as nutrient limitation can result in strengthening of the OM permeability barrier, further decreasing the entry of antibiotics. Elucidation of the pathways responsible for this strengthening will lead to new targets for the development of small molecules that can weaken the OM permeability barrier. The laboratory’s long-term goal is to understand the mechanisms that change the permeability of the OM during periods of clinically relevant stress. Specifically, this project aims to elucidate a novel link between loss of DNA mismatch repair (MMR) and alteration of OM permeability in Escherichia coli K12. MMR is a highly conserved DNA repair mechanism found throughout all domains of life. MMR mutants have been found in clinical antibiotic resistant strains and have been proposed to be an antecedent to the development of resistance mutations facilitated by an increased mutation rate. However, preliminary data demonstrate a second pathway where loss of MMR leads to resistance to a broad range of antibiotics through alteration of OM permeability. Thus, loss of MMR in a host environment would allow bacteria to survive antibiotics treatment longer, while also increasing the probability that a specific resistance mutation can develop due to the increased mutation rate. The SOS DNA damage stress response pathway is not necessary for strengthening the OM permeability barrier demonstrating that a novel pathway connects loss of MMR to OM permeability. The central hypothesis of this work is loss of MMR activates a novel pathway involving signal transduction and transcriptional changes that alter the permeability profile of the OM. This project will elucidate genes involved in this pathway by identifying transcriptional changes that result from pathway activation leading to altered OM permeability (Aim 1) and determining the genes that are necessary for the pathway to altered OM permeability (Aim 2). Completion of the aims will transform understanding of the link between DNA repair and OM permeability and has the potential to uncover new targets for drug discovery.