Environmental and Molecular Control of the Uropathogenic Escherichia coli Extracellular Matrix.

Abstract

Abstract Bacteria are highly social and community-centric, despite being labeled as “single-celled” organisms. The communities that dominate bacterial lifestyles are called biofilms. Cells in a biofilm are protected from a variety of environmental insults that includes toxins, antibiotics, and immune defense mechanisms. Uropathogenic Escherichia coli (UPEC) form biofilms outside the host to prolong survival, inside the host on catheters, and inside of bladder epithelial cells. Bacteria in biofilms self-produce a protective extracellular matrix (ECM) composed of proteins, sugars, and DNA. The transcription of UPEC ECM components, curli and cellulose, is activated via the CsgD protein. The projects presented here detail molecular pathways that influence production of the UPEC ECM. I have found that: 1) The production of both curli and cellulose is activated by the catabolite repressor protein, which activates transcription of csgD in the absence of glucose. 2) YfiR is a disulfide bonded periplasmic protein that misfolds in reducing conditions, which leads to constitutive production of cyclic di-GMP by YfiN and constitutive expression of cellulose. 3) Cysteine auxotrophs have uncoupled ECM production, producing decreased amounts of cellulose and increased amounts of curli compared to WT. Uncoupling of the ECM is due to the lack of thiol reductants in cysteine auxotrophs. Cellulose production is inhibited in cysteine auxotrophs through yfiR, possibly because YfiR is stabilized by the oxidative cellular environment. I also found that UPEC cysteine auxotrophs (including those from 7 patient isolates) produce curli at 37°C, and that the increased cellular oxidation of cysteine auxotrophs affords resistance to the antibiotic mecillinam. Interestingly, sub-inhibitory concentrations of mecillinam and Bactrim inhibit curli production in WT but not in cysteine auxotrophs. Collectively my work describes two regulators of CsgD, the influence of redox on UPEC biofilms, and novel molecular pathways that influence ECM production at 26°C and 37°C.