Dissecting Protein Stratification and Regulation in Uropathogenic Escherichia coli Biofilms

Abstract

Catheter-associated urinary tract infections (CAUTIs) account for ~30% of hospital-acquired infections. Biofilm formation, a process where bacteria assume a temporary multicellular lifestyle, serves as a reservoir for establishment and persistence of CAUTI by many uropathogens. In the biofilm, bacteria can persist for extended periods, owing to protection from antibiotics and immune responses. Biofilm bacteria also alter gene-expression in a location-specific manner, driven by changes in the immediate microenvironment, to create a heterogeneous population that further complicates treatment. Identification of subpopulations crucial to resilience, could present targets for novel anti-biofilm strategies to attenuate CAUTI. This dissertation describes the use of MALDI-TOF imaging mass spectrometry as a tool towards identification of subpopulations based upon unique protein expression, in surface-associated biofilms formed by the primary uropathogen, uropathogenic <i>Escherichia coli<i> (UPEC). My studies revealed environmental-induced segregation of subpopulations expressing different adhesive fibers, including type 1 pili (<i>fim<i>), which are critical for infection establishment in the bladder and on catheters. Type 1 pili were only expressed in subpopulations comprising the biofilm air-exposed region, suggesting expression alters in response to oxygen fluctuations. Type 1 pili expression is controlled by a phase-variable promoter element. My studies revealed that during oxygen-depletion, the <i>fim<i> promoter inverts to a transcription-incompetent state resulting in decreased pilus production. Inversion resulted from an imbalance in expression of the recombinase enzymes that control promoter orientation. In addition, my studies demonstrated a role for the fumarate and nitrate reductase regulator (FNR) as an additional regulator of <i>fim<i> gene expression. Finally, in addition to transcriptional-checkpoints, data point towards a third regulatory mechanism that acts post-translationally to repress pilus elaboration under oxygen-deplete conditions. These data, for the first time, demonstrate regulation of type 1 pili in response to environmental oxygen levels. Analysis of the oxygen environment of the murine bladder, demonstrates conditions suitable for aerobic bacterial respiration and expression of type 1 pili. Together, these data show how type 1 pili are oxygen-regulated to dictate the infectious niche of UPEC, and could lead to the development of new anti-biofilm treatments for the attenuation of CAUTI.