A Comprehensive Transposon Screen Reveals Universal Biofilm Effectors in Uropathogenic Escherichia coli
Date of Award
College of Arts & Sciences
In the urinary tract, bacterial biofilms can develop extracellularly, intracellularly and on virtually all artificial implants, producing chronic and often intractable infections. Understanding the mechanisms of biofilm development will aid in the identification of therapeutic targets for effective disruption or prevention of biofilm formation by uropathogens. Uropathogenic Escherichia coli (UPEC) account for most cases of urinary tract infections (UTI) and can form in vitro and in vivo biofilms. We used UPEC as a model organism to engage fundamental biological questions about the roles of biofilm effectors in niche establishment and pathogenesis. First, we generated a UPEC mutant library by transposon mutagenesis and screened it in multiple conditions for factors that influence biofilm formation, including biofilm assays using luria broth, biofilm assays using Yeast Extract/Casamino Acids (YESCA) broth, hemagglutination assays, and swarming assays. We identified 180 mutants with altered biofilm properties, of which 66 were defective in every condition tested and others were condition-specific. Using a genetic approach, mapping 57/66 transpositions identified 41 factors, the disruption of which affects UPEC biofilm formation in all conditions tested. We categorized these universal biofilm factors into 6 categories: flagellar components, extracellular adhesive fibers, exopolysaccharide formation, cell maintenance, regulators, and hypothetical proteins. We then characterized the corresponding mutants from each class, testing their ability to swim and to form adhesive organelles such as Type 1 pili and curli, factors which have previously been associated with in vivo biofilm formation and UPEC virulence. Our findings revealed mutants, including Tn::yfiLRNB, Tn41::ylbF and Tn97::rfe, that had significant defects in swimming motility and adhesive organelle formation and may also be defective in their ability to cause infection. More importantly, the contribution of these mutants in UPEC pathogenesis has not been investigated. We are currently investigating the role of these 3 factors in UPEC pathogenesis. Collectively, these studies identified important factors involved in biofilm formation in UPEC that could play various roles in the establishment of infection. Future studies will include characterizing the mechanisms by which identified novel factors affect biofilm formation in vitro and initiating studies in the host to assess the potential of targeting these factors for the development of drugs and other therapeutic methods that disrupt or prevent biofilms and urinary tract infections.
Gu, Alice, "A Comprehensive Transposon Screen Reveals Universal Biofilm Effectors in Uropathogenic Escherichia coli" (2011). Undergraduate Theses—Restricted. 11.