Genetic engineering as a means to study the immuno-modulating properties of Uropathogenic Escherichia coli

Abstract

One of the most common bacterial infections in the world is urinary tract infections (UTIs). Women are mostly affected by UTIs and there is a high rate of reoccurrence after the first infection. Several pathogens can lead to UTIs, but the most common causing agent is uropathogenic Escherichia coli (UPEC). Out of several virulence factors, UPEC uses its type 1 pili to bind to bladder epithelial cells, thus avoiding being washed away by voiding the bladder and subsequently causing the epithelial cells to internalize the bacterial cells. The bladder’s innate immune response to UPEC as well as how UPEC manages to avoid this response is very well described. However, limited knowledge is available for the adaptive immune response. A few studies show that the immune system is capable of mounting an adaptive immune response alone or with the aid of vaccines, and another study shows that the adaptive immune response is terminated prematurely, thereby not clearing the infection completely. Dendritic cells (DCs) are antigen-presenting cells that serve as a bridge between the innate and adaptive immune systems. When DCs encounter bacteria, they undergo maturation and activate the adaptive immune response. Remarkably, unpublished data shows that when DCs encounter UPEC they are unable to migrate and activate T cells. UPEC mutants that either express the type 1 pili all the time or not at all were created and used to infect wild-type (wt) DCs. The RNA profile of the DCs after infection was determined and showed an up-regulated gene which will be termed A01. Deletion of this gene from wt DCs followed by infection with UPEC rescue the migration and activation phenotype. In this thesis, a dendritic cell line was engineered to contain an inducible expression of the A01 gene. The cell line was validated for specific DC and maturation markers, such that it can be further used for migration assays. In addition, cell adhesion assays were performed by infecting DCs to either type 1 piliated UPEC or non-piliated UPEC to determine the difference in adhesion. Lastly, both type 1 piliated and non-piliated UPEC were genetically engineered to express YFP and were used in phagocytosis assays, where wt DCs are infected with both YFP expressing UPEC mutants and fixed at certain time points to determine if the type 1 pili cause a difference in the phagocytosis mechanism.