Urinary Tract Infections (UTIs) are a huge public health problem affecting 150 million people each year worldwide. UTIs are predominantly caused by uropathogenic Escherichia coli (UPEC). The roles of flagella in the pathogenesis of UPEC UTI, including immune responses to infections are not well understood. A protective role for IL10 in acute UTI has been shown; however, the UPEC virulence factor(s) responsible for IL10 induction is yet to be identified.
We hypothesised that UPEC flagella induce the production of IL10 during acute UTI. UPEC CFT073 and a derivative strain containing four mutations in genes encoding fimbriae and pili (CFT073-Quad) were deleted for the flagellar filament, fliC, using overlap-extension PCR and gene replacement with λ-red recombinase. The flhDC gene, which regulates flagella biosynthesis, was used in IPTG-inducible pMG600 to overexpress flagella. Extraction and purification of flagellar filaments was performed using a combined ultracentrifugation and Fast Performance Liquid Chromatography (FPLC) approach. Endotoxin was removed using commercial columns to purify the flagellin to homogeneity. Monomerisation assays were performed using temperature gradients between 50oC and 90oC and proteins were visualized in native gels. Flagellin concentration and purity were determined using BCA, coomassie-stained SDS-polyacrylamide gels, western blotting and mass spectrophotometry. Immune modulation by flagellin was assessed using a co-culture model of 5637 uroepithelial cells and U937 monocytes to measure cytokine responses, including IL10 following challenge with either WT, a-flagellate, or hyper-flagellate UPEC CFT073, or purified flagellin protein.
Flagellin protein induced IL10 in addition to several other cytokines in the co-culture model. Knowledge of how Flagellin acts as an immune modulator during UTI, based on the analysis of highly purified protein, could offer new avenues for infection prevention or control.