Journal: Scientific Reports
Article Title: Dual ligand/receptor interactions activate urothelial defenses against uropathogenic E. coli
Figure Lengend Snippet: FimH-mediated adhesion of T1F-UPEC to urothelial surface was a prerequisite for NF-κB activation. ( A ) Effects of different E. coli strains on RelA translocation. Wild-type mice were transurethrally instilled with PBS or that containing the following bacteria: HB101 (a laboratory K12 strain not expressing the type 1 fimbriae); NU14-1 (a FimH deletion strain not expressing the type 1 fimbriae); NU14 (a clinical cystitis T1F-UPEC isolate which is the parental strain of NU14-1); UTI89; and CFT073 (a clinical pyelonephritis T1F-UPEC strain). Immunofluorescent staining was carried out on urinary bladders 1 h post-instillation and nuclei positively stained for RelA was counted and expressed as per 100 umbrella nuclei (see Materials and Methods for details). Note that UPEC strains expressing the type 1 fimbriae (NU14, UTI89 and CFT073), but not those non-type 1-fimbriated ones, induced significant nuclear translocation of RelA. ( B–D ) Effects of FimH-mediated adhesion of UPEC to urothelial cells on NF-κB activation. ( B ) Wild-type mice were transurethrally inoculated with the same number (10 8 cfu) of T1F-UPEC strain UTI89, or a derivative of UTI89 expressing a FimH mutant (e.g., Q133N) (*) that lacks the ability to bind the terminal mannoses; or a derivative of UTI89 with FimH deletion (∆FimH) (∆). ( C–D ) Wild-type mice were transurethrally inoculated with the same number (10 8 cfu) of T1F-UPEC strain UTI89 in the presence of 0, 0.1, 0.5 mg/ml of FimH or 0.5 mg/ml heat-inactivated (Heated) FimH ( C ), or in the presence of 0, 5 or 50 mM of D-mannose ( D ). One hour after inoculation, urinary bladders of all experimental groups ( B–D ) were processed for double-immunofluorescence staining using antibodies against E. coli and RelA. Representative images and semi-quantification of RelA-positive umbrella cell nuclei per 100 total umbrella nuclei were shown. Note that FimH mutation and deletion or addition of free FimH or D-mannose abolished UPEC-urothelial adhesion as well as NF-κB activation. Heat-inactivated FimH, however, failed to block NF-κB activation by UPEC ( C ). Scale bars: 100 μm (B–D).
Article Snippet: Bacterial Strains and Culture The following E. coli strains were used: (1) UTI89, a type 1-fimbriated uropathogenic E. coli (T1F-UPEC) isolate from an acute cystitis patient ; (2) UTI89/Q133N, a recombinant strain engineered from the parental UTI89 strain with a point mutation converting codon 133 of FimH adhesin from Q to N, thus abolishing FimH’s ability to bind terminal mannoses; (3) UTI89/∆FimH, a recombinant strain engineered from the parental UTI89 strain with FimH adhesin deleted; (4) CFT073 (from ATCC), a T1F-UPEC strain isolated from the blood and urine of a patient with acute pyelonephritis ; (5) NU14, a T1F-UPEC strain isolated from an acute cystitis patient ; (6) NU14-1, a recombinant strain derived from NU14 with FimH adhesin deleted ; (7) HB101, a non-fimbriated, K12 laboratory E. coli strain (ATCC) ; and (8) UTI89/FimB, a derivative strain from UTI89 after transfection with a plasmid encoding the FimB gene (pACYC177/FimB), which “locks on” the type 1 fimbrial expression .
Techniques: Activation Assay, Translocation Assay, Mouse Assay, Expressing, Staining, Mutagenesis, Double Immunofluorescence Staining, Blocking Assay