Positively selected FimH residues enhance virulence during urinary tract infection by altering FimH conformation

Abstract

Chaperone-usher pathway pili are a widespread family of extracellular, Gram-negative bacterial fibers with important roles in bacterial pathogenesis. Type 1 pili are important virulence factors in uropathogenic Escherichia coli (UPEC), which cause the majority of urinary tract infections (UTI). FimH, the type 1 adhesin, binds mannosylated glycoproteins on the surface of human and murine bladder cells, facilitating bacterial colonization, invasion, and formation of biofilm-like intracellular bacterial communities. The mannose-binding pocket of FimH is invariant among UPEC. We discovered that pathoadaptive alleles of FimH with variant residues outside the binding pocket affect FimH-mediated acute and chronic pathogenesis of two commonly studied UPEC strains, UTI89 and CFT073. In vitro binding studies revealed that, whereas all pathoadaptive variants tested displayed the same high affinity for mannose when bound by the chaperone FimC, affinities varied when FimH was incorporated into pilus tip-like, FimCGH complexes. Structural studies have shown that FimH adopts an elongated conformation when complexed with FimC, but, when incorporated into the pilus tip, FimH can adopt a compact conformation. We hypothesize that the propensity of FimH to adopt the elongated conformation in the tip corresponds to its mannose binding affinity. Interestingly, FimH variants, which maintain a high-affinity conformation in the FimCGH tip-like structure, were attenuated during chronic bladder infection, implying that FimH's ability to switch between conformations is important in pathogenesis. Our studies argue that positively selected residues modulate fitness during UTI by affecting FimH conformation and function, providing an example of evolutionary tuning of structural dynamics impacting in vivo survival.

Keywords: chronic cystitis; microbial pathogenesis; protein conformations; protein dynamics.

Fig. 1.

FimH positively selected residues. FimH is a two-domain adhesin comprised of a lectin domain of residues 1–150 (green), a pilin domain with residues 159–279 (blue), and a linker loop (yellow) connecting them. Positively selected residues are mapped onto the structures of FimH as red spheres. (A) In the elongated FimH (V27/S62/V163) structure, mannose is observed at the distal binding pocket in white sticks (J96 FimH; PDB ID code: 1KLF; FimC removed for clarity). (B) In the compressed FimH (A27/S62/A163) structure in the absence of mannose, position 133 of the binding pocket is colored white (F18 FimH; PDB ID code: 3JWN). Note the distance of these positively selected residues from the mannose binding pocket.

Fig. 2.

CFT073 and UTI89 acutely infect mouse urinary tracts. (A) IBCs per bladder were enumerated with LacZ stain. (B) Total bladder bacterial counts were determined after homogenization. (C) Total bacteria present in both kidneys were enumerated. n = 2 experiments with five mice per group. C includes kidney titers of mice from A and B.

Fig. 3.

FimH allele modulates acute and chronic pathogenesis. Mice were infected with 10⁷ cfu (range 3.4 × 10⁶ to 1.8 × 10⁷; median 1.02 × 10⁷) of the indicated strains. (A) IBCs were enumerated after LacZ staining. (B) Urine was collected at days 1, 7, 14, and 21, and the number of bacteria present in bladders was determined at 4 wpi. Data points above 10⁴ cfu reflect mice that had persistent bacteriuria and are considered to have chronic cystitis. Red symbols denote mice that resolved bacteriuria and either had a recurrence or high levels of reservoir titers, and were thus included in the resolved category because they did not experience chronic cystitis. The percentage of mice experiencing chronic cystitis is displayed at the top of each column. (C) Kidney titers of the mice from B. (A) n = 2–11 experiments with two to eight mice per group. (B and C) n = 2–8 experiments with 5–10 mice each.

Fig. 4.

FimH::A62/V163 displaces FimH::S62/A163 during chronic coinfection. Coinfections were conducted (A) between UTI89 with FimH::A62/V163 and FimH::S62/A163, (B) between CFT073 with FimH::A62/V163 and FimH::S62/A163, and (C) between UTI89 FimH::A62/V163 and CFT073 FimH::A62/V163. Urine was collected at days 1, 3, 7, 14, and 21 d postinfection (dpi), and bladder and kidney titers were determined at 28 dpi. Log₁₀ of competitive indices of the mice experiencing chronic cystitis is plotted as determined via plating on selective antibiotics. n = 2–3 experiments with 5–10 total mice per group. Wilcoxon signed rank test was conducted to evaluate whether the median value was significantly different from 0; *p < 0.05, **P < 0.01, ***P < 0.005.

Fig. 5.

Effect of FimH on mannose binding and chronic fitness. (A) Representative curves showing FimCH association to, and dissociation from, immobilized BSA-mannose at indicated concentrations. Two biological replicates conducted. (B) Representative 14% SDS/PAGE gel indicating select variants in FimC_HisGH samples. “B” marks the boiled lanes. (C) Representative curves showing FimC_HisGH association to, and dissociation from, immobilized BSA-mannose at the indicated concentrations. Representative of three biological replicates with two technical replicates each with combined K_D data shown in Table 2. (D) Four-week bladder titer is shown for mice experiencing chronic cystitis with coinfections of the listed strains. n = 1–3 experiments with 5–10 mice each with only chronic cystitis mice displayed.

Fig. 6.

Structure–function–treatment model of UPEC pathogenesis. (A) Model of pilus biogenesis including delivery of chaperone–subunit complexes to the N terminal domain (NTD) (blue) of the usher (orange) with transfer to the C-terminal domain (CTD)s (yellow and fuschia). The next subunit reacts with the previous by DSE polymerizing the pilus rod. Insets represent recent crystal structures demonstrating the orientation of FimH as it binds to and exits the usher (47, 71) (PDB ID code: 3RFZ; and PDB ID code: 4J30). (B) UPEC pathogenic cascade showing type 1 piliated UPEC attaching to superficial cells of the bladder, invasion into these cells, and replication in the cytoplasm to form IBCs. Mice can then either resolve the infection with potential recurrences thereafter or develop persistent bacteriuria and chronic cystitis. (C) The dynamics of transmission from the gut to the bladder and vice versa is an intriguing concept that is just now beginning to be studied (6). (D) Mannoside treatment can detach bacteria from the bladder epithelium during chronic cystitis leading to clearance. (E) Pilicides block the ability of the pilus to polymerize.