Conformational ensembles in Klebsiella pneumoniae FimH impact uropathogenesis

Abstract

Klebsiella pneumoniae is an important pathogen causing difficult-to-treat urinary tract infections (UTIs). Over 1.5 million women per year suffer from recurrent UTI, reducing quality of life and causing substantial morbidity and mortality, especially in the hospital setting. Uropathogenic E. coli (UPEC) is the most prevalent cause of UTI. Like UPEC, K. pneumoniae relies on type 1 pili, tipped with the mannose-binding adhesin FimH, to cause cystitis. However, K. pneumoniae FimH is a poor binder of mannose, despite a mannose-binding pocket identical to UPEC FimH. FimH is composed of two domains that are in an equilibrium between tense (low-affinity) and relaxed (high-affinity) conformations. Substantial interdomain interactions in the tense conformation yield a low-affinity, deformed mannose-binding pocket, while domain-domain interactions are broken in the relaxed state, resulting in a high-affinity binding pocket. Using crystallography, we identified the structural basis by which domain-domain interactions direct the conformational equilibrium of K. pneumoniae FimH, which is strongly shifted toward the low-affinity tense state. Removal of the pilin domain restores mannose binding to the lectin domain, thus showing that poor mannose binding by K. pneumoniae FimH is not an inherent feature of the mannose-binding pocket. Phylogenetic analyses of K. pneumoniae genomes found that FimH sequences are highly conserved. However, we surveyed a collection of K. pneumoniae isolates from patients with long-term indwelling catheters and identified isolates that possessed relaxed higher-binding FimH variants, which increased K. pneumoniae fitness in bladder infection models, suggesting that long-term residence within the urinary tract may select for higher-binding FimH variants.

Keywords: UTI; bacterial pathogenesis; mechanism of bacterial colonization.

Fig. 1.

K. pneumoniae FimH is highly invariant and conserved among diverse strains. (A) Phylogeny of K. pneumoniae FimH amino acid sequences (n = 977 strains). Source of strain isolation is annotated by color on outside ring. Minor alleles and strain diversity are shown in SI Appendix, Fig. S1. (B) Amino acid alignment of K. pneumoniae TOP52 FimH and UPEC UTI89 FimH. Blue highlighted residues are identical between sequences, light blue highlighted residues differ but share biochemical properties, and white background denotes different residue with different charge or hydrophobicity. Residues involved in binding to mannose are in orange.

Fig. 2.

K. pneumoniae FimH_LD displays high-affinity binding to mannose with altered binding loop 3 dynamics. (A) Crystal structure of K. pneumoniae TOP52 FimH_LD (cyan) bound to D-mannose (green). (B) Alignment of UPEC FimH_LD bound to D-mannose (gray; PDB 1KLF FimH_LD) to the TOP52 FimH_LD structure (RMSD 0.420). (C) Comparison of binding pocket between UPEC and K. pneumoniae FimH_LD. Binding loops are denoted L1, L2, and L3. (D) Observed BLI K_d of FimH_LD to BSA-mannose. (E) DSF binding of UPEC (black) and K. pneumoniae FimH_LD (blue) to an array of chemically diverse mannosides (n = 3). (F) FAST simulation population distribution of pocket distance from base of L1 to L3 for UPEC FimH_LD (gray) and K. pneumoniae FimH_LD (red). (G) Residue differences at 132 and 141 at the base of binding loop 3 on K. pneumoniae FimH_LD from the residues at these positions in UPEC FimH_LD (PDB 1KLF FimH_LD). (H) DSF binding for TOP52 FimH_LD binding loop three mutants (n = 3). Error bars represent SEM. ANOVA, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 3.

K. pneumoniae TOP52 FimH is allosterically shifted toward a tense low-affinity conformation and variants can shift FimH toward a higher-affinity conformation. (A) HA titers of clinical CAUTI K. pneumoniae isolates with FimH variants (no listed variant means the strain has the same sequence as TOP52; n = 2 to 3). (B) HA titer FimH variants expressed from plasmids in UTI89 LON ΔfimH (n = 3). (C) Positions of S62A and T74P on the structure of UPEC FimH in tense conformation (PDB 5JQI). (D and E). ELISA binding curves of FimC_hisH variants (D) and FimCG_dbH variants (E). Area under the curve measurements are displayed next to each respective graph. (F) HA titers of purified UPEC type 1 pili tipped with FimH variants. All HA titers were completely inhibited by the addition of 100 mM methyl α-D-mannopyranoside. Error bars represent SEM.

Fig. 4.

Relax-shifted FimH variants increase K. pneumoniae type 1 pilus binding. (A) HA titers of chromosomal TOP52 FimH variants. LON indicates FimS promotor locked in on orientation to overexpress type 1 pili. HA titers were completely inhibited by the addition of 100 mM methyl α-D-mannopyranoside (n = 3). (B) FimS on percentage (n = 3). (C). Western blot of FimA and GAPDH loading control of TOP52 FimH variants. (D) 5637-cell attachment and invasion (E) of TOP52 LON FimH variants (n = 6). ANOVA, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 5.

Relaxed-shifted K. pneumoniae FimH variants increase bladder and catheter titers relative to TOP52 FimH. (A and B) C3H/HeN mice infected with 10⁹ CFUs of TOP52 expressing FimH variants and bladder titers enumerated 24 hpi (n = 15 mice total from three independent replicates). (C) Log competitive index 2 dpi from a competitive bladder infection in C3H/HeN mice. Error bars represent geometric SD (n = 10 total from two independent replicates). (D) Bladder titers and percentage of C3H/HeN mice with chronic bladder infection 28 dpi. Orange dots indicate mice which had high bladder titers at 28 dpi but had at least one urine titer below 10⁴ CFUs during the experiment and therefore did not count as a chronically infected mouse (n = 20 over two independent replicates). (E and F) C57BL/6 mice were catheterized and infected with 10⁷ CFUs of TOP52 variants. Catheter titers (E) and bladder titers (F) were enumerated 24 hpi (n = 13 to 14 mice from two independent replicates). Solid lines on all graphs show geometric mean. Dotted lines indicate limit of detection. Mann–Whitney U test (except for C, one-way ANOVA), * P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.