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Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist

Abstract

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually1,2. Despite effective antibiotic therapy, 30–50% of patients experience recurrent UTIs1. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections3,4,5. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs6. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment7. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut5. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.

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Figure 1: Type 1 and F17-like pili promote UPEC intestinal colonization.
Figure 2: Structural analysis of UclDLD.
Figure 3: Mannoside simultaneously reduces the UPEC intestinal reservoir and treats UTI.
Figure 4: Mannoside treatment minimally effects the faecal microbiota configuration and targets human UPEC isolates in mice with different genetic backgrounds.

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Acknowledgements

We thank A. Earl and A. Manson for assistance in acquiring genomic data, J. Nix and Molecular Biology Consortium (Beamline 4.2.2) for collection/processing crystallographic data, L. Vereecke and A. Goncalves at VIB Bio Imaging Core for support with tissue sectioning/microscopy, Z. Han for synthesizing M4284, K. Tamadonfar for assistance with tissue binding studies, and M. Lukaszczyk for help with protein production. This work was supported by grants from the NIH (K08AI113184 (A.L.K.), RO1DK051406 (S.J.H) R01AI048689 (S.J.H.), P50DK064540 (S.J.H.), RC1DK086378 (S.J.H.), DK30292 (J.I.G.) and 1F31DK107057 (C.N.S.)), FWO-Flanders (G030411N), Hercules Foundation (UABR/09/005) and VIB PRJ9.

Author information

Authors and Affiliations

Authors

Contributions

S.J.H., J.I.G. and C.N.S. developed the mouse model and identified relevant CUP pili. S.J.H., J.I.G., C.N.S., J.W.J. and Z.T.C. designed and C.N.S. performed M4284 experiments. J.W.J. provided M4284. S.J.H., J.I.G., A.L.K. and C.N.S. executed microbiota experiments. H.R., S.R., R.D.K. and C.N.S. designed and R.D.K. and S.R. performed in vitro binding assays. H.R., J.S.P. and K.W.D. expressed and purified protein. S.R., H.R., D.H.F., R.D.K. and K.W.D. performed crystallization trials and solved UclD structures. H.R., S.J.H., H.L.S. and C.N.S. designed and H.L.S. performed phylogenetic and genomic analyses. S.J.H., J.I.G., H.R. and C.N.S. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Scott J. Hultgren.

Ethics declarations

Competing interests

J.W.J. and S.J.H. are inventors on US patent US8937167 B2, which covers the use of mannoside-based FimH ligand antagonists for the treatment of disease. J.W.J. and S.J.H. have ownership interest in Fimbrion Therapeutics, and may benefit if the company is successful in marketing mannosides.

Additional information

Reviewer Information Nature thanks E. Pamer, M. Schembri and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Streptomycin treatment allows for persistent UTI89 colonization of the caecum and colon in female C3H/HeN and C57BL/6 mice.

a, Mice were pretreated with streptomycin and subsequently colonized via oral gavage (PO) with UTI89, a prototypical human UPEC cystitis isolate. be, Colonization of UTI89 in C3H/HeN (b, c) or C57BL/6 (d, e) mice from Envigo was assessed by quantifying CFU in faecal samples collected over the course of 21 days from mice who did not receive streptomycin (white circles) or mice pretreated with the antibiotic (black circles). CFU analysis of levels of colonization in the caecum and colon were defined by analysing tissue homogenates prepared 21 days after colonization. Symbols represent geometric mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann–Whitney U test. n = 15 mice, 3 biological replicates (be).

Source data

Extended Data Figure 2 The FimH adhesin is required for type 1 pilus-dependent colonization of the mouse gut and for binding to human intestinal epithelial cells.

a, C3H/HeN mice from Envigo were pretreated with streptomycin and concurrently colonized with 1 × 108 CFU of wild-type UTI89 and UTI89ΔfimH. The wild-type strain outcompetes the strain lacking the FimH adhesin. b, The ability of FimHLD to bind to Caco-2 cells was assessed by a FimH ELISA. Pre-incubation of FimHLD with d-mannose (1 mM) or M4284 (1 mM) results in significant reductions in FimH binding to Caco-2 cells while 10% cyclodextrin (M4284 vehicle) had no significant effect. All data shown are normalized to wells that were not exposed to the purified adhesin. Data in a are mean ± s.e.m, and bars in b represent the median. *P < 0.05, **P < 0.01, ***P < 0.001 by Wilcoxon signed-rank test (a). n = 14 mice, 3 biological replicates (a); n = 4 wells examining FimH binding to Caco-2 cells per protein concentration, 4 technical replicates (b).

Source data

Extended Data Figure 3 F17-like pili are not required for UTI in mice.

C3H/HeN mice received a transurethral inoculation of wild-type UTI89 and UTI89Δucl, concurrently (a, b), or individually (ce). a, UTI89Δucl and wild-type strains persist at similar levels in the urine over 28 days in competitive infections. b, The two strains are also present at equal levels in the bladder and kidney at the time of euthanization (28 days after infection). c, Single infection with the wild-type strain (black circles) or the F17-like mutant strain (white circles) produces similar levels of bacteruria over 28 days. d, Single strain infection also produces similar levels of viable cells in homogenates of whole bladder or kidneys collected at the time of euthanization (28 days after infection). There was no statistically significant difference in the number of mice that resolved bacteriuria while maintaining bladder-associated CFUs after transurethral infection with either wild-type UTI89 or UTI89Δucl (highlighted in red in d), suggesting that both strains are capable of forming similar numbers of quiescent intracellular reservoirs. e, Mice infected transurethrally with wild-type or Δucl strains of UTI89 exhibit a similar number of IBCs at 6 h in the bladder, indicating that loss of the ucl operon does not alter the ability of UTI89 to form IBCs. Error bars represent mean ± s.e.m. (a, b), geometric mean (c, d) or median (e). No significant difference was detected between any samples by Wilcoxon signed-rank test (a, b) or Mann–Whitney U test (ce). n = 10 mice, 2 biological replicates (a, b, e); n = 16 mice, 3 biological replicates (c, d).

Source data

Extended Data Figure 4 Distribution of F17 usher homologues in members of Enterobacteriaceae.

The phylogenetic relationships between F17 homologues were estimated using the sequence of the usher genes. Branch colours indicate host strain and pilus identity, and coloured symbols indicate the annotated pathotype of the E. coli strain for each sequence as determined by publically available annotations. Stars indicate extraintestinal pathogenic E. coli (ExPEC) strains, and circles indicate intestinal pathogenic E. coli strains. Carriage of F17-like pili is enriched in UPEC strains, whereas F17 and ECs1278 pili are more common in intestinal pathogens such as ETEC and EHEC, respectively. The strain names for each sequence and ENA accessions are given. Numbers beneath the branches indicate the percentage of support from 1,000 bootstrap replicates (numbers greater than 80% are shown).

Extended Data Figure 5 Phylogenetic distribution of F17-like carriage in UPEC from patients with recurrent UTI.

The phylogeny of a set of clinical UPEC strains (n = 43 with taxon labels highlighted in green, orange or grey) was contextualized with reference E. coli strains (n = 46, unhighlighted taxon labels) by comparing the concatenated single-copy, core genes of the strains using the RAxML algorithm and the GTRCAT model46. Highlighted taxon labels indicate UPEC isolates collected at enrolment (green) and during recurrent UTI (orange). In all cases, patients cleared each infection before recurrence, no patient exhibited signs of asymptomatic bacteriuria. The study design also allowed for the collection, from cohort participants, of E. coli isolates present in the urine in the days leading up to their clinical visit and recurrent UTI diagnosis (highlighted in grey)17. Branch lines indicate phylogenetic background for strains from clade B2 (red branch lines) and non-B2 clades (blue branch lines). Carriage of F17-like pili (black stars) was limited to the B2 clade and enriched within recurrent UTI UPEC isolates. Bootstrap supports are indicated at internal nodes. Bootstrap values greater than 95 have been removed. The clade to which each strain belongs is indicated in brackets to the right.

Extended Data Figure 6 Testing the effects of more prolonged dosing of M4284 and analysis of the duration of its effects.

a, Experimental design. b, Animals treated as in a show a continued decrease in UTI89 levels in their faeces (samples were processed after 3, 4 and 5 doses of M4284), and at the time of euthanization in the caecum and colon, compared to control mice treated with vehicle alone (control, 10% cyclodextrin). c, d, The effects of mannoside treatment persist 5 days after M4284 exposure. Data are geometric mean ± s.d. *P < 0.05, **P < 0.01 by Mann–Whitney U test. n = 9 mice (control); n = 10 mice (M4284), 2 biological replicates (b); n = 16 mice, 3 biological replicates (d).

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Extended Data Figure 7 The severity of UTI outcome is directly linked to the dose of UTI89 inoculated into the urinary tract.

C3H/HeN mice (Envigo) were given an experimental UTI via transurethral inoculation of either 106 or 108 CFU of UTI89. The doses were chosen to represent the reduction observed in intestinal UTI89 titres before and after treatment with the M4284 mannoside. Mice were euthanized 24 h after inoculation, and UTI89 titres in urine, bladder and kidneys were defined by quantifying CFU. Mice receiving the 106 dose of UTI89 had significantly fewer bacteria in all three biospecimen types, indicating an important relationship between the number of bacteria introduced into the urinary tract and the severity of UTI outcome. Bars represent geometric means. **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann–Whitney U test. n = 10 mice, 2 biological replicates.

Source data

Extended Data Figure 8 16S rRNA-based comparison of faecal bacterial communities in mice obtained from Envigo and CRL and mice of different genetic backgrounds from a common vendor.

a, C3H/HeN mice were treated with M4284 (100 mg kg−1, three doses over 24 h), vehicle alone (10% cyclodextrin, three doses over 24 h), or ciprofloxacin (15 mg kg−1, two doses over 24 h). Untreated mice served as reference controls. Heat maps show the effect of each of the treatments on animals from CRL and Envigo. Each row represents a species-level bacterial taxon, while each column represents a mouse sampled 24 h after the termination of the indicated treatment. Coloured boxes next to the taxon names indicate species whose relative abundance was significantly changed by ciprofloxacin treatment (P < 0.05; Wilcoxon signed-rank test with false discovery rate (FDR) correction). Individual comparisons between untreated and other treatment types did not disclose changes that were statistically significant by Wilcoxon signed-rank test with FDR correction. b, Corresponding faecal samples collected 24 h after treatments (as shown in Extended Data Fig. 8a) were homogenized, diluted serially, and plated on MacConkey medium. The abundance of bacteria capable of growing on the selective medium was similar between faecal samples taken from untreated mice and those collected 24 h after treatment with cyclodextrin and M4284. No colonies were detected from faecal samples collected 24 h after ciprofloxacin treatment. c, Comparison of the representation of bacterial taxa in the faecal microbiota of untreated mice obtained from different vendors or representing different genetic backgrounds. Each row in the heat map represents a species-level taxon, while each column represents a mouse of the indicated genetic background from the indicated vendor. Coloured boxes indicate species whose relative abundances were significantly different (P < 0.05) between all three groups of animals (Kruskal–Wallis test with FDR correction). Rows of each heat map were hierarchically clustered according to pair-wise distances using Pearson correlation. n = 5 mice per treatment type, 1 biological replicate (a); n = 5 mice, 1 biological replicate (b); n = 5 mice per vendor/mouse strain, 1 biological replicate (c). Bars denote median. **P < 0.001, Mann–Whitney U test (b).

Source data

Extended Data Figure 9 The configuration of the faecal microbiota of C3H/HeN mice pretreated with streptomycin and colonized with UTI89 is minimally altered by M4284 treatment.

a, C3H/HeN mice from Envigo were pretreated with streptomycin and 24 h later colonized with UTI89 by oral gavage. Three days after inoculation, animals were treated with three doses of M4284 (100 mg kg−1, three doses over 24 h) or vehicle alone (10% cyclodextrin, 3 doses over 24 h). Faecal samples were collected 24 h after the last dose of M4284 or vehicle. b, Heat map showing the effect of each treatment type. Each row represents a bacterial species-level taxon, while each column represents a mouse 24 h after the indicated treatment. Rows of the heat map were hierarchically clustered according to pair-wise distances using Pearson correlation. No treatments produced changes that were statistically significant, as judged by Wilcoxon signed-rank test with FDR correction. n = 4 mice per treatment type, 1 biological replicate.

Extended Data Figure 10 The percentage reduction in strains by M4284 treatment is similar in mice colonized with genetically distinct human isolates, and in multiple strains of mice colonized with UTI89.

a, The percentage reduction in CFU for the indicated UPEC strains from M4284-treated versus untreated control C3H/HeN mice obtained from Envigo (based on data in Fig. 4c–f). b, CFU data obtained from C3H/HeN mice from Envigo and CRL and C57BL/6 mice from Envigo (based on data in Fig. 4f–h). P values calculated using Kruskal–Wallis test. n = 14 mice, 3 biological replicates (UTI89); n = 10 mice, 2 biological replicates (CFT073, EC958 and 41.4p) (a); n = 14 mice, 3 biological replicates (C3H/HeN from Envigo); n = 10 mice, 2 biological replicates (C3H/HeN from CRL); and n = 9 mice, 2 biological replicates (C57BL/6 from Envigo) (b).

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Supplementary Table 1

The phylogenetic relationships between F17 homologs as determined by comparing relatedness of bacterial usher gene sequences. This file contains an excel worksheet of all the sequence names and accession numbers used to generate the phylogeny presented in Extended Data Figure 4 (47 KB). (XLSX 46 kb)

Supplementary Table 2

The carriage of F17-like pili in rUTI UPEC strains. This file contains an excel worksheet of all the sequence names and corresponding BioProject IDs used to generate the phylogeny presented in Extended Data Figure 5. Accession numbers are currently being assigned to rUTI samples under BioProject ID: PRJNA269984 (50 KB). (XLSX 48 kb)

Supplementary Table 3

UclD X-ray crystallography data. This file contains a PDF consisting of data collection and refinement statistics for the UclD X-ray crystal structures (85 KB). (PDF 144 kb)

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Spaulding, C., Klein, R., Ruer, S. et al. Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist. Nature 546, 528–532 (2017). https://doi.org/10.1038/nature22972

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