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 annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. 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 infections. 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 UTIs. 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 environment. 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 gut. 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.

Extended Data Figure 1. Streptomycin treatment allows for persistent UTI89 colonization of the cecum 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. (b–e) Colonization of UTI89 in C3H/HeN mice from Envigo (b,c) or C57BL/6 mice from Envigo (d,e) was assessed by quantifying colony forming units (CFU) in fecal 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 cecum and colon were defined by analyzing tissue homogenates prepared 21 days post colonization. Symbols represent geometric means ± SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Mann Whitney U test). n=15 mice, 3 biological replicates (b–e).

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 WT UTI89 and UTI89ΔfimH. The WT strain outcompetes the strain lacking the FimH adhesin. (b) The ability of purified FimH lectin domain (FimH^LD) to bind to Caco-2 cells was assessed by a FimH-ELISA. Pre-incubation of FimH^LD with D-mannose (1mM) or M4284 (1mM) 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. Abbreviations CI= competitive index. Ce= cecum, Col= colon. Bars represent mean values ± SEM, *p<0.05, **p<0.01, ***p<0.001, Wilcoxon Signed Ranked test in (a). Bars represent median (b). n=14 mice, 3 biological replicates (a); n=4 wells examing FimH binding to Caco-2 cells per protein concentration, 4 technical replicates (b).

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

C3H/HeN mice received a transurethral inoculation of UTI89 (WT) and UTI89Δucl, concurrently (a, b), or individually (c–e). (a) UTI89Δucl and WT strains persist at similar levels in the urine over 28 d in competitive infections. (b) The two strains are also present at equal levels in the bladder and kidney at the time of sacrifice (28 days post infection). (c) Single infection with the WT 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 harvested at the time of sacrifice (28 days post 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 WT or UTI89Δucl (highlighted in red in d), suggesting that both strains are capable of forming similar numbers of QIRs. (e) Mice infected transurethrally with WT or Δucl strains of UTI89 exhibit a similar number of intracellular bacterial communities (IBCs) at 6 hours in the bladder, indicating that loss of the ucl operon does not alter UTI89’s ability to form IBCs. CI= competitive index. Bars represent mean ± SEM (a,b), geometric mean (c,d) or median (e). No significant difference was detected between any samples by Wilcoxon Signed Ranked test (a,b) or Mann Whitney U test (c–e). n=10 mice, 2 biological replicates (a–b, e). n=16 mice, 3 biological replicates (c, d).

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

The phylogenetic relationships between F17 homologs was estimated using the sequence of the usher genes. Branch colors indicate host strain and pilus identity, while colored 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 while circles indicate intestinal pathogenic E. coli strains. Carriage of F17-like pili is enriched in UPEC strains while F17 and ECs1278 pili are more common in intestinal pathogens such as EHEC. The strain names for each sequence and ENA accession IDs are given. Numbers beneath the branches indicate the percentage of support from 1000 bootstrap replicates (numbers greater than 80% are shown).

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

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 model. Highlighted taxon labels indicate UPEC isolates collected at enrollment (green) and during recurrent UTI (orange). In all cases, patients cleared each infection prior to 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 rUTI diagnosis (highlighted in grey). 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 rUTI UPEC isolates. Bootstrap supports are indicated at internal nodes. Bootstrap values >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 panel (a) show a continued decrease in UTI89 levels in their feces (samples were processed after 3, 4, and 5 doses of M4284), and at the time of sacrifice in the cecum 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. Bars represent geometric mean ± SD, *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).

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 introduction of either 10⁶ or 10⁸ CFU of UTI89. The doses were chosen to represent the reduction observed in intestinal UTI89 titers before and after treatment with the M4284 mannoside. Mice were sacrificed 24 hours after inoculation and UTI89 titers 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.

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

(a) C3H/HeN mice were treated with M4284 (100mg/kg, 3 doses over 24 h), vehicle alone (10% cyclodextrin (Cyclo), 3 doses over 24 h), or ciprofloxacin (Cipro; 15mg/kg, 2 doses over 24 h). Untreated mice served as reference controls. Heatmaps 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 hours after the termination of the indicated treatment. Colored boxes next to the taxon names indicate species whose relative abundance was significantly changed by Cipro treatment (p<0.05;Wilcoxon Signed Rank test with 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 fecal samples collected 24 h after treatments (as shown in Extended Data Figure 8a) were homogenized, diluted serially, and plated on MacConkey medium. The abundance of bacteria capable of growing on the selective medium was similar between fecal samples taken from untreated mice and those collected 24 hours after treatment with cyclodextrin and M4284. No colonies were detected from fecal samples collected 24 hours after ciprofloxacin treatment. (c) Comparison of the representation of bacterial taxa in the fecal microbiota of untreated mice obtained from different vendors or representing different genetic backgrounds. Each row in the heatmap represents a species-level taxon, while each column represents a mouse of the indicated genetic background from the indicated vendor. Colored 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 heatmap 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). bar = median; **p<0.001, Mann Whitney U test (b).

Extended Data Figure 9. The configuration of the fecal 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 3 doses of M4284 (100mg/kg, 3 doses over 24 h) or vehicle alone (10% cyclodextrin (Cyclo), 3 doses over 24 h). Fecal samples were collected 24 h after the last dose of M4284 or vehicle. (b) Heatmap 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 heatmap 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 percent 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 percent reduction in CFU for the indicated UPEC strains from M4284-treated versus untreated control C3H/HeN mice obtained from Envigo (based on data presented in Fig. 4c–f). (b) CFU data obtained from CRL C3H/HeN mice and Envigo C57BL/6 mice (based on data shown 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, 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).

Figure 1. Type 1 and F17-like pili promote UPEC intestinal colonization

Streptomycin pretreated C3H/HeN mice were concurrently (a–j) or singly (k) colonized with WT UTI89 and/or UTI89 lacking one or more CUP operons. (l,m) Purified adhesin lectin domains FimH^LD (type 1 pili) and UclD^LD (F17-like pili) were tested for binding to mouse colonic sections. Sections were stained with hoechst (blue) and antibodies to Muc2, a mucus-associated glycoprotein (green). FimH^LD and UclD^LD binding was lost by pre-treating tissue sections with PNGase and O-glycosidase, respectively. Arrowheads highlight binding by FimH^LD or UclD^LD. (n) UclD^LD does not bind five common monosaccharides. Ce=cecum, Col=colon, CI=competitive index. Bars represent mean values ± SEM (a–j, n), geometric means ± SD (k). *p<0.05, **p<0.01, ***p<0.001 by Wilcoxon Signed Ranked (a–j) or Mann Whitney U test (k,n). n=5 mice, 1 replicate (a, d–g). n=10 mice, 2 replicates (b,h). n=6 mice, 1 replicate (c). n=14 mice, 3 replicates (i). n=8, 2 replicates (j). n=12 mice, 3 replicates (UTI89); n=9 mice, 2 replicates (UTI89Δfim); n=15 mice, 3 replicates (UTI89Δucl); n=10 mice, 2 replicates (UTI89ΔfimΔucl) (k). Replicates are biological (a–k). n=3 tissue sections, 4 represenative images per section (l,m). n=3 wells, 3 technical replicates (n).

Figure 2. Structural analysis of UclD^LD

(a) Superposition of the P2₁ UclD^LD (green) and P2₁2₁2₁ UclD^LD (grey) crystal structures (a-1). F17G adhesin crystal structure (PDB 1OIO) (a-2). Superposition of P2₁ UclD^LD (green) and F17G^LD (cyan) structures (a-3). (b) Comparison of residue positioning and electrostatic surface potential of the putative binding site between the UclD^LD structures and the known binding site of F17G^LD. (c) Structural alignment of UclD^LD and F17G^LD amino acid sequences. Purple residues highlight the putative UclD^LD binding site. Orange and yellow residues highlight insertions in UclD^LD sequence. Starred residues are hypothesized to mediate UclD ligand binding. Beta strands (red arrows), 3/10 helices (coils), and alpa-helices (cylinder) are shown.

Figure 3. Mannoside simultaneously reduces the UPEC intestinal reservoir and treats UTI

(a) M4284 concentration in mouse feces after 1 dose (100mg/kg administered by oral gavage). (b) C3H/HeN mice were intestinally colonized with UTI89 and given 3 oral doses of M4284 (100mg/kg), vehicle alone (10% cyclodextrin, Control), or D-mannose (100mg/kg). (c,d) UTI89 levels in the feces and intestinal segments. (e) UTI89 was introduced into the gut of C3H/HeN mice by oral gavage and into the bladder by transurethral inoculation before receiving 3 doses of M4284. (f,g) UTI89 levels in the gut and urinary tract were assessed. Bars represent median (a), geometric means ± SD (c,d,f); geometric mean (g) *p<0.05, **p<0.01 by Mann Whitney U test. n= 3 mice, 1 replicate (a). n=14 mice (Control); n=15 mice (M4284); 3 replicates (c). n= 10 mice, 2 replicates (d). n=9 mice, 2 replicates (f, g). All replicates biological (a–g).

Figure 4. Mannoside treatment minimally effects the fecal microbiota configuration and targets human UPEC isolates in mice with different genetic backgrounds

Mice were given one of the following oral treatments: (i) M4284 (100mg/kg), (ii) cyclodextrin (Cyclo, 10%), (iii) ciprofloxacin (Cipro, 15mg/kg), or (iv) none (Untreated). Fecal community structure was defined by sequencing bacterial 16S rRNA gene amplicons. (a) For each treatment performed on C3H/HeN mice from Envigo or CRL, the change in microbiota configuration was determined by measuring the unweighted UniFrac distance between samples obtained from each animal before treatment and 24 h after the last dose (larger UniFrac distance equates to a larger shift in community structure). (b–f) Mice were colonized by oral gavage (PO) of one of four different UPEC strains and given three doses of M4284. (g,h) The ability of M4284 to target UTI89 in C3H/HeN mice from CRL (g) and C57BL/6 mice from Envigo Labs (h) was also assessed. Bars represent median (a) or geometric means ± SD (c–h). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 Mann Whitney U test. n=5 mice per vendor, 1 replicate (a). n=10 mice, 2 replicates (c–e, g). n=14 (Control, 10% cyclodextrin); n=15 (M4284); 3 replicates (f). n=10 mice (Control, 10% cyclodextrin); n=9 mice (M4284); 2 replicates (h). All replicates are biological (a–h).