Fig 1.
Fimbriated E. coli 83972 variants; construction and human inoculation.
A. The ABU strain E. coli 83972, does not express functional P or type 1 fimbriae, due to chromosomal PapG point mutations and a fimB-D deletion. In this study, the pap or fim gene clusters were reconstituted in E. coli 83972pap and E. coli 83972fim, respectively. To replace the defective papG gene with a functional copy, a papGX deletion mutant (E. coli 83972ΔpapGX) was generated using lambda red homologous recombination [59]. Briefly, the chloramphenicol acetyltransferase gene (cat) cassette of plasmid pKD3 was amplified with overhangs homologous to the 5´- and 3´-regions of the E. coli 83972 papGX gene fragment and cured upon transformation with plasmid pCP20 [60]. Meanwhile, the functional papGX genes from UPEC strain CFT073 was amplified with homologous overhangs to the tnpA and papF regions of E. coli 83972 and used for electroporation into E. coli 83972ΔpapGX cells. Chromosomal reconstitution of the functional papGX genes in the E. coli 83972 chromosome was achieved via homologous recombination [59]. B. E. coli 83972 carries an internal 4,253-bp fim deletion, comprising the fimEAIC genes and truncated fimB and fimD genes. To reconstitute the fim operon, truncated genes were replaced via lambda red-mediated recombination by a cat cassette [59], flanked by two FRT sites and removed by FLP recombinase-mediated recombination [60]. The resulting 83972Δfim strain was transformed with pCP20 and suicide vector pJZ1, carrying the entire fim operon from pPKL4 [36]. Chromosomal integration of the entire suicide vector including the functional fim operon in E. coli 83972fim resulted in a functional fim copy. C. The human therapeutic inoculation protocol, indicating individual patients (P I–P V) and strains (E. coli 83972, E. coli 83972fim or E. coli 83972pap). Five patients were inoculated with E. coli 83972, which established ABU for a period of at least two weeks [28, 30]. After clearance of the strain by a short course of antibiotics, three patients were re-inoculated with E. coli 83972fim, followed after termination of bacterial carriage by a third inoculation with E. coli 83972pap (P II, P III and P IV). P I received E. coli 83972 followed by E. coli 83972fim but not E. coli 83972pap, and P V received E. coli 83972 followed by E. coli 83972pap but not E. coli 83972fim (S1 Table). D. Kinetics of papA and fimA expression after human inoculation. Bacterial RNA was isolated directly from urine of each patient at the indicated time points and papA or fimA expression was quantified by qRT-PCR. Changes in gene expression were defined relative to frr (ribosome-recycling factor) expression. Values for 0h correspond to the bacterial in vitro culture used for inoculation. E. Kinetics of the urine cytokine response to E. coli 83972fim (P I–P IV, left) P < 0.01 (**) or E. coli 83972pap (P II–P V, right) P < 0.01 (**). Data was normalized by subtraction of the pre-inoculation values in each patient (0h). Mean ± s.e.m. of 4 samples, 2-way ANOVA.
Fig 2.
Signaling pathways activated by E. coli 83972pap.
A. Transcriptomic analysis identifying top regulated canonical pathways in P V at the time of symptoms. B. Maximum activation of type I IFN signaling pathway genes in P V at the time of symptoms. C. Intra-individual comparison of IFN pathway genes expressed in response to E. coli 83972pap, E. coli 83972 or E. coli 83972fim (paired t-test for each patient and time point, two-tailed values). D. Pattern Recognition Receptor (PRR) signaling pathway genes. E. Intra-individual comparison of PRR pathway genes in response to E. coli 83972pap, E. coli 83972 or E. coli 83972fim (paired t-test for each patient and time point, two-tailed values). Red = FC ≥ 2.0 and blue = FC ≤ -2.0.
Fig 3.
IRF7 activation by E. coli 83972pap.
A. An IRF-7-centric gene network was activated in P V, at the time of symptoms (n = 103). B. A more moderate IRF-7 response was detected in P II, 5 days after transient symptoms (n = 45, 2 weeks). C, D. Patients II and IV, who did not experience symptoms, also did not show evidence of IRF-7 activation. Instead, IRF7-dependent genes were inhibited in P IV (n = 39, 2 weeks). E. Heatmap of disease associated, IRF-7-driven genes [8] 2 weeks after E. coli 83972pap inoculation (P II–P V). Genes in the network were activated in P V, at the time of symptoms and a partial response was seen in P II, 5 days after transient symptoms. Red = FC ≥ 2.0 and blue = FC ≤ -2.0.
Fig 4.
IRF-7 activation by the PapG adhesin.
A. Increase in IRF-7 protein levels after infection of human kidney cells with E. coli 83972pap but not E. coli 83972fim (105 cfu/ml, 4 hours). Western blot analysis. B. Increase in nuclear and total IRF-7 staining, quantified by confocal imaging. Mean + s.e.m. of two experiments, 50 cells/experiment. Two-tailed unpaired t-test compared to PBS. Scale bars = 20 μm. C. PapG internalization after infection or stimulation of cells with purified PapG protein (5–25 μg/ml), quantified by confocal imaging, using polyclonal anti-PapGII antibodies. Scale bar = 20 μm. D. The purified PapG adhesin or the PapDG protein complex stimulated an IRF-7 response, in treated cells (5–25 μg/ml). Scale bars = 50 μm. E. Increase in IRF7, IFNB1, MYC and IFIT3 mRNA levels quantified by qRT-PCR. Cells were infected with E. coli 83972pap or stimulated with the PapG adhesin or the PapDG protein complex (5 or 25 ug/ml). Mean + s.e.m. of two experiments, multiple unpaired t-test compared to PBS. F. IRF7 gene and promoter map. IRF7 promoter DNA (1563bp, -1308 to +255) was used as a probe, in an electrophoretic mobility shift assay (EMSA). G. Extracts from uninfected or E. coli 83972pap infected cells were mixed with the indicated IRF7 promoter fragment. By polyacrylamide gel electrophoresis, one band shift was detected in uninfected cells (band 1) and a second band in E. coli 83972pap infected cells (band 2). Specificity for PapG was supported by two super-shifted bands (bands 3 and 4), in the presence of anti-PapG antibody. Bands 3 and 4 were inhibited by using anti-IRF3 antibody. All bands were attenuated by combining anti-IFNβ with anti-IRF-3 or anti-MYC antibodies. H. Model of IRF7 activation by PapG, including IRF-3, IFNβ and MYC. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).
Fig 5.
Functional analysis of the early response to E. coli 83972fim inoculation.
A. Volcano plot of gene sets regulated by E. coli 83972fim after 3 hours compared to pre-inoculation samples in P I–P IV (Gene Ontology). Identified gene sets are plotted as the -log (p-value) against their Normalized Enrichment Score (NES) and functionally annotated (see S2 Table). Most inhibited gene sets were involved in RNA processing and translation (purple). Activated genes were mainly involved in ion channel (green)- and neuropeptide (red) regulation as well as immune signaling. B. Top five gene sets identified at the time of maximum response to E. coli 83972fim (3 hours). C. Top 15 activated gene sets at the time of maximum response to E. coli 83972fim (3 hours).
Fig 6.
E. coli 83972fim and FimH regulate ion channel expression.
A. Early activation of potassium channels in patients inoculated with E. coli 83972fim (n = 47). GSEA, 3-hour samples from P I–P IV. B. Kinetics of ion channel expression in patients inoculated with E. coli 83972fim, showing a rapid Ca2+ and K+ channels response at 3 hours and a sustained K+ channels response at 24- and 48 hours. C-E. Increased expression of K+ channels (TWIK, TRAAK and KCNJ11 but not KCNJ2, C) and cation channels (TRPC1 and TRPV6, D and E) in bladder epithelial cells infected with E. coli 83972 or E. coli 83972fim (105 cfu/ml, 4 hours). Confocal imaging (C,D) and Western blot analysis (E). Scale bars = 20 μm. F. The increase in TRPC1 and TRPV6 expression was effectively blocked by addition of the soluble FimH antagonist α-D-methyl-mannopyranoside (α-D-man., 2.5%). G,H. Purified FimCH protein (1.25–5 μg/ml, 4 hours) increased TRPC1 expression in a dose dependent manner. Confocal imaging (G) and Western blot analysis (H). Scale bars = 20 μm. I. Activation of K+, Ca2+ and Zn2+ fluxes by purified FimCH (5 μg/ml) as determined by fluorescence spectrometry with repeated 20 second-measurements for 16-20 minutes (mean of three experiments). The responses were effectively blocked by addition of α-D-methyl-mannopyranoside (α-D-man., 2.5%). Mean + s.e.m. of three experiments, 50 cells/experiment. Two-tailed unpaired t-test compared to PBS. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).
Fig 7.
Neuronal sensing and nervous system development regulated by E. coli 83972fim.
A. GSEA analysis identified 22 significantly regulated gene sets, involved in neuro-transmitter receptor expression and neuropeptide binding (nominal P-value < 0.01). These gene sets were identified compared to the pre-inoculation sample in each patient, as exemplified in P I after 3 hours. NES = normalized enrichment score. B. Significant NES data (P < 0.01) for gene-sets activated at different time points in patients inoculated with E. coli 83972fim (P I–P IV). Regulated categories included genes involved in neurotransmission, nervous system development and taste receptors. C. Increased levels of SP (red) and its receptor NK1R (green) in bladder epithelial cells infected with E. coli 83972fim or E. coli 83972 (105 cfu/ml, 4 hours). D. The purified FimCH protein complex (1.25–5 ug/mL) stimulated SP (red) and NK1R (green) responses, as shown by confocal imaging. Mean + s.e.m. of three experiments, 50 cells/experiment. Two-tailed unpaired t-test compared to PBS. Scale bars = 20 μm. P < 0.05 (*), P < 0.01 (**).
Fig 8.
Predicted upstream regulators of transcription.
Tentative upstream regulators of the transcriptional response to E. coli 83972pap and E. coli 83972fim were identified, using the IPA upstream regulator analysis. The -log (p-value) describes the prevalence of regulator-associated genes in data set and a positive z-score predicts activation while a negative score predicts inhibition. A. Prediction of IRF-7 as a transcriptional regulator of the response to E. coli 83972pap by activated downstream genes. B. Top predicted transcriptional regulators for the E. coli 83972pap dataset from P V at the time of symptoms red = activated, blue = inhibited). C,D. Predicted transcriptional regulators for E. coli 83972fim in the dataset from P I, 3 hours (C) or 24 hours (D) after inoculation. E. Prediction of MYC as a transcriptional regulator of the response to E. coli 83972fim by inhibited downstream genes.
Fig 9.
Bacterial adhesins, cell surface receptors and signaling pathways.
A. Innate immune recognition of P-fimbriated E. coli. The PapG adhesin binds Galα1-4Galβ-oligosaccharide motifs in glycosphingolipid receptors. Release of ceramide, the membrane anchor of the glycolipids, activates TLR4 signaling [37, 61, 62], and phosphorylation of the TLR4 adaptor proteins TRAM (TIR domain-containing adapter molecule 2 or TICAM2) and TRIF (TIR domain-containing adapter molecule 1 or TICAM1) activates downstream signaling, involving the phosphorylation of mitogen-activated protein (MAP) kinases, phospholipase C, p38, activating JNK (c-Jun N-terminal kinases), CREB (cyclic AMP response element-binding) and FOS-JUN (AP1), leading to IRF3- and IRF-7-dependent and AP-1 dependent transcription of cytokine- and chemokine genes, as well as type I interferons (IFN) including IFN-β [7]. Activation results in inflammatory cell recruitment and symptoms depend on the genetic make up of the host. In this study, PapG is defined as an agonist of IRF-7, in hosts and cells inoculated with E. coli 83972pap. B. Type 1 fimbriae recognize several mannosylated host cell glycoconjugate receptors. The FimH adhesin binds to uroplakins [63], to integrins through N-oligosaccharides [64], to the Tamm-Horsfall protein (or uromodulin) [65] and to immunoglobulins [66] as well as CD48 on mucosal mast cells [67]. Downstream, type 1 fimbriae have been proposed to stimulate the innate immune response, trigger apoptosis, and stimulate mast cell degranulation as well as promoting actin rearrangement in bladder epithelial cells [2, 68, 69]. In this study, FimH is defined as a broad, mostly inhibitory regulator of RNA processing and translation and inducer of ion channel- and solute carrier expression as well as neurokinin ligand-receptor networks. C. ABU strains like E. coli 83972 have developed successful adaptation strategies, which include the deletion or inactivation of virulence genes, leading to a reduction in genome size [33]. In addition, we have shown that ABU strains actively create a calm, non-reactive environment in the host by inhibiting gene expression. An intriguing mechanism is the suppression of by RNA polymerase II (Pol II) phosphorylation, which results in a protected phenotype [23].