Bacterial control of host gene expression through RNA polymerase II

The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coil develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II-dependent (Pol II-dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coil 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II-dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur

The Genetics of Urinary Tract Infections and the Innate Defense of the Kidney and Urinary tract

The urinary tract is a sterile organ system. Urinary tract infections (UTIs) are common and often serious infections. Research has focused on uropathogen, environment, and host factors leading to UTI pathogenesis. A growing body of evidence exists implicating genetic factors that can contribute to UTI risks. In this review, we highlight genetic variations in aspects of the innate immune system critical to the host response to uropathogens. This overview includes genetic variations in pattern recognition receptor molecules, chemokines/cytokines, and neutrophil activation. We also comprehensively cover murine knockout models of UTI, genetic variations involved in renal scarring as a result of ascending UTIs, and asymptomatic bacteriuria

Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis

Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain

IRF7 inhibition prevents destructive innate immunity-A target for nonantibiotic therapy of bacterial infections

Boosting innate immunity represents an important therapeutic alternative to antibiotics. However, the molecular selectivity of this approach is a major concern because innate immune responses often cause collateral tissue damage. We identify the transcription factor interferon regulatory factor 7 (IRF-7), a heterodimer partner of IRF-3, as a target for non-antibiotics-based therapy of bacterial infections. We found that the efficient and self-limiting innate immune response to bacterial infection relies on a tight balance between IRF-3 and IRF-7. Deletion of Irf3 resulted in overexpression of Irf7 and led to an IRF-7-driven hyperinflammatory phenotype, which was entirely prevented if Irf7 was deleted. We then identified a network of strongly up-regulated, IRF-7-dependent genes in Irf3-/- mice with kidney pathology, which was absent in Irf7-/- mice. IRF-3 and IRF-7 from infected kidney cell nuclear extracts were shown to bind OAS1, CCL5, andIFNB1 promoter oligonucleotides. These data are consistent in children with lowIRF7 expression in the blood: attenuating IRF7 promoter polymorphisms (rs3758650-T and rs10902179-G) negatively associated with recurrent pyelonephritis. Finally, we identified IRF-7 as a target for immunomodulatory therapy. Administering liposomal Irf7 siRNA to Irf3-/- mice suppressed mucosal IRF-7 expression, and the mice were protected against infection and renal tissue damage. These findings offer a response to the classical but unresolved question of "good versus bad inflammation" and identify IRF7 as a therapeutic target for protection against bacterial infection

Molecular Basis of Acute Cystitis Reveals Susceptibility Genes and Immunotherapeutic Targets

Tissue damage is usually regarded as a necessary price to pay for successful elimination of pathogens by the innate immune defense. Yet, it is possible to distinguish protective from destructive effects of innate immune activation and selectively attenuate molecular nodes that create pathology. Here, we identify acute cystitis as an Interleukin-1 beta (IL-1β)-driven, hyper-inflammatory condition of the infected urinary bladder and IL-1 receptor blockade as a novel therapeutic strategy. Disease severity was controlled by the mechanism of IL-1β processing and mice with intact inflammasome function developed a moderate, self-limiting form of cystitis. The most severe form of acute cystitis was detected in mice lacking the inflammasome constituents ASC or NLRP-3. IL-1β processing was hyperactive in these mice, due to a new, non-canonical mechanism involving the matrix metalloproteinase 7- (MMP-7). ASC and NLRP-3 served as transcriptional repressors of MMP7 and as a result, Mmp7 was markedly overexpressed in the bladder epithelium of Asc-/- and Nlrp3-/- mice. The resulting IL-1β hyper-activation loop included a large number of IL-1β-dependent pro-inflammatory genes and the IL-1 receptor antagonist Anakinra inhibited their expression and rescued susceptible Asc-/- mice from bladder pathology. An MMP inhibitor had a similar therapeutic effect. Finally, elevated levels of IL-1β and MMP-7 were detected in patients with acute cystitis, suggesting a potential role as biomarkers and immunotherapeutic targets. The results reproduce important aspects of human acute cystitis in the murine model and provide a comprehensive molecular framework for the pathogenesis and immunotherapy of acute cystitis, one of the most common infections in man. Trial Registration: The clinical studies were approved by the Human Ethics Committee at Lund University (approval numbers LU106-02, LU236-99 and Clinical Trial Registration RTP-A2003, International Committee of Medical Journal Editors, www.clinicaltrials.gov)

Regulation of <i>MMP7</i> expression by ASC and NLRP-3.

<p>(<b>A</b>) MMP-7, ASC and NLRP-3 responses to infection were visualized by confocal microscopy. An increase in MMP-7 and decrease in ASC staining were detected after infection of HTB-9 cells with CY-17 and CY-92 for 1 hour, compared to uninfected control cells. NLRP-3 staining was weakly affected. (<b>B</b>) Quantification of total fluorescence intensity (open pin-hole) after subtraction of the background staining in uninfected cells (PBS). Medians ± SEMs of 50 cells, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, compared to PBS control, two-tailed unpaired <i>t</i>-test (MMP-7 and NLRP-3) or two-tailed Mann Whitney test (ASC). One of three experiments is shown. (<b>C</b>) Western blot confirming the change in cellular content of MMP-7, ASC and NLRP-3, 1 hour after infection with the indicated strains. Fold change compared to PBS of normalized values (against GAPDH). One experiment out of 2 is shown. (<b>D</b>) Increase in MMP-7 expression in HTB-9 cells transfected with siRNAs specific for <i>ASC</i> or <i>NLRP3</i> and infected with CY-17 (4 hours, scale bars = 20 μm). (<b>E</b>) Western blot confirming the knock-down of ASC or NLRP-3 with siRNAs. A further reduction in ASC expression was detected after CY-17 infection (4 hours, quantified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005848#ppat.1005848.s008" target="_blank">S8B Fig</a>, one experiment out of 2 is shown.). (<b>F</b>) PCR amplification of a 259 bp fragment in the <i>MMP7</i> promoter (P1, -18/-276 relative to the transcription start site). (<b>G</b>) EMSA of the amplified fragment and nuclear extract from CY-17 infected HTB-9 cells (4 hours). Binding of ASC and NLRP-3 to P1 was identified as a band shift (arrow indicating protein-DNA complex). The band shift was inhibited by ASC- or NLRP-3-specific antibody. Free DNA formed a single low molecular weight band (arrow indicating free probe). The band shift was not affected by the IgG isotype control. One of three similar experiments is shown. (<b>H</b>) EMSA of the 259 bp <i>MMP7</i> promoter fragment P1 and recombinant ASC. Dose-dependent formation of an ASC-P1 complex is shown as a band shift (arrow indicating ASC-DNA complex), which was inhibited by 0.5 and 1.0 μg of anti-ASC antibodies. The band shift was not affected by negative control murine IgG control. One of three similar experiments is shown.</p

Hyper-activation of IL-1β dependent gene expression and bladder pathology in <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice.

<p>Transcriptomic analysis of whole bladder RNA from infected mice (CFT073, 7 days), compared to uninfected controls of each genotype (cut off FC 1.41, <i>P</i> < 0.05). (<b>A</b>) Heatmap of regulated genes in <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice with the highest bladder pathology score, defined by neutrophil infiltration, loss of tissue structure and epithelial thickness in H&E stained bladder tissue sections. Scale FC -4 to 4, red = upregulated, blue = downregulated. A distinct gene set distinguished the <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice with a high histopathology score from C57BL/6 WT mice or <i>Il1b</i>^<i>-/-</i> mice without pathology. (<b>B</b>) Top up-regulated genes in the pathology-associated gene set, compared to uninfected controls of each genotype. Means ± SEMs of 5 mice for <i>Asc</i>^<i>-/-</i> mice and 2 <i>Nlrp3</i>^<i>-/-</i> mice. (<b>C</b>) Analysis of IL-1β, inflammasome activators and effectors in <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice, detecting massive over-expression compared to <i>Il1b</i>^<i>-/-</i> and WT mice. Red = upregulated, blue = suppressed. The data set included gene expression profiles from 7 <i>Asc</i>^<i>-/-</i> and 5 <i>Nlrp3</i>^<i>-/-</i> mice, and two each of the C57BL/6 WT and <i>Il1b</i>^<i>-/-</i> controls. Uninfected control RNA of each genotype were used to define significantly regulated genes (≥ 2 mice per genotype). Histopathology scores and group numbers for individual mice (see also Experiments 1, 2 and 3 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005848#ppat.1005848.s011" target="_blank">S1 Table</a>).</p

IL-1β processing by MMP-7.

<p>(<b>A</b>) Gene expression profiling identified <i>Mmp7</i> as one of the top up-regulated gene in <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice with bladder pathology (CFT073 infected mice, 7 days). Log₂ fold change of <i>Mmp7</i> expression levels in individual mice are shown relative to the H&E pathology score. <i>Mmp7</i> was not regulated in C57BL/6 WT mice or in <i>Il1b</i>^<i>-/-</i> or <i>Casp1</i>^<i>-/-</i> mice. (<b>B</b>) Strong epithelial MMP-7 staining in <i>Asc</i>^<i>-/-</i> and <i>Nlrp3</i>^<i>-/-</i> mice with bladder pathology. MMP-7 staining was very low in C57BL/6 WT, <i>Il1b</i>^<i>-/-</i> and <i>Casp1</i>^<i>-/-</i> mice. Scale bars = 50 μm. (<b>C</b>) Phenotype of <i>Mmp7</i>^-/- mice, 7 days after infection with CFT073. Intact mucosal tissue structure with inflammatory cell infiltration. Low bacterial and neutrophil counts in urine compared to <i>Asc</i>^-/- mice (<i>n</i> = 5 mice per group, means ± SEMs, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, two-tailed unpaired <i>t</i>-test). Scale bar = 1 mm. IL-1β levels were elevated in the urine of <i>Asc</i>^-/- mice but not in <i>Mmp7</i>^-/- mice, as detected by ELISA. (<b>D</b>) Proteolytic cleavage of pro-IL-1β by MMP-7 <i>in vitro</i>, using purified enzyme and GST-tagged pro-IL-1β. The IL-1β fragments generated by proteolysis were 18 and 16 kDa, defined by Western blot using an antibody specific for the mature form of IL-1β. Recombinant mature IL-1β and GST-tagged pro-IL-1β were used as controls, as well as recombinant MMP-7. One representative experiment out of three, see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005848#ppat.1005848.s007" target="_blank">S7A Fig</a>. (<b>E</b>) Bioassay for IL-1β activity, measuring the PGE₂ response of human bladder epithelial cells to the IL-1β fragments generated by MMP-7 proteolysis of GST-tagged pro-IL-1β. The cleaved products activated PGE₂ but MMP-7 and pro-IL-1β had no effect (means ± SEMs of two experiments, ** <i>P</i> < 0.01, two-tailed Mann Whitney test).</p

Elevated concentrations of IL-1β and MMP-7 in the urine of patients with acute cystitis.

<p>(<b>A</b>) IL-1β concentrations in urine samples from patients with acute cystitis (<i>n</i> = 9). (<b>B</b>) IL-1β concentrations in consecutive urine samples from patients with ABU, who were long-term asymptomatic carriers of <i>E</i>. <i>coli</i> 83972 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005848#ppat.1005848.ref021" target="_blank">21</a>] (means ± SEMs, 20 patients, 161 urine samples). Elevated levels of IL-1β in the cystitis patients compared to the ABU group. Pie chart (inset) depicts the distribution of IL-1β concentrations in each patient group. (<b>C</b>) Histogram compares IL-1β concentrations between the two patient groups (means ± SEMs, *** <i>P <</i> 0.001, two-tailed Mann Whitney test). (<b>D</b>) MMP-7 concentrations were higher in urine samples from the patients with acute cystitis than in patients with long-term ABU (means ± SEMs, *** <i>P</i> < 0.001, two-tailed unpaired <i>t</i>-test). Urine samples were obtained from patients with sporadic acute cystitis at the time of diagnosis. The patients with ABU participated in a prospective study of therapeutic inoculation with <i>E</i>. <i>coli</i> 83972 and were subjected to long-term follow up [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005848#ppat.1005848.ref021" target="_blank">21</a>]. Multiple samples were obtained during asymptomatic carriage (3–14 samples per patient).</p