| 1. |
- Ambite, Ines, et al.
(författare)
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Bacterial suppression of RNA polymerase II-dependent host gene expression
- 2016
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Ingår i: Pathogens. - : MDPI AG. - 2076-0817. ; 5:3
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Forskningsöversikt (refereegranskat)abstract
- Asymptomatic bacteriuria (ABU) is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli) strains and therefore elicit weak innate immune responses in the urinary tract. In addition, ABU strains are active modifiers of the host environment, which they influence by suppressing RNA polymerase II (Pol II)-dependent host gene expression. In patients inoculated with the ABU strain E. coli 83972, gene expression was markedly reduced after 24 h (>60% of all regulated genes). Specific repressors and activators of Pol II-dependent transcription were modified, and Pol II Serine 2 phosphorylation was significantly inhibited, indicating reduced activity of the polymerase. This active inhibition included disease–associated innate immune response pathways, defined by TLR4, IRF-3 and IRF-7, suggesting that ABU strains persist in human hosts by active suppression of the antibacterial defense. In a search for the mechanism of inhibition, we compared the whole genome sequences of E. coli 83972 and the uropathogenic strain E. coli CFT073. In addition to the known loss of virulence genes, we observed that the ABU strain has acquired several phages and identified the lytic Prophage 3 as a candidate Pol II inhibitor. Intact phage particles were released by ABU during in vitro growth in human urine. To address if Prophage 3 affects Pol II activity, we constructed a Prophage 3 negative deletion mutant in E. coli 83972 and compared the effect on Pol II phosphorylation between the mutant and the E. coli 83972 wild type (WT) strains. No difference was detected, suggesting that the Pol II inhibitor is not encoded by the phage. The review summarizes the evidence that the ABU strain E. coli 83972 modifies host gene expression by inhibition of Pol II phosphorylation, and discusses the ability of ABU strains to actively create an environment that enhances their persistence.
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| 2. |
- Ambite, Ines, et al.
(författare)
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Fimbriae reprogram host gene expression - Divergent effects of P and type 1 fimbriae
- 2019
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 15:6, s. 1007671-1007671
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Tidskriftsartikel (refereegranskat)abstract
- Pathogens rely on a complex virulence gene repertoire to successfully attack their hosts. We were therefore surprised to find that a single fimbrial gene reconstitution can return the virulence-attenuated commensal strain Escherichia coli 83972 to virulence, defined by a disease phenotype in human hosts. E. coli 83972pap stably reprogrammed host gene expression, by activating an acute pyelonephritis-associated, IRF7-dependent gene network. The PapG protein was internalized by human kidney cells and served as a transcriptional agonist of IRF-7, IFN-β and MYC, suggesting direct involvement of the fimbrial adhesin in this process. IRF-7 was further identified as a potent upstream regulator (-log (p-value) = 61), consistent with the effects in inoculated patients. In contrast, E. coli 83972fim transiently attenuated overall gene expression in human hosts, enhancing the effects of E. coli 83972. The inhibition of RNA processing and ribosomal assembly indicated a homeostatic rather than a pathogenic end-point. In parallel, the expression of specific ion channels and neuropeptide gene networks was transiently enhanced, in a FimH-dependent manner. The studies were performed to establish protective asymptomatic bacteriuria in human hosts and the reconstituted E. coli 83972 variants were developed to improve bacterial fitness for the human urinary tract. Unexpectedly, P fimbriae were able to drive a disease response, suggesting that like oncogene addiction in cancer, pathogens may be addicted to single super-virulence factors.
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| 3. |
- Ambite, Ines, et al.
(författare)
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Molecular Basis of Acute Cystitis Reveals Susceptibility Genes and Immunotherapeutic Targets
- 2016
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 12:10
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Tidskriftsartikel (refereegranskat)abstract
- 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).
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| 4. |
- Ambite, Ines
(författare)
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Molecular determinants of disease severity in urinary tract infection
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the urinary tract, Escherichia coli infection may result in life-threatening disease, or asymptomatic bacterial carriage, comparable to bacterial commensalism in the gut. Pathogenic strains trigger a disease provoking host response which differs depending on the infected organ. The diversity of the response reflects the virulence repertoire of the infecting strain and by the susceptibility of the host. During asymptomatic bacteriuria (ABU), the lack of clinical symptoms has been attributed to the loss of virulence by the strains. ABU strains have a reduced genome size and carry point mutations or deletions in virulence genes. In the prototype strain E. coli 83972 fimbrial gene clusters are affected by multiple point mutations (the papG adhesin gene) or large deletions (the fim gene cluster). As a result, the innate immune response to ABU strains is reduced. In addition, we made the discovery that asymptomatic carrier strains actively inhibit RNA Polymerase II (Pol II) Ser2 phosphorylation. Pol II is the enzyme required for the transcription of all mRNA in eukaryotes and its function is tightly regulated. E. coli 83972 suppresses Pol II-dependent host gene expression, including genes involved in immune activation. Compared to a broad chemical inhibitor, DRB, inhibition was more limited, suggesting a degree of specificity for a certain repertoire of host genes. Through this suppression of transcription, ABU strains may promote tissue integrity while inhibiting potentially destructive immune activation. NlpD was identified as a protein released by asymptomatic carrier strains with potent host gene expression inhibitory capacity. NlpD targeted the Pol II phosphorylation machinery by interacting with the biggest Pol II subunit, RPB1, and PAF1C in host cells. In treated mice, NlpD inhibited the destructive arm of innate immune activation and reduced bacterial loads. Our findings suggest that molecules of bacterial origin may be explored as therapies to reproduce the beneficial effects associated with ABU.The symptoms of acute cystitis, in contrast, are caused by an exaggerated inflammatory response triggered by infection. We now define acute cystitis as an IL-1β-driven, hyper-inflammatory disorder with atypical IL-1β processing through MMP-7. The results also suggest a genetic susceptibility factor in patients with severe and chronic cystitis, through mutations affecting ASC or NLRP3 expression. ASC and NLRP-3 were identified as negative regulators of MMP-7 expression and the identified molecular determinants and IL-1β and MMP-7 as novel targets for immunomodulatory therapy with potent effects in vivo as well as biomarkers for acute cystitis.In contrast to acute cystitis, acute pyelonephritis is a severe, sometimes life-threatening kidney infection with systemic involvement and risk of developing bacteremia. Uropathogenic E. coli strains initiate tissue attack of the renal pelvis thus starting disease pathogenesis. A normally protective innate immune signalling cascade, controlled by toll like receptor (TLR)4, is exaggerated explaining the acute disease and sometimes destructive, long-term effects. The response is determined, in part, by bacterial P fimbriae, their receptors and the quality of the signalling cascade that they activate, including several transcription factors. The transcription factor IRF-3 controls the protective arm of the innate immune response to kidney infection, and as a result, mice lacking Irf3 develop severe infection accompanied by urosepsis and renal abscess formation. We identify IRF-7 as a driving force for the disease response in Irf3-/- mice and development of renal pathology. We also define Irf7 as an immuno-therapeutic target that can be controlled with small interfering RNA (siRNA) to restore the balance of resistance versus pathology and prevent kidney damage.Finally, we show that P fimbriae influence the IRF-7 expression and the repertoire of downstream genes associated with acute pyelonephritis (APN). Specifically, P fimbriae were shown to act as IRF-7 agonists. The expression of functional P fimbriae was sufficient to reprogram host gene expression, through effects of PapG on the transcriptional machinery of the host. Paradoxically, the results suggest that a single “super virulence” factor may be sufficient to tilt the balance from peaceful coexistence to disease and for the host to recognize and respond to a strain that lacks most other virulence factors.
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| 5. |
- Ambite, Ines, et al.
(författare)
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Susceptibility to urinary tract infection : Benefits and hazards of the antibacterial host response
- 2016
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Ingår i: Microbiology spectrum. - Washington, DC, USA : ASM Press. - 2165-0497. ; 4:3
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Forskningsöversikt (refereegranskat)abstract
- A paradigm shift is needed to improve and personalize the diagnosis of infectious disease and to select appropriate therapies. For many years, only the most severe and complicated bacterial infections received more detailed diagnostic and therapeutic attention as the efficiency of antibiotic therapy has guaranteed efficient treatment of patients suffering from the most common infections. Indeed, treatability almost became a rationale not to analyze bacterial and host parameters in these larger patient groups. Due to the rapid spread of antibiotic resistance, common infections like respiratory tract- or urinary-tract infections (UTIs) now pose new and significant therapeutic challenges. It is fortunate and timely that infectious disease research can offer such a wealth of new molecular information that is ready to use for the identification of susceptible patients and design of new suitable therapies. Paradoxically, the threat of antibiotic resistance may become a window of opportunity, by encouraging the implementation of new diagnostic and therapeutic approaches. The frequency of antibiotic resistance is rising rapidly in uropathogenic organisms and the molecular and genetic understanding of UTI susceptibility is quite advanced. More bold translation of the new molecular diagnostic and therapeutic tools would not just be possible but of great potential benefit in this patient group. This chapter reviews the molecular basis for susceptibility to UTI, including recent advances in genetics, and discusses the consequences for diagnosis and therapy. By dissecting the increasingly well-defined molecular interactions between bacteria and host and the molecular features of excessive bacterial virulence or host-response malfunction, it is becoming possible to isolate the defensive from the damaging aspects of the host response. Distinguishing "good" from "bad" inflammation has been a long-term quest of biomedical science and in UTI, patients need the "good" aspects of the inflammatory response to resist infection while avoiding the "bad" aspects, causing chronicity and tissue damage.
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| 6. |
- Ambite, Ines, et al.
(författare)
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The Genetics of Urinary Tract Infections and the Innate Defense of the Kidney and Urinary tract
- 2016
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Ingår i: Journal of pediatric genetics. - : Georg Thieme Verlag KG. - 2146-4596 .- 2146-460X. ; 5:1, s. 25-32
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 7. |
- Butler, Daniel S.C., et al.
(författare)
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Neuroepithelial control of mucosal inflammation in acute cystitis
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- The nervous system is engaged by infection, indirectly through inflammatory cascades or directly, by bacterial attack on nerve cells. Here we identify a neuro-epithelial activation loop that participates in the control of mucosal inflammation and pain in acute cystitis. We show that infection activates Neurokinin-1 receptor (NK1R) and Substance P (SP) expression in nerve cells and bladder epithelial cells in vitro and in vivo in the urinary bladder mucosa. Specific innate immune response genes regulated this mucosal response, and single gene deletions resulted either in protection (Tlr4−/− and Il1b−/− mice) or in accentuated bladder pathology (Asc−/− and Nlrp3−/− mice), compared to controls. NK1R/SP expression was lower in Tlr4−/− and Il1b−/− mice than in C56BL/6WT controls but in Asc−/− and Nlrp3−/− mice, NK1R over-activation accompanied the exaggerated disease phenotype, due, in part to transcriptional de-repression of Tacr1. Pharmacologic NK1R inhibitors attenuated acute cystitis in susceptible mice, supporting a role in disease pathogenesis. Clinical relevance was suggested by elevated urine SP levels in patients with acute cystitis, compared to patients with asymptomatic bacteriuria identifying NK1R/SP as potential therapeutic targets. We propose that NK1R and SP influence the severity of acute cystitis through a neuro-epithelial activation loop that controls pain and mucosal inflammation.
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| 8. |
- Godaly, Gabriela, et al.
(författare)
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Innate immunity and genetic determinants of urinary tract infection susceptibility.
- 2015
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Ingår i: Current Opinion in Infectious Diseases. - 1473-6527. ; 28:1, s. 88-96
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Forskningsöversikt (refereegranskat)abstract
- Urinary tract infections (UTIs) are common, dangerous and interesting. Susceptible individuals experience multiple, often clustered episodes, and in a subset of patients, infections progress to acute pyelonephritis (APN), sometimes accompanied by uro-sepsis. Others develop asymptomatic bacteriuria (ABU). Here, we review the molecular basis for these differences, with the intention to distinguish exaggerated host responses that drive disease from attenuated responses that favour protection and to highlight the genetic basis for these extremes, based on knock-out mice and clinical studies.
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| 9. |
- Godaly, Gabriela, et al.
(författare)
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Urinary Tract Infection Molecular Mechanisms and Clinical Translation.
- 2016
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Ingår i: Pathogens. - : MDPI AG. - 2076-0817. ; 5:1
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Forskningsöversikt (refereegranskat)abstract
- Rapid developments in infection biology create new and exciting options for individualized diagnostics and therapy. Such new practices are needed to improve patient survival and reduce morbidity. Molecular determinants of host resistance to infection are being characterized, making it possible to identify susceptible individuals and to predict their risk for future morbidity. Immunotherapy is emerging as a new strategy to treat infections worldwide and controlled boosting of the host immune defense represents an important therapeutic alternative to antibiotics. In proof of concept studies, we have demonstrated that this approach is feasible. The long-term goal is not just to remove the pathogens but to also develop technologies that restore resistance to infection in disease-prone patients and devise personalized therapeutic interventions. Here, we discuss some approaches to reaching these goals, in patients with urinary tract infection (UTI). We describe critical host signaling pathways that define symptoms and pathology and the genetic control of innate immune responses that balance protection against tissue damage. For some of these genes, human relevance has been documented in clinical studies, identifying them as potential targets for immune-modulatory therapies, as a complement to antibiotics.
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| 10. |
- Puthia, Manoj, et al.
(författare)
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IRF7 inhibition prevents destructive innate immunity-A target for nonantibiotic therapy of bacterial infections
- 2016
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Ingår i: Science Translational Medicine. - : American Association for the Advancement of Science (AAAS). - 1946-6234 .- 1946-6242. ; 8:336
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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