Fimbriae reprogram host gene expression – Divergent effects of P and type 1 fimbriae

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

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

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Molecular determinants of disease severity in urinary tract infection

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

Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression

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 (&gt;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

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

A scientific journey from discovery to validation of efficacy in cancer patients : HAMLET and alpha1-oleate

The protein-lipid complex alpha1-oleate, derived from HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), is identified as a molecular entity with significant therapeutic potential. Structural characterization of the complex and results of a successful placebo-controlled clinical trial are presented

Susceptibility to urinary tract infection : Benefits and hazards of the antibacterial host response

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

Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression

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 (&gt;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

Immunomodulation—A Molecular Solution to Treating Patients with Severe Bladder Pain Syndrome?

Background: Patients with bladder pain syndrome experience debilitating pain and extreme frequency of urination. Numerous therapeutic approaches have been tested, but as the molecular basis of disease has remained unclear, specific therapies are not available. Objective: Recently, a systematic gene deletion strategy identified interleukin-1 (IL-1) hyperactivation as a cause of severe cystitis in a murine model. Treatment with an IL-1 receptor antagonist (IL-1RA) restored health in genetically susceptible mice, linking IL-1–dependent inflammation to pain and pathology in the bladder mucosa. The study objective was to investigate whether IL-1RA treatment might be beneficial in patients with bladder pain syndrome. Design, setting, and participants: Patients diagnosed with bladder pain syndrome were invited to participate and subjected to daily IL-1RA injections for 1 wk, followed by a treatment break. Patients with other urological disorders accompanied by pain were included as controls. Outcome measurements and statistical analysis: When symptoms returned, treatment was resumed and responding patients were maintained on treatment long term, with individualized dosing regimens. Symptom scores were recorded and molecular effects were quantified by neuropeptide and gene expression analysis. DNA samples were subjected to exome genotyping. Results and limitations: IL-1RA treatment reduced bladder pain and the frequency of urination in 13/17 patients (p < 0.001). Substance P levels in urine were lowered, and responders returned to a more normal lifestyle. Neuroinflammatory-dependent and IL-1–dependent gene networks were inhibited, as well as regulators of innate immunity. Genotyping revealed disease-associated IL1R1, NLRP3, and IL1RN DNA sequence variants in the responders. Controls did not benefit from IL-1RA treatment, except for one patent with cystitis cystica. Conclusions: In this clinical study, IL-1RA treatment is proposed to reduce chronic bladder pain, immediately and in the long term. Despite the limited number of study patients, the potent acute effect and lasting symptom relief indicate that this therapeutic approach may be worth exploring in controlled clinical trials. Patient summary: Treatment with an interleukin-1 (IL-1) receptor antagonist is proposed for treating bladder pain syndrome, as it can result in symptom relief and increase quality of life. Reduced neuroinflammation and IL-1 signaling provided molecular evidence of the treatment effects. Take Home Message: Interleukin-1 (IL-1) receptor antagonist immunotherapy is proposed as a new approach to treating bladder pain syndrome, a debilitating disorder. Treated patients experienced symptom relief and increased quality of life. Reduced neuroinflammation and IL-1 signaling provided molecular evidence of the treatment effects