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Journal articleHegade VS, Pechlivanis A, McDonald JA, et al., 2019,
Autotaxin, bile acid profile and effect of IBAT inhibition in primary biliary cholangitis patients with pruritus
, Liver International, Vol: 39, Pages: 967-975, ISSN: 1478-3223BACKGROUND &AIMS: Pruritus is a common symptom in patients with primary biliary cholangitis (PBC) for which ileal bile acid transporter (IBAT) inhibition is emerging as a potential therapy. We explored the serum metabonome and gut microbiota profile in PBC patients with pruritus and investigated the effect of GSK2330672, an IBAT inhibitor. METHODS: We studied fasting serum bile acids (BAs), autotaxin and faecal microbiota in 22 PBC patients with pruritus at baseline and after two weeks of GSK2330672 treatment. Control group included 31 asymptomatic PBC patients and 18 healthy volunteers. BA profiling was done by ultraperformance liquid chromatography coupled to a mass spectrometry (UPLC-MS). Faecal microbiomes were analysed by 16S ribosomal RNA gene sequencing. RESULTS: In PBC patients with pruritus serum levels of total and glyco-conjugated primary BAs and autotaxin were significantly elevated. Autotaxin activity correlated significantly with tauro- and glyco-conjugated cholic acid (CA) and chenodeoxycholic acid (CDCA), both at baseline and after GSK2330672. GSK2330672 significantly reduced autotaxin and all tauro- and glyco- conjugated BAs and increased faecal levels of CA (p=0.048) and CDCA (p=0.027). Gut microbiota of PBC patients with pruritus was similar to control groups. GSK2330672 increased relative abundance of Firmicutes (p=0.033) and Clostridia (p=0.04) and decreased Bacteroidetes (p=0.033) and Bacteroidia (p=0.04). CONCLUSIONS: Pruritus in PBC does not show a distinct gut bacterial profile but is associated with elevated serum bile acid and autotaxin levels which decrease after IBAT inhibition. In cholestatic pruritus, a complex interplay between BAs and ATX is likely and may be modified by IBAT inhibition. This article is protected by copyright. All rights reserved.
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Journal articleAllegretti JR, Hurtado J, Carrellas M, et al., 2019,
7 – The icon study: Inflammatory bowel disease and recurrent clostridium difficile infection: Outcomes after fecal microbiota transplantation
, Gastroenterology, Vol: 156, Pages: S-2-S-3, ISSN: 0016-5085 -
Journal articleMcDonald JA, Perez JL, Mullish BH, et al., 2019,
Mo1953 – Growth inhibition of clostridioides difficile by short and medium chain fatty acids
, Gastroenterology, Vol: 156, Pages: S-898-S-898, ISSN: 0016-5085 -
Journal articleRoy RB, Sambou B, Uhia I, et al., 2019,
An auto-luminescent fluorescent BCG whole blood assay to enable evaluation of paediatric mycobacterial responses using minimal blood volumes
, Frontiers in Pediatrics, Vol: 7, ISSN: 2296-2360Introduction: Understanding protective human immunity against mycobacteria is critical to developing and evaluating new vaccines against tuberculosis. Children are the most susceptible population to infection, disease, and death from tuberculosis, but also have the strongest evidence of BCG-inducible protection. Limited amounts of blood can be obtained for research purposes in paediatrics and therefore there is a need for high-yield, low-volume, human immunology assays.Methods: We transformed BCG Danish with plasmids encoding luciferase full operon derived from Photorhabdus luminescens together with Green Fluorescent Protein and antibiotic selection markers. We characterised the luminescent and fluorescent properties of this recombinant BCG strain (BCG-GFP-LuxFO) using a luminometer and flow cytometry and developed a paediatric whole blood in vitro infection model.Results: Luminescence of BCG-GFP-LuxFO correlated with optical density (Spearman Rank Correlation coefficient r = 0.985, p < 0.0001) and colony forming units (CFUs) in liquid culture medium (r = 0.971, p < 0.0001). Fluorescence of BCG-GFP-LuxFO in paediatric whole blood was confirmed by flow cytometry in granulocytes and monocytes 1 h following infection. Luminescence of BCG-GFP-LuxFO in whole blood corresponded with CFUs (r = 0.7123, p < 0.0001).Conclusion: The BCG-GFP-LuxFO assay requires 225 μL whole blood per sample, from which serial luminescence measurements can be obtained, together with biochemical analysis of supernatants and cellular assay applications using its fluorescent properties. This offers the opportunity to study human-mycobacterial interactions using multiple experimental modalities with only minimal blood volumes. It is therefore a valuable method for investigating paediatric immunity to tuberculosis.
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Journal articleBerry J-L, Gurung I, Anonsen JH, et al., 2019,
Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis.
, J Biol Chem, Vol: 294, Pages: 6796-6808Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins, i.e. a methylated N terminus; (iii) are found in the same heteropolymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin-fold with a highly unusual flexible C terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than bona fide Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that S. sanguinis Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.
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Journal articleGoddard P, Sanchez Garrido J, Slater S, et al., 2019,
Enteropathogenic E. coli stimulates effector-driven rapid caspase-4 activation in human macrophages
, Cell Reports, Vol: 27, Pages: 1008-1017.e6, ISSN: 2211-1247Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.
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Journal articleCalderazzo MA, Trujillo-Torralbo M-B, Finney LJ, et al., 2019,
Inflammation and infections in unreported chronic obstructive pulmonary disease exacerbations
, International Journal of Chronic Obstructive Pulmonary Disease, Vol: 2019, Pages: 823-832, ISSN: 1176-9106Purpose: COPD patients often do not report acute exacerbations to healthcare providers – unreported exacerbations. It is not known whether variances in symptoms, airway obstruction, aetiology and inflammatory responses account for differences in reporting of COPD exacerbations. The aims of the study were to compare symptoms, lung function changes, aetiology and inflammatory markers between exacerbations that were reported to healthcare providers or treated, with those that were unreported and untreated.Patients and methods: We recruited a cohort of COPD patients and collected clinical data and blood and airway samples when stable and during acute exacerbations. Virological and bacterial analyses were carried out and inflammatory markers measured.Results: We found no differences in symptoms, lung function, incidence of infection and inflammatory markers between reported and unreported exacerbations. Subjects who reported all exacerbations had higher BODE scores, lower FEV1 and more exacerbations compared with those who did not.Conclusion: The failure to report exacerbations is not related to the severity, aetiology or inflammatory profile of the exacerbation. Patients with less severe COPD and less frequent exacerbations are less likely to report exacerbations. The decision to report an exacerbation is not an objective marker of exacerbation severity and therefore studies that do not count unreported exacerbations will underestimate the frequency of clinically significant exacerbations. A better understanding of the factors that determine non-reporting of exacerbations is required to improve exacerbation reporting.
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Journal articleGallego del Sol F, Penades JR, Marina A, 2019,
Deciphering the molecular mechanism underpinning phage arbitrium communication systems
, Molecular Cell, Vol: 74, Pages: 59-72.e3, ISSN: 1097-2765Bacillus phages use a communication system, termed “arbitrium,” to coordinate lysis-lysogeny decisions. Arbitrium communication is mediated by the production and secretion of a hexapeptide (AimP) during lytic cycle. Once internalized, AimP reduces the expression of the negative regulator of lysogeny, AimX, by binding to the transcription factor, AimR, promoting lysogeny. We have elucidated the crystal structures of AimR from the Bacillus subtilis SPbeta phage in its apo form, bound to its DNA operator and in complex with AimP. AimR presents intrinsic plasticity, sharing structural features with the RRNPP quorum-sensing family. Remarkably, AimR binds to an unusual operator with a long spacer that interacts nonspecifically with the receptor TPR domain, while the HTH domain canonically recognizes two inverted repeats. AimP stabilizes a compact conformation of AimR that approximates the DNA-recognition helices, preventing AimR binding to the aimX promoter region. Our results establish the molecular basis of the arbitrium communication system.
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Journal articleSharrock J, Estacio Gomez A, Jacobson J, et al., 2019,
fs(1)h controls metabolic and immune function and enhances survival via AKT and FOXO in Drosophila
, Disease Models & Mechanisms, Vol: 12, ISSN: 1754-8403The Drosophila fat body is the primary organ of energy storage as well as being responsible for the humoral response to infection. Its physiological function is of critical importance to the survival of the organism; however, many molecular regulators of its function remain ill-defined. Here, we show that the Drosophila melanogaster bromodomain-containing protein FS(1)H is required in the fat body for normal lifespan as well as metabolic and immune homeostasis. Flies lacking fat body fs(1)h exhibit short lifespan, increased expression of immune target genes, an inability to metabolize triglyceride, and low basal AKT activity, mostly resulting from systemic defects in insulin signalling. Removal of a single copy of the AKT-responsive transcription factor foxo normalises lifespan, metabolic function, uninduced immune gene expression and AKT activity. We suggest that the promotion of systemic insulin signalling activity is a key in vivo function of fat body fs(1)h.
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Journal articleHopkins E, Roumeliotis TI, Mullineaux-Sanders C, et al., 2019,
Intestinal epithelial cells and the microbiome undergo swift reprogramming at the inception of colonic Citrobacter rodentium infection
, mBio, Vol: 10, ISSN: 2150-7511We used the mouse attaching and effacing (A/E) pathogen Citrobacter rodentium, which models the human A/E pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC), to temporally resolve intestinal epithelial cell (IEC) responses and changes to the microbiome during in vivo infection. We found the host to be unresponsive during the first 3 days postinfection (DPI), when C. rodentium resides in the caecum. In contrast, at 4 DPI, the day of colonic colonization, despite only sporadic adhesion to the apex of the crypt, we observed robust upregulation of cell cycle and DNA repair processes, which were associated with expansion of the crypt Ki67-positive replicative zone, and downregulation of multiple metabolic processes (including the tricarboxylic acid [TCA] cycle and oxidative phosphorylation). Moreover, we observed dramatic depletion of goblet and deep crypt secretory cells and an atypical regulation of cholesterol homeostasis in IECs during early infection, with simultaneous upregulation of cholesterol biogenesis (e.g., 3-hydroxy-3-methylglutaryl–coenzyme A reductase [Hmgcr]), import (e.g., low-density lipoprotein receptor [Ldlr]), and efflux (e.g., AbcA1). We also detected interleukin 22 (IL-22) responses in IECs (e.g., Reg3γ) on the day of colonic colonization, which occurred concomitantly with a bloom of commensal Enterobacteriaceae on the mucosal surface. These results unravel a new paradigm in host-pathogen-microbiome interactions, showing for the first time that sensing a small number of pathogenic bacteria triggers swift intrinsic changes to the IEC composition and function, in tandem with significant changes to the mucosa-associated microbiome, which parallel innate immune responses.
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