Moderate-intensity aerobic and resistance exercise is safe and favorably influences body composition in patients with quiescent Inflammatory Bowel Disease: a randomized controlled cross-over trial.

BACKGROUND: Overweight and metabolic problems now add to the burden of illness in patients with Inflammatory Bowel Disease. We aimed to determine if a program of aerobic and resistance exercise could safely achieve body composition changes in patients with Inflammatory Bowel Disease. METHODS: A randomized, cross-over trial of eight weeks combined aerobic and resistance training on body composition assessed by Dual Energy X-ray Absorptiometry was performed. Patients in clinical remission and physically inactive with a mean age of 25 ± 6.5 years and Body Mass Index of 28.9 ± 3.8 were recruited from a dedicated Inflammatory Bowel Disease clinic. Serum cytokines were quantified, and microbiota assessed using metagenomic sequencing. RESULTS: Improved physical fitness was demonstrated in the exercise group by increases in median estimated VO2max (Baseline: 43.41mls/kg/min; post-intervention: 46.01mls/kg/min; p = 0.03). Improvement in body composition was achieved by the intervention group (n = 13) with a median decrease of 2.1% body fat compared with a non-exercising group (n = 7) (0.1% increase; p = 0.022). Lean tissue mass increased by a median of 1.59 kg and fat mass decreased by a median of 1.52 kg in the exercising group. No patients experienced a deterioration in disease activity scores during the exercise intervention. No clinically significant alterations in the α- and ß-diversity of gut microbiota and associated metabolic pathways were evident. CONCLUSIONS: Moderate-intensity combined aerobic and resistance training is safe in physically unfit patients with quiescent Inflammatory Bowel Disease and can quickly achieve favourable body compositional changes without adverse effects. TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov; Trial number: NCT02463916 .

A Repeated-measures Case Series of Physiological Responses to a Transoceanic Rowing Race.

This repeated-measures case series describes the changes in cardiorespiratory fitness, body composition and systemic inflammation in 4 well-trained athletes pre- and post-completion of an unsupported transatlantic rowing race. The acute effects of endurance exercise have been well described previously, but the enduring consequences of ultra-endurance on the cardiorespiratory, metabolic and immune systems are largely unknown. This study explores these physiological adaptations following 2 weeks of recovery. Cardiorespiratory fitness testing, body composition analysis, and blood sampling for inflammatory cytokines were recorded immediately before race departure and repeated 14 days following race completion. Mean VO2max (ml/kg/min) was similar pre- (48.2±2.8) and post-race (46.7±1.5). Heart rate responses were equivalent at incremental workloads. Mean blood lactate (mmol/L) was higher at low to moderate power outputs and lower at maximal effort (14.6±1.85 vs. 13.1±2.5). Percentage body fat (17.7 ± 7.9 vs. 16.2±7.4) was analogous to pre-race analysis. Low-grade inflammation persisted, indicated by an increase in IL-1ß (69%), IL-8 (10%), TNF-α (8%), IL-6 (5.4%), and C-reactive protein (22.4%). VO2max and heart rate responses were similar pre- and post-race, but sub-maximal efficiency measures of cardiorespiratory fitness were consistent with persistent fatigue. Body composition had returned to baseline but low-grade systemic inflammation persisted. Persistent pro-inflammatory cytokinaemia is known to exert deleterious consequences on immune, metabolic, and psychological function. Adequate recovery is necessary to re-establish inflammatory homeostasis, and the results of this study may inform these decisions.

Tissue and Bronchoalveolar Lavage Biomarkers in Idiopathic Pulmonary Fibrosis Patients on Pirfenidone.

BACKGROUND: Pirfenidone is a novel anti-fibrotic agent in idiopathic pulmonary fibrosis with proven clinical benefit. Better human tissue models to demonstrate the immunomodulatory and anti-fibrotic effect of pirfenidone are required. OBJECTIVES: The purpose of the study was to use transbronchial lung cryobiopsy (TBLC), a novel technique which provides substantial tissue samples, and a large panel of biomarkers to temporally assess disease activity and response to pirfenidone therapy. METHODS: Thirteen patients with confirmed idiopathic pulmonary fibrosis (IPF) underwent full physiological and radiological assessment at diagnosis and after 6-month pirfenidone therapy. They underwent assessment for a wide range of potential serum and bronchoalveolar lavage biomarkers of disease activity. Finally, they underwent TBLC before and after treatment. Tissue samples were assessed for numbers of fibroblast foci, for Ki-67, a marker of tissue proliferation and caspase-3, a marker of tissue apoptosis. RESULTS: All patients completed treatment and investigations without significant incident. There was no significant fall in number of fibroblast foci per unit tissue volume after treatment (pre-treatment: 0.14/mm2 vs. post-treatment 0.08/mm2, p = 0.1). Likewise, there was no significant change in other markers of tissue proliferation, Ki-67 or Caspase-3 with pirfenidone treatment. We found an increase in three bronchoalveolar lavage angiogenesis cytokines, Placental Growth Factor, Vascular Endothelial Growth Factor-A, and basic Fibroblast Growth Factor, two anti-inflammatory cytokines Interleukin-10 and Interleukin-4 and Surfactant Protein-D. CONCLUSIONS: TBLC offers a unique opportunity to potentially assess the course of disease activity and response to novel anti-fibrotic activity in IPF.

A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis.

Cyclic guanosine 3',5'-monophosphate (cyclic GMP) is a second messenger whose role in bacterial signalling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified that XC_0250, which encodes a protein with a class III nucleotidyl cyclase domain, is required for cyclic GMP synthesis. Purified XC_0250 was active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide-binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di-GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 bound cyclic GMP and a structure-function analysis, directed by determination of the crystal structure of the holo-complex, demonstrated the site of cyclic GMP binding that modulates cyclic di-GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector that directly links cyclic GMP and cyclic di-GMP signalling.

The DSF Family of Cell-Cell Signals: An Expanding Class of Bacterial Virulence Regulators.

Many pathogenic bacteria use cell-cell signaling systems involving the synthesis and perception of diffusible signal molecules to control virulence as a response to cell density or confinement to niches. Bacteria produce signals of diverse structural classes. Signal molecules of the diffusible signal factor (DSF) family are cis-2-unsaturated fatty acids. The paradigm is cis-11-methyl-2-dodecenoic acid from Xanthomonas campestris pv. campestris (Xcc), which controls virulence in this plant pathogen. Although DSF synthesis was thought to be restricted to the xanthomonads, it is now known that structurally related molecules are produced by the unrelated bacteria Burkholderia cenocepacia and Pseudomonas aeruginosa. Furthermore, signaling involving these DSF family members contributes to bacterial virulence, formation of biofilms and antibiotic tolerance in these important human pathogens. Here we review the recent advances in understanding DSF signaling and its regulatory role in different bacteria. These advances include the description of the pathway/mechanism of DSF biosynthesis, identification of novel DSF synthases and new members of the DSF family, the demonstration of a diversity of DSF sensors to include proteins with a Per-Arnt-Sim (PAS) domain and the description of some of the signal transduction mechanisms that impinge on virulence factor expression. In addition, we address the role of DSF family signals in interspecies signaling that modulates the behavior of other microorganisms. Finally, we consider a number of recently reported approaches for the control of bacterial virulence through the modulation of DSF signaling.

Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence.

Bis-(3',5') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d)∼2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

The PAS domain-containing histidine kinase RpfS is a second sensor for the diffusible signal factor of Xanthomonas campestris.

A cell-cell signalling system mediated by the fatty acid signal DSF controls the virulence of Xanthomonas campestris pv. campestris (Xcc) to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon the sensor RpfC and regulator RpfG. Detailed analyses of the regulatory roles of different Rpf proteins have suggested the occurrence of further sensors for DSF. Here we have used a mutagenesis approach coupled with high-resolution transcriptional analysis to identify XC_2579 (RpfS) as a second sensor for DSF in Xcc. RpfS is a complex sensor kinase predicted to have multiple Per/Arnt/Sim (PAS) domains, a histidine kinase domain and a C-terminal receiver (REC) domain. Isothermal calorimetry showed that DSF bound to the isolated N-terminal PAS domain with a Kd of 1.4 µM. RpfS controlled expression of a sub-set of genes distinct from those controlled by RpfC to include genes involved in type IV secretion and chemotaxis. Mutation of XC_2579 was associated with a reduction in virulence of Xcc to Chinese Radish when assayed by leaf spraying but not by leaf inoculation, suggesting a role for RpfS-controlled factors in the epiphytic phase of the disease cycle.

Crystal structure of an HD-GYP domain cyclic-di-GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre.

Bis-(3',5') cyclic di-guanylate (c-di-GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c-di-GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD-GYP domains. Here, we have determined the structure of an enzymatically active HD-GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c-di-GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c-di-GMP. This structure completes the picture of all domains involved in c-di-GMP metabolism and reveals that the HD-GYP family splits into two distinct subgroups containing bi- and trinuclear metal centres.

XbmR, a new transcription factor involved in the regulation of chemotaxis, biofilm formation and virulence in Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker. Biofilm formation on citrus leaves plays an important role in epiphytic survival of Xcc. Biofilm formation is affected by transposon insertion in XAC3733, which encodes a transcriptional activator of the NtrC family, not linked to a gene encoding a sensor protein, thus could be considered as an 'orphan' regulator whose function is poorly understood in Xanthomonas spp. Here we show that mutation of XAC3733 (named xbmR) resulted in impaired structural development of the Xcc biofilm, loss of chemotaxis and reduced virulence in grapefruit plants. All defective phenotypes were restored to wild-type levels by the introduction of PA2567 from Pseudomonas aeruginosa, which encodes a phosphodiesterase active in the degradation of cyclic diguanosine monophosphate (c-di-GMP). A knockout of xbmR led to a substantial downregulation of fliA that encodes a σ(28) transcription factor, as well as fliC and XAC0350 which are potential member of the σ(28) regulon. XAC0350 encodes an HD-GYP domain c-di-GMP phosphodiesterase. These findings suggest that XbmR is a key regulator of flagellar-dependent motility and chemotaxis exerting its action through a regulatory pathway that involves FliA and c-di-GMP.

High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signalling reveals novel genes that contribute to Xanthomonas phytopathogenesis.

The bacterium Xanthomonas campestris is an economically important pathogen of many crop species and a model for the study of bacterial phytopathogenesis. In X. campestris, a regulatory system mediated by the signal molecule DSF controls virulence to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon a system comprising the sensor RpfC and regulator RpfG. Here we have addressed the action and role of Rpf/DSF signalling in phytopathogenesis by high-resolution transcriptional analysis coupled to functional genomics. We detected transcripts for many genes that were unidentified by previous computational analysis of the genome sequence. Novel transcribed regions included intergenic transcripts predicted as coding or non-coding as well as those that were antisense to coding sequences. In total, mutation of rpfF, rpfG and rpfC led to alteration in transcript levels (more than fourfold) of approximately 480 genes. The regulatory influence of RpfF and RpfC demonstrated considerable overlap. Contrary to expectation, the regulatory influence of RpfC and RpfG had limited overlap, indicating complexities of the Rpf signalling system. Importantly, functional analysis revealed over 160 new virulence factors within the group of Rpf-regulated genes.

Proteomics analysis of the regulatory role of Rpf/DSF cell-to-cell signaling system in the virulence of Xanthomonas campestris.

The black rot pathogen Xanthomonas campestris utilizes molecules of the diffusible signal factor (DSF) family as signals to regulate diverse processes contributing to virulence. DSF signal synthesis and transduction requires proteins encoded by the rpf gene cluster. RpfF catalyzes DSF synthesis, whereas the RpfCG two-component system links the perception of DSF to alteration in the level of the second messenger cyclic di-GMP. As this nucleotide can exert a regulatory influence at the post-transcriptional and post-translational levels, we have used comparative proteomics to identify Rpf-regulated processes in X. campestris that may not be revealed by transcriptomics. The abundance of a number of proteins was altered in rpfF, rpfC, or rpfG mutants compared with the wild type. These proteins belonged to several functional categories, including biosynthesis and intermediary metabolism, regulation, oxidative stress or antibiotic resistance, and DNA replication. For many of these proteins, the alteration in abundance was not associated with alteration in transcript level. A directed mutational analysis allowed us to describe a number of new virulence factors among these proteins, including elongation factor P and a putative outer membrane protein, which are both widely conserved in bacteria.

Dynamic complex formation between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris.

RpfG is a member of a class of wide spread bacterial two-component regulators with an HD-GYP cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris, RpfG together with the sensor kinase RpfC regulates multiple factors as a response to the cell-to-cell Diffusible Signalling Factor (DSF). A dynamic physical interaction of RpfG with two diguanylate cyclase (GGDEF) domain proteins controls motility. Here we show that, contrary to expectation, regulation of motility by the GGDEF domain proteins does not depend upon their cyclic di-GMP synthetic activity. Furthermore we show that the complex of RpfG and GGDEF domain proteins recruits a specific PilZ domain 'adaptor' protein, and this complex then interacts with the pilus motor proteins PilU and PiIT. The results support a model in which DSF signalling influences motility through the highly regulated dynamic interaction of proteins that affect pilus action. A specific motif that we identify to be required for HD-GYP domain interaction is conserved in a number of GGDEF domain proteins, suggesting that regulation via interdomain interactions is of broad relevance.

Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris.

RpfG is a paradigm for a class of widespread bacterial two-component regulators with a CheY-like receiver domain attached to a histidine-aspartic acid-glycine-tyrosine-proline (HD-GYP) cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc), a two-component system comprising RpfG and the complex sensor kinase RpfC is implicated in sensing and responding to the diffusible signaling factor (DSF), which is essential for cell-cell signaling. RpfF is involved in synthesizing DSF, and mutations of rpfF, rpfG, or rpfC lead to a coordinate reduction in the synthesis of virulence factors such as extracellular enzymes, biofilm structure, and motility. Using yeast two-hybrid analysis and fluorescence resonance energy transfer experiments in Xcc, we show that the physical interaction of RpfG with two proteins with diguanylate cyclase (GGDEF) domains controls a subset of RpfG-regulated virulence functions. RpfG interactions were abolished by alanine substitutions of the three residues of the conserved GYP motif in the HD-GYP domain. Changing the GYP motif or deletion of the two GGDEF-domain proteins reduced Xcc motility but not the synthesis of extracellular enzymes or biofilm formation. RpfG-GGDEF interactions are dynamic and depend on DSF signaling, being reduced in the rpfF mutant but restored by DSF addition. The results are consistent with a model in which DSF signal transduction controlling motility depends on a highly regulated, dynamic interaction of proteins that influence the localized expression of cyclic di-GMP.

ROCK STUDY in CF: sustained anti-inflammatory effects of lumacaftor-ivacaftor in sputum and peripheral blood samples of adult patients with cystic fibrosis-an observational study.

BACKGROUND: Previous studies showed that the combination of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) corrector and potentiator, lumacaftor-ivacaftor (LUMA-IVA) provides meaningful clinical benefits in patients with cystic fibrosis who are homozygous for the Phe508del CFTR mutation. However, little is known about the effect of LUMA-IVA on Proinflammatory Cytokines (PICs). OBJECTIVES: To investigate the impact of LUMA-IVA CFTR modulation on circulatory and airway cytokines before and after 12 months of LUMA-IVA treatment in a real-world setting. METHODS: We assessed both plasma and sputum PICs, as well as standard clinical outcomes including Forced Expiratory Volume in one second (FEV1) %predicted, Body Mass Index (BMI), sweat chloride and pulmonary exacerbations at baseline and prospectively for one year post commencement of LUMA-IVA in 44 patients with cystic fibrosis aged 16 years and older homozygous for the Phe508del CFTR mutation. RESULTS: Significant reduction in plasma cytokines including interleukin (IL)-8 (p<0.05), tumour necrosis factor (TNF)-α (p<0.001), IL-1ß (p<0.001) levels were observed while plasma IL-6 showed no significant change (p=0.599) post-LUMA-IVA therapy. Significant reduction in sputum IL-6 (p<0.05), IL-8 (p<0.01), IL-1ß (p<0.001) and TNF-α (p<0.001) levels were observed after LUMA-IVA therapy. No significant change was noted in anti-inflammatory cytokine IL-10 levels in both plasma and sputum (p=0.305) and (p=0.585) respectively. Clinically significant improvements in FEV1 %predicted (mean+3.38%, p=0.002), BMI (mean+0.8 kg/m2, p<0.001), sweat chloride (mean -19 mmol/L, p<0.001), as well as reduction in intravenous antibiotics usage (mean -0.73, p<0.001) and hospitalisation (mean -0.38, p=0.002) were observed after initiation of LUMA-IVA therapy. CONCLUSION: This real-world study demonstrates that LUMA-IVA has significant and sustained beneficial effects on both circulatory and airway inflammation. Our findings suggest that LUMA-IVA may improve inflammatory responses, which could potentially contribute to improved standard clinical outcomes.

The Ax21 protein is a cell-cell signal that regulates virulence in the nosocomial pathogen Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia encodes proteins related to the Rax proteins of Xanthomonas oryzae, which are required for the synthesis and secretion of the Ax21 protein. Here we show that Ax21 acts as a cell-cell signal to regulate a diverse range of functions, including virulence, in this nosocomial pathogen.

A sensor kinase recognizing the cell-cell signal BDSF (cis-2-dodecenoic acid) regulates virulence in Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic human pathogen that uses cis-2-dodecenoic acid (BDSF) as a quorum-sensing signal to control expression of virulence factors. BDSF is a signal molecule of the diffusible signal factor (DSF) family that was first described in the plant pathogen Xanthomonas campestris. The mechanism of perception of this signal and the range of functions regulated in B. cenocepacia are, however, unknown. A screen for transposon mutants unable to respond to exogenous signal identified BCAM0227 as a potential BDSF sensor. BCAM0227 is a histidine sensor kinase with an input domain unrelated to that of RpfC, the DSF sensor found in xanthomonads. Transcriptome profiling established the scope of the BDSF regulon and demonstrated that the sensor controls expression of a subset of these genes. A chimeric sensor kinase in which the input domain of BCAM0227 replaced the input domain of RpfC was active in BDSF signal perception when expressed in X. campestris. Mutation of BCAM0227 gave rise to reduced cytotoxicity to Chinese hamster ovary cells and reduced virulence to Wax moth larvae and in the agar-bead mouse model of pulmonary infection. The findings identify BCAM0227 as a novel BDSF sensor and a potential target for interference in virulence-related signalling in B. cenocepacia.

Extended-culture and culture-independent molecular analysis of the airway microbiota in cystic fibrosis following CFTR modulation with ivacaftor.

BACKGROUND: Treatment with Ivacaftor provides a significant clinical benefit in people with cystic fibrosis (PWCF) with the class III G551D-CFTR mutation. This study determined the effect of CFTR modulation with ivacaftor on the lung microbiota in PWCF. METHODS: Using both extended-culture and culture-independent molecular methods, we analysed the lower airway microbiota of 14 PWCF, prior to commencing ivacaftor treatment and at the last available visit within the following year. We determined total bacterial and Pseudomonas aeruginosa densities by both culture and qPCR, assessed ecological parameters and community structure and compared these with biomarkers of inflammation and clinical outcomes. RESULTS: Significant improvement in FEV1, BMI, sweat chloride and levels of circulating inflammatory biomarkers were observed POST-ivacaftor treatment. Extended-culture demonstrated a higher density of strict anaerobic bacteria (p = 0.024), richness (p = 1.59*10-4) and diversity (p = 0.003) POST-treatment. No significant difference in fold change was observed by qPCR for either total bacterial 16S rRNA copy number or P. aeruginosa density for oprL copy number with treatment. Culture-independent (MiSeq) analysis revealed a significant increase in richness (p = 0.03) and a trend towards increased diversity (p = 0.07). Moreover, improvement in lung function, richness and diversity displayed an inverse correlation with the main markers of inflammation (p < 0.05). CONCLUSIONS: Following treatment with ivacaftor, significant improvements in clinical parameters were seen. Despite modest changes in overall microbial community composition, there was a shift towards a bacterial ecology associated with less severe CF lung disease. Furthermore, a significant correlation was observed between richness and diversity and levels of circulating inflammatory markers.

Intraspecies signaling involving the diffusible signal factor BDSF (cis-2-dodecenoic acid) influences virulence in Burkholderia cenocepacia.

Burkholderia cenocepacia produces a diffusible fatty acid signal molecule, cis-2-dodecenoic acid (BDSF), that has been implicated in interspecies and interkingdom communication. Here, we show that BDSF also acts as an intraspecies signal in B. cenocepacia to control factors contributing to virulence of this major opportunistic pathogen.