Brg1/PRMT5 nuclear complex epigenetically regulates FOXO1 in IPF mesenchymal progenitor cells.

We have discovered intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the human idiopathic pulmonary fibrosis (IPF) lung. IPF MPCs display a durably distinct transcriptome, suggesting that they have undergone epigenetic modifications. Prior studies indicate that the chromatin remodeler Brg1 associates with the arginine methyltransferase PRMT5 to epigenetically regulate transcription factors. We hypothesize that a Brg1/PRMT5 nuclear complex epigenetically regulates critical nodes in IPF MPC self-renewal signaling networks. IPF and control MPCs were isolated from primary mesenchymal cell lines established from IPF and control patients. RNA-sequencing identified increased expression of the FOXO1 transcription factor in IPF MPCs compared with controls, a result we confirmed by Q-PCR and Western blot analysis. Immunoprecipitation identified a CD44/Brg1/PRMT5 nuclear complex in IPF MPCs. Chromatin immunoprecipitation assays showed that PRMT5 and its methylation mark H3R2me2 are enriched on the FOXO1 promoter. We show that loss of Brg1 and PRMT5 function decreases FOXO1 expression and impairs IPF MPC self-renewal, and that loss of FOXO1 function decreases IPF MPC self-renewal and expression of the SOX2 and OCT4 stemness markers. Our findings indicate that the FOXO1 gene is overexpressed in IPF MPCs in a CD44/Brg1/PRMT5 nuclear complex-dependent manner. Our data suggest that Brg1 alters chromatin accessibility, enriching PRMT5 occupancy on the FOXO1 promoter, and PRMT5 methylates histone H3 arginine 2 (H3R2) on the FOXO1 promoter, increasing its expression. Our data are in accord with the concept that this coordinated interplay is responsible for promoting IPF MPC self-renewal and maintaining a critical pool of fibrogenic MPCs that drive IPF progression.NEW & NOTEWORTHY Our research offers valuable understanding regarding the epigenetic control of IPF MPC. The data we obtained strongly support the idea that the coordination between chromatin remodeling and histone methylation plays a key role in regulating transcription factors. Specifically, our findings indicate that FOXO1, an essential transcription factor, likely governs the self-renewal of IPF MPC, which is crucial for maintaining a critical pool of fibrogenic MPCs. This interplay could be an important therapeutic target.

Evaluation of Expanded 2-Aminobenzothiazole Library for Inhibition of Pseudomonas aeruginosa Virulence Phenotypes.

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. The high sequence conservation in the catalytic and adenosine triphosphate-binding (CA) domain of histidine kinases and their essential role in bacterial signal transduction could enable broad-spectrum antibacterial activity. Through this signal transduction, histidine kinases regulate multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the CA domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain of histidine kinases. We found these compounds have anti-virulence activities in Pseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

Agmatine accumulation by Pseudomonas aeruginosa clinical isolates confers antibiotic tolerance and dampens host inflammation.

PURPOSE: In the cystic fibrosis (CF) airways, Pseudomonas aeruginosa undergoes diverse physiological changes in response to inflammation, antibiotic pressure, oxidative stress and a dynamic bioavailable nutrient pool. These include loss-of-function mutations that result in reduced virulence, altered metabolism and other phenotypes that are thought to confer a selective advantage for long-term persistence. Recently, clinical isolates of P. aeruginosa that hyperproduce agmatine (decarboxylated arginine) were cultured from individuals with CF. Sputum concentrations of this metabolite were also shown to correlate with disease severity. This raised the question of whether agmatine accumulation might also confer a selective advantage for P. aeruginosa during chronic colonization of the lung. METHODOLOGY AND RESULTS: We screened a library of P. aeruginosa CF clinical isolates and found that ~5 % of subjects harboured isolates with an agmatine hyperproducing phenotype. Agmatine accumulation was a direct result of mutations in aguA, encoding the arginine deiminase that catalyses the conversion of agmatine into various polyamines. We also found that agmatine hyperproducing isolates (aguA-) had increased tolerance to the cationic antibiotics gentamicin, tobramycin and colistin relative to their chromosomally complemented strains (aguA+). Finally, we revealed that agmatine diminishes IL-8 production by airway epithelial cells in response to bacterial infection, with a consequent decrease in neutrophil recruitment to the murine airways in an acute pneumonia model. CONCLUSION: These data highlight a potential new role for bacterial-derived agmatine that may have important consequences for the long-term persistence of P. aeruginosa in the CF airways.

Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa.

A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled (13)C5,(15)N4-agmatine (synthesized by decarboxylation of uniformly labeled (13)C6,(15)N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1% (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 µM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients.

The arginine decarboxylase pathways of host and pathogen interact to impact inflammatory pathways in the lung.

The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.