Chemical Analysis of Cellular and Extracellular Carbohydrates of a Biofilm-Forming Strain Pseudomonas aeruginosa PA14

Background: Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A\ufffdL) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the \ufffd\ufffdscaffolding\ufffd\ufffd polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A\ufffdL biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated. Principal Findings: In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanyi type O:2a,c (Lanyi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-a-L-GalNAcA-(1\ufffd3)-a-D-QuiNAc-(1\ufffd3)- a-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic b-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ,20% of dry weight) of LPS-like material. Conclusions: We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is an abundant extracellular carbohydrate of PA14. We present evidence that LPS-like material is found as a component of a biofilm matrix of P. aeruginosa.Peer reviewed: YesNRC publication: Ye

Hydrodilatation, corticosteroids and adhesive capsulitis: A randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Hydrodilatation of the glenohumeral joint is by several authors reported to improve shoulder pain and range of motion for patients with adhesive capsulitis. Procedures described often involve the injection of corticosteroids, to which the reported treatment effects may be attributed. Any important contribution arising from the hydrodilatation procedure itself remains to be demonstrated.</p> <p>Methods</p> <p>In this randomized trial, a hydrodilatation procedure including corticosteroids was compared with the injection of corticosteroids without dilatation. Patients were given three injections with two-week intervals, and all injections were given under fluoroscopic guidance. Outcome measures were the Shoulder Pain and Disability Index (SPADI) and measures of active and passive range of motion. Seventy-six patients were included and groups were compared six weeks after treatment. The study was designed as an open trial.</p> <p>Results</p> <p>The groups showed a rather similar degree of improvement from baseline. According to a multiple regression analysis, the effect of dilatation was a mean improvement of 3 points (confidence interval: -5 to 11) on the SPADI 0–100 scale. T-tests did not demonstrate any significant between-group differences in range of motion.</p> <p>Conclusion</p> <p>This study did not identify any important treatment effects resulting from three hydrodilatations that included steroid compared with three steroid injections alone.</p> <p>Trial registration</p> <p>The study is registered in Current Controlled Trials with the registration number ISRCTN90567697.</p

RNA Modulators of Complex Phenotypes in Mammalian Cells

RNA-mediated gene silencing, in the form of RNA interference (RNAi) or microRNAs (miRNAs) has provided novel tools for gene discovery and validation in mammalian cells. Here, we report on the construction and application of a random small RNA expression library for use in identifying small interfering RNA (siRNA) effectors that can modify complex cellular phenotypes in mammalian cells. The library is based in a retroviral vector and uses convergent promoters to produce unique small complementary RNAs. Using this library, we identify a range of small RNA-encoding gene inserts that overcome resistance to 5-fluorouracil (5-FU)- or tumour necrosis factor alpha (TNF-α)- induced cell death in colorectal cancer cells. We demonstrate the utility of this technology platform by identifying a key RNA effector, in the form of a siRNA, which overcomes cell death induced by the chemotherapeutic 5-FU. The technology described has the potential to identify both functional RNA modulators capable of altering physiological systems and the cellular target genes altered by these modulators

Protein Phosphatase-1α Interacts with and Dephosphorylates Polycystin-1

Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme

Multiple FadD Acyl-CoA Synthetases Contribute to Differential Fatty Acid Degradation and Virulence in Pseudomonas aeruginosa

A close interconnection between nutrient metabolism and virulence factor expression contributes to the pathophysiology of Pseudomonas aeruginosa as a successful pathogen. P. aeruginosa fatty acid (FA) degradation is complicated with multiple acyl-CoA synthetase homologs (FadDs) expressed in vivo in lung tissue during cystic fibrosis infections. The promoters of two genetically linked P. aeruginosa fadD genes (fadD1 and fadD2) were mapped and northern blot analysis indicated they could exist on two different transcripts. These FadDs contain ATP/AMP signature and FA-binding motifs highly homologous to those of the Escherichia coli FadD. Upon introduction into an E. coli fadD-/fadR- double mutant, both P. aeruginosa fadDs functionally complemented the E. coli fadD-/fadR- mutant, allowing degradation of different chain-length FAs. Chromosomal mutagenesis, growth analysis, induction studies, and determination of kinetic parameters suggested that FadD1 has a substrate preference for long-chain FAs while FadD2 prefers shorter-chain FAs. When compared to the wild type strain, the fadD2 mutant exhibited decreased production of lipase, protease, rhamnolipid and phospholipase, and retardation of both swimming and swarming motilities. Interestingly, fadD1 mutant showed only increased swarming motility. Growth analysis of the fadD mutants showed noticeable deficiencies in utilizing FAs and phosphatidylcholine (major components of lung surfactant) as the sole carbon source. This defect translated into decreased in vivo fitness of P. aeruginosa in a BALB/c mouse lung infection model, supporting the role of lipids as a significant nutrient source for this bacterium in vivo

Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1)

International audienceRif1 is a conserved protein that plays essential roles in orchestrating DNA replication timing, controlling nuclear architecture, telomere length and DNA repair. However, the relationship between these different roles, as well as the molecular basis of Rif1 function is still unclear. The association of Rif1 with insoluble nuclear lamina has thus far hampered exhaustive characterization of the associated protein complexes. We devised a protocol that overcomes this problem, and were thus able to discover a number of novel Rif1 interactors, involved in chromatin metabolism and phosphorylation. Among them, we focus here on PP1. Data from different systems have suggested that Rif1-PP1 interaction is conserved and has important biological roles. Using mutagenesis, NMR, isothermal calorimetry and surface plasmon resonance we demonstrate that Rif1 is a high-affinity PP1 adaptor, able to out-compete the well-established PP1-inhibitor I2 in vitro. Our conclusions have important implications for understanding Rif1 diverse roles and the relationship between the biological processes controlled by Rif1

Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion

Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal c-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here we identified an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase WarB. We established that WarA binds to c-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bi-functional Escherichia coli LPS O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.BBSRC & Wellcome Trus