Reversal of TGF-β1 stimulation of α-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's - derived fibroblasts

<p>Abstract</p> <p>Background</p> <p>Myofibroblasts, a derived subset of fibroblasts especially important in scar formation and wound contraction, have been found at elevated levels in affected Dupuytren's tissues. Transformation of fibroblasts to myofibroblasts is characterized by expression of alpha- smooth muscle actin (α-SMA) and increased production of extracellular matrix (ECM) components, both events of relevance to connective tissue remodeling. We propose that increasing the activation of the cyclic AMP (cAMP)/protein kinase A signaling pathway will inhibit transforming growth factor-beta1 (TGF-β₁)-induced ECM synthesis and myofibroblast formation and may provide a means to blunt fibrosis.</p> <p>Methods</p> <p>Fibroblasts derived from areas of Dupuytren's contracture cord (DC), from adjacent and phenotypically normal palmar fascia (PF), and from palmar fascia from patients undergoing carpal tunnel release (CTR; CT) were treated with TGF-β₁(2 ng/ml) and/or forskolin (10 μM) (a known stimulator of cAMP). Total RNA and protein extracted was subjected to real time RT-PCR and Western blot analysis.</p> <p>Results</p> <p>The basal mRNA expression levels of fibronectin- extra domain A (FN1-EDA), type I (COL1A2) and type III collagen (COL3A1), and connective tissue growth factor (CTGF) were all significantly increased in DC- and in PF-derived cells compared to CT-derived fibroblasts. The TGF-β₁stimulation of α-SMA, CTGF, COL1A2 and COL3A1 was greatly inhibited by concomitant treatment with forskolin, especially in DC-derived cells. In contrast, TGF-β₁stimulation of FN1-EDA showed similar levels of reduction with the addition of forskolin in all three cell types.</p> <p>Conclusion</p> <p>In sum, increasing cAMP levels show potential to inhibit the formation of myofibroblasts and accumulation of ECM components. Molecular agents that increase cAMP may therefore prove useful in mitigating DC progression or recurrence.</p

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence.

BACKGROUND: The polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur. RESULTS: Here, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater. CONCLUSIONS: We identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential

Streptococcus pneumoniae in Biofilms Are Unable to Cause Invasive Disease Due to Altered Virulence Determinant Production

It is unclear whether Streptococcus pneumoniae in biofilms are virulent and contribute to development of invasive pneumococcal disease (IPD). Using electron microscopy we confirmed the development of mature pneumococcal biofilms in a continuous-flow-through line model and determined that biofilm formation occurred in discrete stages with mature biofilms composed primarily of dead pneumococci. Challenge of mice with equal colony forming units of biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Biofilm pneumococci of numerous serotypes were hyper-adhesive and bound to A549 type II pneumocytes and Detroit 562 pharyngeal epithelial cells at levels 2 to 11-fold greater than planktonic counterparts. Using genomic microarrays we examined the pneumococcal transcriptome and determined that during biofilm formation S. pneumoniae down-regulated genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide (CPS) production, and virulence. We confirmed these changes by measuring CPS by ELISA and immunoblotting for the toxin pneumolysin and the bacterial adhesins phosphorylcholine (ChoP), choline-binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP). We conclude that biofilm pneumococci were avirulent due to reduced CPS and pneumolysin production along with increased ChoP, which is known to bind C-reactive protein and is opsonizing. Likewise, biofilm pneumococci were hyper-adhesive due to selection for the transparent phase variant, reduced CPS, and enhanced production of PsrP, CbpA, and ChoP. These studies suggest that biofilms do not directly contribute to development of IPD and may instead confer a quiescent mode of growth during colonization

Pyruvate Oxidase Influences the Sugar Utilization Pattern and Capsule Production in Streptococcus pneumoniae:A Membrane Active Peptide Killing Bacterial Cells

<p>Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence, oxidative stress survival and death in stationary phase. Under semi-aerobic conditions, transcriptomic and metabolite profiling analysis of a spxB mutant grown on glucose showed minor changes compared to the wild type, apart from the significant induction of two operons involved in carbohydrate uptake and processing. This induction leads to a change in the sugar utilization capabilities of the bacterium, as indicated by the analysis of the growth profiles of the D39 parent and spxB mutant on alternative carbohydrates. Metabolic analysis and growth experiments showed that inactivation of SpxB has no effect on the glucose fermentation pattern, except under aerobic conditions. More importantly, we show that mutation of spxB results in the production of increased amounts of capsule, the major virulence factor of S. pneumoniae. Part of this increase can be attributed to induction of capsule operon (cps) transcription. Therefore, we propose that S. pneumoniae utilizes pyruvate oxidase as an indirect sensor of the oxygenation of the environment, resulting in the adaption of its nutritional capability and the amount of capsule to survive in the host.</p>