Extracellular matrix formation enhances the ability of streptococcus pneumoniae to cause invasive disease

Extent: 17p.During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100- fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease.Claudia Trappetti, Abiodun D. Ogunniyi, Marco R. Oggioni and James C. Pato

Isoprenaline can activate the acetylcholine-induced K+ current in canine atrial myocytes via Gs-derived βγ subunits

G protein βγ subunits activate the acetylcholine-induced potassium current Ik,ach. There is no evidence of specificity at the level of the βγ subunits. Therefore all G protein-coupled receptors in atrial myocytes should be able to activate Ik,ach. Paradoxically, it is often stated that isoprenaline does not activate Ik,ach. Rationales to explain this negative result include insufficient concentrations of Gs in the atrium or restricted access of Gs-derived βγ subunits to the Ik,ach channel. We took advantage of a non-specific increase in Gs that results after infection with adenovirus.Adenoviral infection unmasked a 1 μm isoprenaline-induced Ik,ach which was prevented by propranolol. Isoprenaline occasionally activated Ik,ach in uninfected and freshly dissociated atrial myocytes but the effect was larger and more consistent in infected myocytes.Pertussis toxin pretreatment (100 ng ml−1 overnight) did not block the effect of isoprenaline. The effect of isoprenaline became persistent if cells were pretreated with cholera toxin (200 ng nl−1).Signal transduction events distal to adenylyl cyclase were not involved in isoprenaline-induced Ik,ach. Forskolin (10 μm) did not activate Ik,ach. Inhibition of adenylyl cyclase with cytoplasmic application of 300 μm 2′-deoxyadenosine 3′-monophosphate did not prevent the activation of Ik,ach by isoprenaline.Cytoplasmic application of a βγ binding peptide derived from the C terminus of β-adrenergic receptor kinase 1 (50 μm) prevented the effect of isoprenaline on Ik,ach. The peptide did not prevent the stimulation of the L-type calcium current by isoprenaline.The results indicate that β-adrenoceptors can activate Ik,ach in atrial myocytes through the release of βγ subunits from Gs

Upregulation of phospholipase d expression and activation in ventricular pressure-overload hypertrophy

Evidence for a role of phospholipase D (PLD) in cellular proliferation and differentiation is accumulating. We studied PLD activity and expression in normal and hypertrophic rat and human hearts. In rat heart, abdominal aortic banding (constriction to 50% of original lumen) caused hypertrophy in the left ventricle (as shown by weight index and ANP expression) by about 15% after 30 days without histological evidence of fibrosis or signs of decompensation and in the right ventricle after 100 days. The hypertrophy was accompanied by small increases of basal PLD activity and strong potentiation of stimulated PLD activity caused by 4beta-phorbol-12beta,13alpha-dibutyrate (PDB) and by phenylephrine. The mRNA expressions of both PLD1 and PLD2 determined by semiquantitative competitive RT-PCR were markedly enhanced after aortic banding. In the caveolar fraction of the rat heart, PLD2 protein determined by Western blot analysis was upregulated in parallel with the expression of caveolin-3. A similar induction of PLD mRNA and protein expression was observed in hypertrophied human hearts of individuals (39-45-year-old) who had died from non-cardiac causes. In conclusion, PLD1 and PLD2 expressions were strongly enhanced both in rat and human heart hypertrophy, which may be responsible for the coincident potentiation of the PLD activation by alpha-adrenoceptor and protein kinase C stimulation. These results are compatible with a significant role of PLD activation in cell signaling of ventricular pressure-overload hypertroph