A dual role of extracellular DNA during biofilm formation of Neisseria meningitidis

Major pathogenic clonal complexes (cc) of Neisseria meningitidis differ substantially in their point prevalence among healthy carriers. We show that frequently carried pathogenic cc (e.g. sequence type ST‐41/44 cc and ST‐32 cc) depend on extracellular DNA (eDNA) to initiate in vitro biofilm formation, whereas biofilm formation of cc with low point prevalence (ST‐8 cc and ST‐11 cc) was eDNA‐independent. For initial biofilm formation, a ST‐32 cc type strain, but not a ST‐11 type strain, utilized eDNA. The release of eDNA was mediated by lytic transglycosylase and cytoplasmic N‐acetylmuramyl‐l ‐alanine amidase genes. In late biofilms, outer membrane phospholipase A‐dependent autolysis, which was observed in most cc, but not in ST‐8 and ST‐11 strains, was required for shear force resistance of microcolonies. Taken together, N. meningitidis evolved two different biofilm formation strategies, an eDNA‐dependent one yielding shear force resistant microcolonies, and an eDNA‐independent one. Based on the experimental findings and previous epidemiological observations, we hypothesize that most meningococcal cc display a settler phenotype, which is eDNA‐dependent and results in a stable interaction with the host. On the contrary, spreaders (ST‐11 and ST‐8 cc) are unable to use eDNA for biofilm formation and might compensate for poor colonization properties by high transmission rates.     

Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in a novel human model of the blood-cerebrospinal fluid barrier

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.     

Inhibition of cyclic amp phosphodiesterase by 5'-deoxy-5'-S-isobutylthioadenosine at biologically active concentrations of drug

5'-Deoxy-5'-S-isobutylthioadenosine (SIBA), a synthetic analogue of S-adenosylhomocysteine, has been reported by others to inhibit a number of biological processes and these effects of SIBA have been attributed generally to inhibition of methyltransferases. However, the present studies with mouse lymphocytes show that SIBA also acts as a competitive inhibitor (K_i = 130 μM) of the high-affinity cyclic AMP phosphodiesterase and potentiates the cyclic AMP response of intact cells to several activators of adenylate cyclase. Moreover, SIBA has been found to inhibit lymphocyte-mediated cytolysis, a cellular function known to be sensitive to elevated lymphocyte levels of cyclic AMP, at concentrations (IC₅₀ = 250 μM) similar to those which inhibit cyclic AMP phosphodiesterase. These results indicate the need for caution in attributing biological effects of SIBA singularly to inhibition of methyltransferases and suggest the possible agency of cyclic AMP in the mechanism of SIBA action.     

Meningococcal biofilm formation: structure, development and phenotypes in a standardized continuous flow system

We show that in a standardized in vitro flow system unencapsulated variants of genetically diverse lineages of Neisseria meningitidis formed biofilms, that could be maintained for more than 96 h. Biofilm cells were resistant to penicillin, but not to rifampin or ciprofloxacin. For some strains, microcolony formation within biofilms was observed. Microcolony formation in strain MC58 depended on a functional copy of the pilE gene encoding the pilus subunit pilin, and was associated with twitching of cells. Nevertheless, unpiliated pilE mutants formed biofilms showing that attachment and accumulation of cells did not depend on pilus expression. Mutation and complementation analysis revealed that the type IV pilus-associated protein PilX, which was recently shown to mediate interbacterial aggregation, indirectly supported microcolony formation by contributing to pilus expression. A large number of PilX alleles was identified among genetically diverse meningococcal strains. PilX alleles differed in their propensity to support autoaggregation of cells in suspension, but not in their ability to support microcolony formation within biofilms in the continuous flow system.     

GABA(A) receptor function is regulated by lipid bilayer elasticity

Docosahexaenoic acid (DHA) and other polyunsaturated fatty acids (PUFAs) promote GABA(A) receptor [(3)H]-muscimol binding, and DHA increases the rate of GABA(A) receptor desensitization. Triton X-100, a structurally unrelated amphiphile, similarly promotes [(3)H]-muscimol binding. The mechanism(s) underlying these effects are poorly understood. DHA and Triton X-100, at concentrations that affect GABA(A) receptor function, increase the elasticity of lipid bilayers measured as decreased bilayer stiffness using gramicidin channels as molecular force transducers. We have previously shown that membrane protein function can be regulated by amphiphile-induced changes in bilayer elasticity and hypothesized that GABA(A) receptors could be similarly regulated. We therefore studied the effects of four structurally unrelated amphiphiles that decrease bilayer stiffness (Triton X-100, octyl-beta-glucoside, capsaicin, and DHA) on GABA(A) receptor function in mammalian cells. All the compounds promoted GABA(A) receptor [(3)H]-muscimol binding by increasing the binding capacity of high-affinity binding without affecting the associated equilibrium binding constant. A semiquantitative analysis found a similar quantitative relation between the effects on bilayer stiffness and [(3)H]-muscimol binding. Membrane cholesterol depletion, which also decreases bilayer stiffness, similarly promoted [(3)H]-muscimol binding. In whole-cell voltage-clamp experiments, Triton X-100, octyl-beta-glucoside, capsaicin, and DHA all reduced the peak amplitude of the GABA-induced currents and increased the rate of receptor desensitization. The effects of the amphiphiles did not correlate with the expected changes in monolayer spontaneous curvature. We conclude that GABA(A) receptor function is regulated by lipid bilayer elasticity. PUFAs may generally regulate membrane protein function by affecting the elasticity of the host lipid bilayer.     

Interaction between nitric oxide and ethylene in the induction of alternative oxidase in ozone-treated tobacco plants

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain, an alternative pathway that terminates with a single homodimeric protein, the alternative oxidase (AOX). We recorded temporary inhibition of cytochrome capacity respiration and activation of AOX pathway capacity in tobacco plants (Nicotiana tabacum L. cv BelW3) fumigated with ozone (O(3)). The AOX1a gene was used as a molecular probe to investigate its regulation by signal molecules such as hydrogen peroxide, nitric oxide (NO), ethylene (ET), salicylic acid, and jasmonic acid (JA), all of them reported to be involved in the O(3) response. Fumigation leads to accumulation of hydrogen peroxide in mitochondria and early accumulation of NO in leaf tissues. Although ET accumulation was high in leaf tissues 5 h after the start of O(3) fumigation, it declined during the recovery period. There were no differences in the JA and 12-oxo-phytodienoic acid levels of treated and untreated plants. NO, JA, and ET induced AOX1a mRNA accumulation. Using pharmacological inhibition of ET and NO, we demonstrate that both NO- and ET-dependent pathways are required for O(3)-induced up-regulation of AOX1a. However, only NO is indispensable for the activation of AOX1a gene expression.     

Heregulin-induced epigenetic regulation of the utrophin-A promoter

Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin is of therapeutic interest since its over-expression can compensate dystrophin's absence. Utrophin is enriched at neuromuscular junctions due to heregulin-mediated utrophin-A promoter activation. We demonstrate that heregulin activated MSK1/2 and phosphorylated histone H3 at serine 10 in cultured C2C12 muscle cells, in an ERK-dependent manner. MSK1/2 inhibition suppressed heregulin-mediated utrophin-A activation. MSK1 over-expression potentiated heregulin-mediated utrophin-A activation and chromatin remodeling at the utrophin-A promoter. These results identify MSK1/2 as key effectors modulating utrophin-A expression as well as identify novel targets for DMD therapy.     

The look-ahead effect of phenotypic mutations

Background  

The evolution of complex molecular traits such as disulphide bridges often requires multiple mutations. The intermediate steps in such evolutionary trajectories are likely to be selectively neutral or deleterious. Therefore, large populations and long times may be required to evolve such traits.