Identification of a novel anti-σ<Superscript>E</Superscript> factor in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>

Background  

Fine tuning expression of genes is a prerequisite for the strictly human pathogen Neisseria meningitidis to survive hostile growth conditions and establish disease. Many bacterial species respond to stress by using alternative σ factors which, in complex with RNA polymerase holoenzyme, recognize specific promoter determinants. σ^E, encoded by rpoE (NMB2144) in meningococci, is known to be essential in mounting responses to environmental challenges in many pathogens. Here we identified genes belonging to the σ^E regulon of meningococci.     

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.     

Outer-Inner Membrane Vesicles Naturally Secreted by Gram-Negative Pathogenic Bacteria

Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles.     

[Ni(L)(MeCN)]complex cation as a template for the assembly of extended I3 · I5 and I5 · I7 polyiodide networks {L=2,5,8-trithia[9](2,9)-1,10-phenanthrolinophane}. Synthesis and structures of [Ni(L)(MeCN)]I8 and [Ni(L)(MeCN)]I12

The compounds [Ni(L)(MeCN)]I₈ (1) and [Ni(L)(MeCN)]I₁₂ (2) have been obtained from the reactions of the complexes [Ni(L)(L^′)][BF₄]_(2 + n) {L=2,5,8-trithia[9](2,9)-1,10-phenanthrolinophane; L^′=MeCN, Cl⁻, Br⁻, I⁻; n=charge of L^′} with an excess of I₂ (molar ratios of 6, 10 and 20 have been used), in the presence of the stoichiometric amount of I⁻ (as Bu₄ⁿNI) necessary to balance the charge of the complex cation [Ni(L)(L^′)]^(2 + n)+. An X-ray diffraction analysis shows that, independently of the nature of L^′, both 1 and 2 contain the complex cation [Ni(L)(MeCN)]²⁺, which is therefore capable of templating two different polyiodide networks based on interacting I₃⁻/I₅⁻ and I₅⁻/I₇ units, respectively. The solid state FT-Raman spectra of 1 and 2 are discussed based on their structural features.     

Thiodisaccharides with galactofuranose or arabinofuranose as terminal units: Synthesis and inhibitory activity of an exo β-d-galactofuranosidase

Thiodisaccharides having β-d-Galf or α-l-Araf units as non-reducing end have been synthesized by the SnCl₄- or MoO₂Cl₂-promoted thioglycosylation of per-O-benzoyl-d-galactofuranose (1), its 1-O-acetyl analogue 4, or per-O-acetyl-α-l-arabinofuranose (16) with 6-thioglucose or 6-thiogalactose derivatives. After convenient removal of the protecting groups, the free thiodisaccharides having the basic structure β-d-Galf(1→6)-6-thio-α-d-Glcp-OMe (5) or β-d-Galf(1→6)-6-thio-α-d-Galp-OMe (15) were obtained. The respective α-l-Araf analogues 18 and 20 were prepared similarly from 16. Alternatively, β-d-Galf(1→4)-4-thio-3-deoxy-α-l-Xylp-OiPr was synthesized by Michael addition to a sugar enone of 1-thio-β-d-Galf derivative, generated in situ from the glycosyl isothiourea derivative of 1. The free S-linked disaccharides were evaluated as inhibitors of the β-galactofuranosidase from Penicillium fellutanum, being 15 and 20 the more active inhibitors against this enzyme.     

Altered insulin receptor signalling and β-cell cycle dynamics in type 2 diabetes mellitus

Insulin resistance, reduced β-cell mass, and hyperglucagonemia are consistent features in type 2 diabetes mellitus (T2DM). We used pancreas and islets from humans with T2DM to examine the regulation of insulin signaling and cell-cycle control of islet cells. We observed reduced β-cell mass and increased α-cell mass in the Type 2 diabetic pancreas. Confocal microscopy, real-time PCR and western blotting analyses revealed increased expression of PCNA and down-regulation of p27-Kip1 and altered expression of insulin receptors, insulin receptor substrate-2 and phosphorylated BAD. To investigate the mechanisms underlying these findings, we examined a mouse model of insulin resistance in β-cells--which also exhibits reduced β-cell mass, the β-cell-specific insulin receptor knockout (βIRKO). Freshly isolated islets and β-cell lines derived from βIRKO mice exhibited poor cell-cycle progression, nuclear restriction of FoxO1 and reduced expression of cell-cycle proteins favoring growth arrest. Re-expression of insulin receptors in βIRKO β-cells reversed the defects and promoted cell cycle progression and proliferation implying a role for insulin-signaling in β-cell growth. These data provide evidence that human β- and α-cells can enter the cell-cycle, but proliferation of β-cells in T2DM fails due to G1-to-S phase arrest secondary to defective insulin signaling. Activation of insulin signaling, FoxO1 and proteins in β-cell-cycle progression are attractive therapeutic targets to enhance β-cell regeneration in the treatment of T2DM.     

Spectroscopic and kinetic analyses reveal the pyridoxal 5'-phosphate binding mode and the catalytic features of Treponema denticola cystalysin

To obtain insight into the functional properties of Treponema denticola cystalysin, we have analyzed the pH- and ligand-induced spectral transitions, the pH dependence of the kinetic parameters, and the substrate specificity of the purified enzyme. The absorption spectrum of cystalysin has maxima at 418 and 320 nm. The 320 nm band increases at high pH, while the 418 nm band decreases; the apparent pK(spec) of this spectral transition is about 8.4. Cystalysin emitted fluorescence at 367 and 504 nm upon excitation at 320 and 418 nm, respectively. The pH profile for the 367 nm emission intensity increases above a single pK of approximately 8.4. On this basis, the 418 and 320 nm absorbances have been attributed to the ketoenamine and substituted aldamine, respectively. The pH dependence of both log k(cat) and log k(cat)/K(m) for alpha,beta-elimination reaction indicates that a single ionizing group with a pK value of approximately 6.6 must be unprotonated to achieve maximum velocity. This implies that cystalysin is more catalytically competent in alkaline solution where a remarkable portion of its coenzyme exists as inactive aldamine structure. Binding of substrates or substrate analogues to the enzyme over the pH range 6-9.5 converts both the 418 and 320 nm bands into an absorbing band at 429 nm, assigned to the external aldimine in the ketoenamine form. All these data suggest that the equilibrium from the inactive aldamine form of the coenzyme shifts to the active ketoenamine form on substrate binding. In addition, reinvestigation of the substrate spectrum of alpha,beta-elimination indicates that cystalysin is a cyst(e)ine C-S lyase rather than a cysteine desulfhydrase as claimed previously.     

Effects of brain ischemia on intermediate filaments of rat hippocampus

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [¹⁴Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of³²P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.     

Nutrient recycling by insect and fish communities in high-elevation tropical streams

Hydrobiologia - High- to mid-elevation streams are often oligotrophic, but harbor diverse groups of aquatic animals that can satisfy a substantial proportion of nutrient demand. Therefore, we...