Oldrich Benada

Segmented filamentous bacteria in a defined bacterial cocktail induce intestinal inflammation in SCID mice reconstituted with CD45RBhigh CD4+ T cells

Background: The aim was to analyze the influence of intestinal microbiota on the development of intestinal inflammation. We used the model of chronic inflammation that develops spontaneously in the colon of conventional severe combined immunodeficiency (SCID) mice restored with the CD45 RBhigh subset of CD4+T cells isolated from the spleen of normal BALB/c mice. Methods: A CD4+CD45RBhigh subpopulation of T cells was purified from the spleen of conventional BALB/c mice by magnetic separation (MACS) and transferred into immunodeficient SCID mice. Germ‐free (GF) SCID mice or SCID mice monoassociated with Enterococcus faecalis, SFB (segmented filamentous bacteria), Fusobacterium mortiferum, Bacteroides distasonis, and in combination Fusobacterium mortiferum + SFB or Bacteroides distasonis + SFB were used as recipients. SCID mice were colonized by a defined cocktail of specific pathogen‐free (SPF) bacteria. Mice were evaluated 8–12 weeks after the cell transfer for clinical and morphological signs of inflammatory bowel disease (IBD). Results: After the transfer of the CD4+CD45RBhigh T‐cell subpopulation to SCID mice severe colitis was present in conventional animals and in mice colonized with a cocktail of SPF microflora plus SFB. Altered intestinal barrier in the terminal ileum of mice with severe colitis was documented by immunohistology using antibodies to ZO‐1 (zona occludens). Conclusions: Only SFB bacteria together with a defined SPF mixture were effective in triggering intestinal inflammation in the model of IBD in reconstituted SCID mice, while no colitis was detected in GF mice or in mice colonized either with SPF microflora or monoassociated only with SFB or colonized by Bacteroides distasonis + SFB or Fusobacterium mortiferum + SFB. (Inflamm Bowel Dis 2007)

Adenylate Cyclase Toxin Subverts Phagocyte Function by RhoA Inhibition and Unproductive Ruffling

Adenylate cyclase toxin (CyaA or ACT) is a key virulence factor of pathogenic Bordetellae. It penetrates phagocytes expressing the αMβ2 integrin (CD11b/CD18, Mac-1 or CR3) and paralyzes their bactericidal capacities by uncontrolled conversion of ATP into a key signaling molecule, cAMP. Using pull-down activity assays and transfections with mutant Rho family GTPases, we show that cAMP signaling of CyaA causes transient and selective inactivation of RhoA in mouse macrophages in the absence of detectable activation of Rac1, Rac2, or RhoG. This CyaA/cAMP-induced drop of RhoA activity yielded dephosphorylation of the actin filament severing protein cofilin and massive actin cytoskeleton rearrangements, which were paralleled by rapidly manifested macrophage ruffling and a rapid and unexpected loss of macropinocytic fluid phase uptake. As shown in this study for the first time, CyaA/cAMP signaling further caused a rapid and near-complete block of complement-mediated phagocytosis. Induction of unproductive membrane ruffling, hence, represents a novel sophisticated mechanism of down-modulation of bactericidal activities of macrophages and a new paradigm for action of bacterial toxins that hijack host cell signaling by manipulating cellular cAMP levels.

Stress-response sigma factor σΗ is essential for morphological differentiation of Streptomyces coelicolor A3(2)

Abstract. We previously cloned the sigH gene encoding a stress-response sigma factor σΗ in Streptomyces coelicolor A3(2), located in an operon with the gene encoding proposed anti-sigma factor UshX. To clarify the in vivo function of σΗ, a stable null mutant of sigH was prepared by homologous recombination. This mutation appeared to have no obvious effect on vegetative growth, but dramatically affected morphological differentiation. Microscopy showed that the sigH mutant produced undifferentiated hyphae with rare spore chains, giving the colony a pale gray color compared to the dark gray wild-type spores. The sigH mutation partially affected growth under conditions of high osmolarity. Expression of the sigH operon was investigated in the S. coelicolor sigH mutant. Out of four promoters directing expression of the sigH operon, the sigH-P2 promoter – the only promoter preferentially induced by salt-stress conditions – was inactive in the sigH mutant. The results indicated that the sigH-P2 promoter is dependent (directly or indirectly) upon σΗ and that the operon is autocatalytically activated. We propose that in S. coelicolor σΗ has a dual role, regulating the osmotic response and morphological differentiation.

Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin

The Bordetella adenylate cyclase‐hemolysin (CyaA, ACT, or AC‐Hly) is a multifunctional toxin. Simultaneously with promoting calcium ion entry, CyaA delivers into host cells an adenylate cyclase enzyme (AC) and permeabilizes cell membrane by forming small cation‐selective pores. Indirect evidence suggested that these two activities were accomplished by different membraneinserted CyaA conformers, one acting as an AC‐delivering monomer and the other as an uncharacterized poreforming oligomer. We tested this model by directly detecting toxin oligomers in cell membrane and by assessing oligomerization of specific mutants with altered poreforming properties. CyaA oligomers were revealed in sheep erythrocyte membranes by immunogold labeling and directly demonstrated by pulldown of membraneinserted CyaA together with biotinylated CyaAAC‐ toxoid. Membrane oligomers of CyaAcould also be resolved by nondenaturing electrophoresis of mild detergent extracts of erythrocytes. Furthermore, CyaA mutants exhibiting enhanced (E581K) or reduced (E570K+E581P) specific hemolytic and pore‐forming activity were found to exhibit also a correspondingly enhanced or reduced propensity to form oligomers in erythrocyte membranes. On the other hand, processed CyaA, with the AC domain cleaved off by erythrocyte proteases, was detected only in a monomeric form excluded from the oligomers of unprocessed CyaA. These results provide the first direct evidence that oligomerization is involved in formation of CyaApores in target membranes and that translocation of the AC domain across cell membrane may be accomplished by monomelic CyaA.—Vojtova‐Vodolanova, J., Basler, M., Osicka, R., Knapp, O., Maier, E., Cerny, J., Benada, O., Benz, R., Sebo, P. Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin. FASEB J. 23, 2831–2843 (2009). www.fasebj.org

Heterologous expression of full-length capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies

Abstract A capsid protein of porcine circovirus 2 (PCV 2) serves as a diagnostic antigen for the detection of PCV 2-associated disease known as a postweaning multisystemic wasting syndrome (PMWS). In this report, a bacterial expression system was developed for the expression and purification of the full-length PCV 2 capsid (Cap) protein from a codon-optimized cap gene. Replacement of rare arginine codons located at the 5′ end of the cap reading frame with codons optimal for E. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. The Cap protein was purified to greater than 95% homogeneity by using a single cation-exchange chromatography at a yield of 10mg per litre of bacterial culture. Despite the failure of the E. coli-expressed Cap protein to self-assemble into virus-like particles (VLPs), the immunization of mice with recombinant Cap yielded antibodies with the same specificity as those raised against native PCV 2 virions. In addition, the antigenic properties of the purified Cap protein were employed in a subunit-based indirect ELISA to monitor the levels of PCV 2 specific antibodies in piglets originating from a herd which was experiencing PCV 2 infection. These results pave the way for a straightforward large-scale production of the recombinant PCV 2 capsid protein and its use as a diagnostic antigen or a PCV 2 subunit vaccine.

β-Cyclodextrin-modified monolithic stationary phases for capillary electrochromatography and nano-HPLC chiral analysis of ephedrine and ibuprofen

ABSTRACT Chiral monolithic capillary columns for reversed-phase capillary electrochromatography (CEC) and for nano-HPLC were prepared by linking β-cyclodextrin modifier into the acrylic monolithic phase. Columns with the physically and chemically bonded β-cyclodextrin derivatives were tested under CEC and nano-HPLC conditions; enatioselective separation of (−)-ephedrine/(+)-pseudoephedrine and (+/−)-ibuprofen was successfully performed. The separation efficiency of CEC and HPLC was examined and compared; resolution of (+/−) ibuprophen was 2.45 and 2.97 for CEC and HPLC respectively, number theoretical plates of thiourea were 41,600 N/m in CEC.

Terracidiphilus gabretensis gen. nov., sp. nov., an Abundant and Active Forest Soil Acidobacterium Important in Organic Matter Transformation

ABSTRACT Understanding the activity of bacteria in coniferous forests is highly important, due to the role of these environments as a global carbon sink. In a study of the microbial biodiversity of montane coniferous forest soil in the Bohemian Forest National Park (Czech Republic), we succeeded in isolating bacterial strain S55T, which belongs to one of the most abundant operational taxonomic units (OTUs) in active bacterial populations, according to the analysis of RNA-derived 16S rRNA amplicons. The 16S rRNA gene sequence analysis showed that the species most closely related to strain S55T include Bryocella elongata SN10T (95.4% identity), Acidicapsa ligni WH120T (95.2% identity), and Telmatobacter bradus TPB6017T (95.0% identity), revealing that strain S55T should be classified within the phylum Acidobacteria, subdivision 1. Strain S55T is a rod-like bacterium that grows at acidic pH (3 to 6). Its phylogenetic, genotypic, phenotypic, and chemotaxonomic characteristics indicate that strain S55T corresponds to a new genus within the phylum Acidobacteria; thus, we propose the name Terracidiphilus gabretensis gen. nov., sp. nov. (strain S55T = NBRC 111238T = CECT 8791T). This strain produces extracellular enzymes implicated in the degradation of plant-derived biopolymers. Moreover, analysis of the genome sequence of strain S55T also reveals the presence of enzymatic machinery required for organic matter decomposition. Soil metatranscriptomic analyses found 132 genes from strain S55T being expressed in the forest soil, especially during winter. Our results suggest an important contribution of T. gabretensis S55T in the carbon cycle in the Picea abies coniferous forest.

Intrinsically Disordered Enamel Matrix Protein Ameloblastin Forms Ribbon-like Supramolecular Structures via an N-terminal Segment Encoded by Exon 5

Background: Ameloblastin plays a key role in the complex biomineralization process that forms tooth enamel, the hardest tissue of the body. Results: Ameloblastin self-associates into ribbon-like supramolecular structures via a short segment encoded by exon 5. Conclusion: Ameloblastin self-association may be essential for correct structural organization and mineralization of the enamel in vivo. Significance: The results provide molecular insight into biology of tooth enamel formation. Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.

Characterization of pseudacyclins A-E, a suite of cyclic peptides produced by Pseudallescheria boydii.

Pseudallescheria boydii sensu lato is an emerging fungal pathogen causing fatal infections in both immunocompromised and immunocompetent hosts. In this work, two P. boydii isolates (human and animal origin) have been identified as being producers of cyclic peptides. Five putative nonribosomal peptides with a unique structure, which have been named pseudacyclins, were characterized by nuclear magnetic resonance spectroscopy and mass spectrometry. The most abundant representative of the pseudacyclins was quantified also on fungal spores. The presence of these peptides on inhaled fungal spores creates the possibility for exploitation of pseudacyclins as early indicators of fungal infections caused by Pseudallescheria species.

The mechanism of gingiva metallic pigmentations formation

The occurrence of blue-grey areas in the soft tissue represents one of the problems affecting patients whose teeth have been restored using metallic materials. It is generally accepted that it is caused by mechanical penetration of a metallic material into the soft tissue. Several facts indicate that this mechanism is not general. The aim of the study was to determine an alternative mechanism of the origin of pigmentations, based on the corrosion interaction of metallic materials used in prosthodontics with the oral environment. The study was comprised of an analysis of pigment particles, determination of exposure conditions of metallic materials in vivo and laboratory evaluation of corrosion properties of the studied materials. Particles containing silver, sulphur and/or selenium could be seen in ultra-thin sections in the lamina propria gingivae. Comparison of the corrosion laboratory results with the results of in vivo measurements indicated the intensification of corrosion under these conditions. Amalgams and silver-containing alloys used for teeth restorations may release silver under the conditions of the oral cavity. The formation of soluble silver compounds in the sulcular area or in a crevice between the crown and the cast post-and-core reconstruction facilitates their transport to the soft tissue and subsequent deposition.