Zinc metalloproteinase ProA directly activates Legionella pneumophila PlaC glycerophospholipid:cholesterol acyltransferase

Enzymes secreted by Legionella pneumophila, such as phospholipases A (PLAs) and glycerophospholipid:cholesterol acyltransferases (GCATs), may target host cell lipids and therefore contribute to the establishment of Legionnaires disease. L. pneumophila possesses three proteins, PlaA, PlaC, and PlaD, belonging to the GDSL family of lipases/acyltransferases. We have shown previously that PlaC is the major GCAT secreted by L. pneumophila and that the zinc metalloproteinase ProA is essential for GCAT activity. Here we characterized the mode of PlaC GCAT activation and determined that ProA directly processes PlaC. We further found that not only cholesterol but also ergosterol present in protozoa was palmitoylated by PlaC. Such ester formations were not induced by either PlaA or PlaD. PlaD was shown here to possess lysophospholipase A activity, and interestingly, all three GDSL enzymes transferred short chain fatty acids to sterols. The three single putative catalytic amino acids (Ser-37, Asp-398, and His-401) proved essential for all PlaC-associated PLA, lysophospholipase A, and GCAT activities. A further four cysteine residues are important for the PLA/GCAT activities as well as their oxidized state, and we therefore conclude that PlaC likely forms at least one disulfide loop. Analysis of cleavage site and loop deletion mutants suggested that for GCAT activation deletion of several amino acids within the loop is necessary rather than cleavage at a single site. Our data therefore suggest a novel enzyme inhibition/activation mechanism where a disulfide loop inhibits PlaC GCAT activity until the protein is exported to the external space where it is ProA-activated.     

Localization of endothelin receptors in bleomycin-induced pulmonary fibrosis in the rat

Pulmonary fibrosis is characterized by excessive extracellular matrix deposition with concomitant loss of gas exchange units, and endothelin-1 (ET-1) has been implicated in its pathogenesis. Increased levels of ET-1 from tissues and bronchoalveolar lavage have been reported in patients with pulmonary fibrosis and in animal models after intratracheal bleomycin. We characterized the cellular distribution of alveolar ET receptors by immunohistochemistry in bleomycin-induced pulmonary fibrosis in the rat and determined the regulation by bleomycin of ET receptor mRNA expression in isolated alveolar macrophages and rat lung fibroblasts. We found significant increases in the numbers of fibroblasts and macrophages at day 7 compared to day 28 and control animals. ET_B receptor immunoreactivity was observed on fibroblasts and invading monocytes. Isolated fibroblasts expressed both ET_A and ET_B receptor mRNA, and ET_A receptor mRNA was upregulated by bleomycin. Isolated resident alveolar macrophages expressed neither ET_A nor ET_B receptor mRNA which were also not induced by bleomycin. We conclude that, while ET_B receptor stimulation of fibroblasts and monocytes recruited during bleomycin-induced lung injury exerts antagonistic effects on fibroblast collagen synthesis, the observed increase in the number of fibroblasts in vivo and upregulation of fibroblast ET_A receptor mRNA by bleomycin in vitro point to a predominance of the profibrotic effects of ET receptor engagement.     

Porcine E. coli: Virulence-Associated Genes,Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars.Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation.In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.     

Methane-Carbon Flow into the Benthic Food Web at Cold Seeps – A Case Study from the Costa Rica Subduction Zone

Cold seep ecosystems can support enormous biomasses of free-living and symbiotic chemoautotrophic organisms that get their energy from the oxidation of methane or sulfide. Most of this biomass derives from animals that are associated with bacterial symbionts, which are able to metabolize the chemical resources provided by the seeping fluids. Often these systems also harbor dense accumulations of non-symbiotic megafauna, which can be relevant in exporting chemosynthetically fixed carbon from seeps to the surrounding deep sea. Here we investigated the carbon sources of lithodid crabs (Paralomis sp.) feeding on thiotrophic bacterial mats at an active mud volcano at the Costa Rica subduction zone. To evaluate the dietary carbon source of the crabs, we compared the microbial community in stomach contents with surface sediments covered by microbial mats. The stomach content analyses revealed a dominance of epsilonproteobacterial 16S rRNA gene sequences related to the free-living and epibiotic sulfur oxidiser Sulfurovum sp. We also found Sulfurovum sp. as well as members of the genera Arcobacter and Sulfurimonas in mat-covered surface sediments where Epsilonproteobacteria were highly abundant constituting 10% of total cells. Furthermore, we detected substantial amounts of bacterial fatty acids such as i-C15∶0 and C17∶1ω6c with stable carbon isotope compositions as low as −53‰ in the stomach and muscle tissue. These results indicate that the white microbial mats at Mound 12 are comprised of Epsilonproteobacteria and that microbial mat-derived carbon provides an important contribution to the crab''s nutrition. In addition, our lipid analyses also suggest that the crabs feed on other ¹³C-depleted organic matter sources, possibly symbiotic megafauna as well as on photosynthetic carbon sources such as sedimentary detritus.     

Amlexanox provides a potential therapy for nonsense mutations in the lysosomal storage disorder Aspartylglucosaminuria

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by mutations in the gene for aspartylglucosaminidase (AGA). This enzyme participates in glycoprotein degradation in lysosomes. AGU results in progressive mental retardation, and no curative therapy is currently available. We have here characterized the consequences of AGA gene mutations in a compound heterozygous patient who exhibits a missense mutation producing a Ser72Pro substitution in one allele, and a nonsense mutation Trp168X in the other. Ser72 is not a catalytic residue, but is required for the stabilization of the active site conformation. Thus, Ser72Pro exchange impairs the autocatalytic activation of the AGA precursor, and results in a considerable reduction of the enzyme activity and in altered AGA precursor processing. Betaine, which can partially rescue the AGA activity in AGU patients carrying certain missense mutations, turned out to be ineffective in the case of Ser72Pro substitution. The Trp168X nonsense allele results in complete lack of AGA polypeptide due to nonsense-mediated decay (NMD) of the mRNA. Amlexanox, which inhibits NMD and causes a translational read-through, facilitated the synthesis of a full-length, functional AGA protein from the nonsense allele. This could be demonstrated as presence of the AGA polypeptide and increased enzyme activity upon Amlexanox treatment. Furthermore, in the Ser72Pro/Trp168X expressing cells, Amlexanox induced a synergistic increase in AGA activity and polypeptide processing due to enhanced processing of the Ser72Pro polypeptide. Our data show for the first time that Amlexanox might provide a valid therapy for AGU.     

NMR spectroscopic and theoretical study of the complexation of the inhibitor allosamidin in the binding pocket of the plant chitinase hevamine

Based on NMR spectroscopic information about the allosamidin-hevamine complex, ab initio MO calculations of the ring current effect of the aromatic moieties of Trp255, Tyr183 and Tyr6 of hevamine were carried out to investigate the role of these amino acid residues in binding interactions with allosamidin in solution. In addition, the intermolecular steric compression effect on the 13C chemical shifts of the allosamizoline carbon atoms and the hydrogen bonding to Glu127 was identified. It can be inferred that the binding forces are strongest in the allosamizoline moiety of allosamidin.     

Caenorhabditis elegans Semi-Automated Liquid Screen Reveals a Specialized Role for the Chemotaxis Gene cheB2 in Pseudomonas aeruginosa Virulence

Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity.     

DDX6 recruits translational silenced human reticulocyte 15-lipoxygenase mRNA to RNP granules

Erythroid precursor cells lose the capacity for mRNA synthesis due to exclusion of the nucleus during maturation. Therefore, the stability and translation of mRNAs that code for specific proteins, which function in late stages of maturation when reticulocytes become erythrocytes, are controlled tightly. Reticulocyte 15-lipoxygenase (r15-LOX) initiates the breakdown of mitochondria in mature reticulocytes. Through the temporal restriction of mRNA translation, the synthesis of r15-LOX is prevented in premature cells. The enzyme is synthesized only in mature reticulocytes, although r15-LOX mRNA is already present in erythroid precursor cells. Translation of r15-LOX mRNA is inhibited by hnRNP K and hnRNP E1, which bind to the differentiation control element (DICE) in its 3' untranslated region (3'UTR). The hnRNP K/E1-DICE complex interferes with the joining of the 60S ribosomal subunit to the 40S subunit at the AUG. We took advantage of the inducible human erythroid K562 cell system that fully recapitulates this process to identify so far unknown factors, which are critical for DICE-dependent translational regulation. Applying RNA chromatography with the DICE as bait combined with hnRNP K immunoprecipitation, we specifically purified the DEAD-box RNA helicase 6 (DDX6) that interacts with hnRNP K and hnRNP E1 in a DICE-dependent manner. Employing RNA interference and fluorescence in situ hybridization, we show that DDX6 colocalizes with endogenous human (h)r15-LOX mRNA to P-body-like RNP granules, from which 60S ribosomal subunits are excluded. Our data suggest that in premature erythroid cells translational silencing of hr15-LOX mRNA is maintained by DDX6 mediated storage in these RNP granules.     

Secreted modular calcium-binding protein-1 localization during mouse embryogenesis

BM-40 is an extracellular matrix-associated protein and is characterized by an extracellular calcium-binding domain as well as a follistatin-like domain. Secreted modular calcium-binding protein-1 (SMOC-1) is a new member of the BM-40 family. It consists of two thyroglobulin-like domains, a follistatin-like domain and a new domain without known homologues and is expressed ubiquitously in many adult murine tissues. Immunofluorescence studies, as well as immunogold electron microscopy, have confirmed the localization of SMOC-1 in or around basement membranes of adult murine skin, blood vessels, brain, kidney, skeletal muscle, and the zona pellucida surrounding the oocyte. In the present work, light microscopic immunohistochemistry has revealed that SMOC-1 is localized in the early mouse embryo day 7 throughout the entire endodermal basement membrane zone of the embryo proper. SMOC-1 mRNA is synthesized, even in early stages of mouse development, by mesenchymal as well as epithelial cells deriving from all three germ layers. In embryonic stage day 12, and fetal stages day 14, 16, and 18, the protein is present in the basement membrane zones of brain, blood vessels, skin, skeletal muscle, lung, heart, liver, pancreas, intestine, and kidney. This broad and organ-specific distribution suggests multifunctional roles of SMOC-1 during mouse embryogenesis.