NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Bacterial-induced protection against allergic inflammation through a multicomponent immunoregulatory mechanism.

Background Airborne microbial products have been reported to promote immune responses that suppress asthma, yet how these beneficial effects take place remains controversial and poorly understood. Methods We exposed mice to the bacterium Escherichia coli and subsequently induced allergic airway inflammation through sensitization and intranasal challenge with ovalbumin. Results Pulmonary exposure to the bacterium Escherichia coli leads to a suppression of allergic airway inflammation. This immune modulation was neither mediated by the induction of a T helper 1 (Th1) response nor regulatory T cells; however, it was dependent on Toll-like receptor 4 (TLR4) but did not involve TLR desensitisation. Dendritic cell migration to the draining lymph nodes and activation of T cells was unaffected by prior exposure to E.coli, while dendritic cells in the lung displayed a less activated phenotype and had impaired antigen presentation capacity. Consequently, in situ Th2 cytokine production was abrogated. The suppression of airway hyper-responsiveness was mediated through the recruitment of gd T cells; however, the suppression of dendritic cells and T cells was mediated through a distinct mechanism that could not be overcome by the local administration of activated dendritic cells, or by the in vivo administration of tumour necrosis factor a. Conclusion Our data reveal a localized immunoregulatory pathway that acts to protect the airways from allergic inflammation.

Exploring the mechanisms regulating nutrient bioavailability and lipid metabolism through a nutrigenomics approach

SUMMARY Following the complete sequencing of the human genome, the field of nutrition has begun utilizing this vast quantity of information to comprehensively explore the interactions between diet and genes. This approach, coined nutrigenomics, aims to determine the influence of common dietary ingredients on the genome, and attempts to relate the resulting different phenotypes to differences in the cellular and/or genetic response of the biological system. However, complementary to defining the biological outcomes of dietary ingredients, we must also understand the influence of the multiple factors (such as the microbiota, bile, and function of transporters) that may contribute to the bioavailability, and ultimately bioefficacy, of these ingredients. The gastrointestinal tract (GIT) is the body's foremost tissue boundary, interacting with nutrients, exogenous compounds and microbiota, and whose condition is influenced by the complex interplay between these environmental factors and genetic elements. In order to understand GIT nutrient-gene interactions, our goal was to comprehensively elucidate the region-specific gene expression underlying intestinal functions. We found important regional differences in the expression of members of the ATP-binding cassette family of transporters in the mouse intestine, suggesting that absorption of dietary compounds may vary along the GIT. Furthermore, the influence of the microbiota on host gene expression indicated that this luminal factor predominantly influences immune function and water transport throughout the GIT; however, the identification of region-specific functions suggest distinct host-bacterial interactions along the GIT. Thus, these findings reinforce that to understand nutrient bioavailability and GIT function, one must consider the physiologically distinct regions of the gut. Nutritional molecules absorbed by the enterocytes of the GIT enter circulation and will be selectively absorbed and metabolised by tissues throughout the body; however, their bioefficacy in the body will depend on the unique and shared molecular mechanisms of the various tissues. Using a nutrigenomic approach, the biological responses of the liver and hippocampus of mice fed different long chain-polyunsaturated fatty acids diets revealed tissue-specific responses. Furthermore, we identified stearoyl-CoA desaturase as a hepatic target for arachidonic acid, suggesting a potentially novel molecular mechanism that may protect against diet-induced obesity. In summary, this work begins to unveil the fundamentally important role that nutrigenomics will play in unravelling the molecular mechanisms, and those exogenous factors capable of influencing these mechanisms, that regulate the bioefficacy of nutritional molecules. RÉSUMÉ Suite au séquençage complet du génome humain, le domaine de la nutrition a commencé à utiliser cette vaste quantité d'information pour explorer de manière globale les interactions entre la nourriture et les gènes. Cette approche, appelée « nutrigenomics », a pour but de déterminer l'influence d'ingrédients couramment utilisés dans l'alimentation sur le génome, et d'essayer de relier ces différents phénotypes, ainsi révélés, à des différences de réponses cellulaires et/ou génétiques. Cependant, en plus de définir les effets biologiques d'ingrédients alimentaires, il est important de comprendre l'influence des multiples facteurs (telle que la microflore, la bile et la fonction des transporteurs) pouvant contribuer à la bio- disponibilité et par conséquent à l'efficacité de ces ingrédients. Le tractus gastro-intestinal (TGI), qui est la première barrière vers les tissus, interagit avec les nutriments, les composés exogènes et la microflore. La fonction de cet organe est influencée par les interactions complexes entre les facteurs environnementaux et les éléments génétiques. Dans le but de comprendre les interactions entre les nutriments et les gènes au niveau du TGI, notre objectif a été de décrire de manière globale l'expression génique spécifique de chaque région de l'intestin définissant leurs fonctions. Nous avons trouvé d'importantes différences régionales dans l'expression des transporteurs de la famille des « ATP-binding cassette transporter » dans l'intestin de souris, suggérant que l'absorption des composés alimentaires puisse varier le long de l'intestin. De plus, l'étude des effets de la microflore sur l'expression des gènes hôtes a indiqué que ce facteur de la lumière intestinale influence surtout la fonction immunitaire et le transport de l'eau à travers l'intestin. Cependant, l'identification des fonctions spécifiques de chaque région suggère des interactions distinctes entre l'hôte et les bactéries le long de l'intestin. Ainsi, ces résultats renforcent l'idée que la compréhension de la bio-disponibilité des nutriments, et par conséquent la fonction du TGI, doit prendre en considération les différences régionales. Les molécules nutritionnelles transportées par les entérocytes jusqu'à la circulation sanguine, sont ensuite sélectivement absorbées et métabolisées par les différents tissus de l'organisme. Cependant, leur efficacité biologique dépendra du mécanisme commun ou spécifique de chaque tissu. En utilisant une approche « nutriogenomics », nous avons pu mettre en évidence les réponses biologiques spécifiques du foie et de l'hippocampe de souris nourris avec des régimes supplémentés avec différents acides gras poly-insaturés à chaîne longue. De plus, nous avons identifié la stearoyl-CoA desaturase comme une cible hépatique pour l'acide arachidonique, suggérant un nouveau mécanisme moléculaire pouvant potentiellement protéger contre le développement de l'obésité. En résumé, ce travail a permis de dévoiler le rôle fondamental qu'une approche telle que la « nutrigenomics » peut jouer dans le décryptage des mécanismes moléculaires et de leur régulation par des facteurs exogènes, qui ensemble vont contrôler l'efficacité biologique des nutriments.

Intracellular excision and reintegration dynamics of the ICEclc genomic island of Pseudomonas knackmussii sp. strain B13.

Genomic islands are DNA elements acquired by horizontal gene transfer that are common to a large number of bacterial genomes, which can contribute specific adaptive functions, e.g. virulence, metabolic capacities or antibiotic resistances. Some genomic islands are still self-transferable and display an intricate life-style, reminiscent of both bacteriophages and conjugative plasmids. Here we studied the dynamical process of genomic island excision and intracellular reintegration using the integrative and conjugative element ICEclc from Pseudomonas knackmussii B13 as model. By using self-transfer of ICEclc from strain B13 to Pseudomonas putida and Cupriavidus necator as recipients, we show that ICEclc can target a number of different tRNA(Gly) genes in a bacterial genome, but only those which carry the GCC anticodon. Two conditional traps were designed for ICEclc based on the attR sequence, and we could show that ICEclc will insert with different frequencies in such traps producing brightly fluorescent cells. Starting from clonal primary transconjugants we demonstrate that ICEclc is excising and reintegrating at detectable frequencies, even in the absence of recipient. Recombination site analysis provided evidence to explain the characteristics of a larger number of genomic island insertions observed in a variety of strains, including Bordetella petri, Pseudomonas aeruginosa and Burkholderia.

Pyochelin-mediated signalling in Pseudomonas aeruginosa

SUMMARY: Iron is an essential element for nearly all organisms but it is poorly available in most environments and not sufficient to support microbial growth. Bacteria have evolved a range of strategies to acquire this important metal, the most common of these being siderophore-mediated iron uptake. Siderophores are high-affinity iron chelators which are released to the extracellular environment where they complex iron and deliver it to the bacterial cell, via specific uptake systems. The Gram-negative bacterium Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, which both contribute to the virulence of this opportunistic human pathogen. The genes responsible for pyochelin-mediated iron uptake are grouped in the P. aeruginosa chromosome. The pyochelin biosynthetic genes are organized in two divergent operons, pchDCBA and pchEFGHI, which flank the regulatory gene pchR. The fptA gene, encoding the ferric pyochelin outer membrane receptor, occurs immediately downstream of the pchEFGHI genes. The biosynthesis of the siderophore and its receptor is subjected to dual regulation enabling P. aeruginosa to respond not only to the intracellular iron level but also to the presence of the siderophore in the extracellular environment. Negative regulation is mediated by the widespread Fur protein which employs ferrous iron as a corepressor and binds to a consensus sequence in the promoter region of iron-regulated genes. Positive regulation occurs during iron starvation and requires the AraC-type transcriptional regulator PchR. This regulator, together with pyochelin, induces the expression of pyochelin biosynthesis and uptake genes via a mechanism which was partly unraveled during this thesis. A 32-bp conserved sequence element (PchR-box) was identified in promoter regions of pyochelin-controlled genes. The PchR-box in the pchR-pchDCBA intergenic region was found to be essential for the induction of the pchDCBA operon and for the repression of the divergently transcribed pchR gene. PchR was purified as a fusion with maltose-binding protein (MBP). Mobility shift assays demonstrated specific binding of MBP-PchR to the PchR-box in the presence, but not in the absence of pyochelin. PchR-box mutations which interfered with pyochelin-dependent regulation in vivo, also affected pyochelin-dependent PchR-box recognition in vitro. These results show that pyochelin is the intracellular effector required for PchR-mediated regulation. The fact that extracellular pyochelin triggers this regulation implies that the siderophore can enter the cytoplasm. This conclusion was corroborated by analysing the importance of known and putative pyochelin uptake genes for pyochelin-dependent gene regulation. The pyochelin receptor gene fptA is followed by three genes, fptB, fptC, and fptX, which were shown here to be co-transcribed with fPtA. While fPtX encodes an inner membrane pen-I-lease, the functions of FptB and FptC are currently unknown. FptA and FptX, which are both required for pyochelin-mediated iron uptake, were found to be also needed for pyochelin-dependent gene regulation. FptB and FptC however, were not required and their role, if any, in the uptake of the PchR effector pyochelin remains elusive. RESUME Le fer est un élément essentiel pour la quasi-totalité des organismes, mais dans la plupart des environnements, il est difficilement accessible et insuffisant à la croissance microbienne. Les bactéries ont développé de multiples stratégies pour acquérir ce précieux métal, la plus commune étant l'acquisition au moyen de sidérophores. Les sidérophores sont des petites molécules dotées d'une forte affinité pour le fer qui, une fois relâchées dans l'environnement extracellulaire, vont complexer le fer et le délivrer à la cellule bactérienne par l'intermédiaire de systèmes d'acquisition spécifiques. La bactérie Gram-négative Pseudomonas aeruginosa produit deux sidérophores, la pyoverdine et la pyochéline, qui contribuent également à la virulence de ce pathogène opportuniste. Les gènes impliqués dans l'acquisition du fer à l'aide de la pyochéline sont regroupés sur t. le chromosome de P. aeruginosa. Les gènes de biosynthèse de la pyochéline sont organisés en deux opérons divergents, pchDCBA et pchEFGHI, qui flanquent le gène régulateur pchR. Le gène fptA, codant pour le récepteur de la pyochéline dans la membrane externe, est situé immédiatement en aval des gènes pchEFGHL La biosynthèse du sidérophore et de son récepteur est soumise à une double régulation permettant à P. aeruginosa de réagir non seulement à la quantité de fer intracellulaire, mais également à la présence du sidérophore dans le milieu extracellulaire. La répression se fait par l'intermédiaire de la protéine Fur, qui nécessite le fer ferreux comme co-répresseur et se lie à une séquence consensus dans la région promotrice des gènes régulés par le fer. L'induction se produit lorsque le fer est limitant, et requiert PchR, un régulateur transcriptionnel de la famille AraC. En présence de pyochéline, ce régulateur induit l'expression des gènes de biosynthèse et du récepteur de la pyochéline par l'intermédiaire d'un mécanisme partiellement résolu dans ce travail. Une séquence conservée (PchR-box) a été identifiée dans la région promotrice des gènes régulés par la pyochéline. La PchR-box située dans la région intergénique pchR-pchDCBA s'est révélée être importante pour l'induction de l'opéron pchDCBA et la répression du gène divergent pchR. PchR a été purifiée en tant que protéine de fusion avec une protéine liant le maltose (MBP). Des expériences de gel retard ont démontré la liaison spécifique de la protéine MBP-PchR sur la PchR-box en présence, mais non en absence de pyochéline. Les mutations de la PchR-box qui ont affecté la régulation pyochéline-dépendante in vivo, ont également eu un effet sur la liaison de la protéine in vitro. Ces résultats démontrent que la pyochéline est l'effecteur intracellulaire nécessaire à la régulation par PchR. Le fait que la pyochéline extracellulaire soit capable d'activer cette régulation implique que le sidérophore entre dans le cytoplasme. Cette conclusion a été corroborée par l'évaluation du rôle des gènes connus ou putatifs de l'incorporation du fer via la pyochéline sur la régulation pyochéline-dépendente. Le gène fPtA, codant pour le récepteur de la pyochéline, est suivi de trois gènes, fptB,fptC, et fptX, co-transcrits avec,ffitA. Si sffitX code pour une perméase de la membrane interne, la fonction de FptB et FptC reste obscure. FptA et FptX, nécessaires à l'acquisition du fer par l'intermédiaire de la pyochéline, se sont également révélés être requis pour la régulation pyochéline-dépendante des gènes pchDCBA, pchEFGHI et fptABCX. FptB et FptC n'ont quant à eux vraisemblablement pas de rôle majeur à jouer, si ce n'est aucun, dans l'incorporation de la pyochéline.

Current cytogenetic map of the common shrew, Sorex araneus L.: localization of 7 genes and 4 microsatellites

Here we present information on the assignment of 7 genes, ACADVL, ADORA3, ATP7A, MTMR4, MYH2, HBB, TSPAN-3, and 4 common shrew microsatellites to chromosomes of the common shrew (Sorex araneus) and on the current status of its cytogenetic map. Comparative mapping data were used for the analysis of evolutionary chromosomal rearrangements in the common shrew genome.

Seasonal fluctuations of bacterial community diversity in agricultural soil and experimental validation by laboratory disturbance experiments.

Natural fluctuations in soil microbial communities are poorly documented because of the inherent difficulty to perform a simultaneous analysis of the relative abundances of multiple populations over a long time period. Yet, it is important to understand the magnitudes of community composition variability as a function of natural influences (e.g., temperature, plant growth, or rainfall) because this forms the reference or baseline against which external disturbances (e.g., anthropogenic emissions) can be judged. Second, definition of baseline fluctuations in complex microbial communities may help to understand at which point the systems become unbalanced and cannot return to their original composition. In this paper, we examined the seasonal fluctuations in the bacterial community of an agricultural soil used for regular plant crop production by using terminal restriction fragment length polymorphism profiling (T-RFLP) of the amplified 16S ribosomal ribonucleic acid (rRNA) gene diversity. Cluster and statistical analysis of T-RFLP data showed that soil bacterial communities fluctuated very little during the seasons (similarity indices between 0.835 and 0.997) with insignificant variations in 16S rRNA gene richness and diversity indices. Despite overall insignificant fluctuations, between 8 and 30% of all terminal restriction fragments changed their relative intensity in a significant manner among consecutive time samples. To determine the magnitude of community variations induced by external factors, soil samples were subjected to either inoculation with a pure bacterial culture, addition of the herbicide mecoprop, or addition of nutrients. All treatments resulted in statistically measurable changes of T-RFLP profiles of the communities. Addition of nutrients or bacteria plus mecoprop resulted in bacteria composition, which did not return to the original profile within 14 days. We propose that at less than 70% similarity in T-RFLP, the bacterial communities risk to drift apart to inherently different states.

High bacterial load in negative pressure wound therapy (NPWT) foams used in the treatment of chronic wounds.

No earlier study has investigated the microbiology of negative pressure wound therapy (NPWT) foam using a standardized manner. The purpose of this study is to investigate the bacterial load and microbiological dynamics in NPWT foam removed from chronic wounds (>3 months). To determine the bacterial load, a standardized size of the removed NPWT foam was sonicated. The resulting sonication fluid was cultured, and the colony-forming units (CFU) of each species were enumerated. Sixty-eight foams from 17 patients (mean age 63 years, 71% males) were investigated. In 65 (97%) foams, â0/00¥âeuro0/001 and in 37 (54%) â0/00¥2 bacterial types were found. The bacterial load remained high during NPWT treatment, ranging from 10(4) to 10(6) CFU/ml. In three patients (27%), additional type of bacteria was found in subsequent foam cultures. The mean bacterial countâeuro0/00±âeuro0/00standard deviation was higher in polyvinyl alcohol foam (6.1âeuro0/00±âeuro0/000.5 CFU/ml) than in polyurethane (5.5âeuro0/00±âeuro0/000.8 CFU/ml) (pâeuro0/00=âeuro0/000.02). The mean of log of sum of CFU/ml in foam from 125âeuro0/00mmHg (5.5âeuro0/00±âeuro0/000.8) was lower than in foam from 100âeuro0/00mmHg pressure (5.9âeuro0/00±âeuro0/000.5) (pâeuro0/00=âeuro0/000.01). Concluding, bacterial load remains high in NPWT foam, and routine changing does not reduce the load.

Discovery of catalases in members of the Chlamydiales order.

Catalase is an important virulence factor for survival in macrophages and other phagocytic cells. In Chlamydiaceae, no catalase had been described so far. With the sequencing and annotation of the full genomes of Chlamydia-related bacteria, the presence of different catalase-encoding genes has been documented. However, their distribution in the Chlamydiales order and the functionality of these catalases remain unknown. Phylogeny of chlamydial catalases was inferred using MrBayes, maximum likelihood, and maximum parsimony algorithms, allowing the description of three clade 3 and two clade 2 catalases. Only monofunctional catalases were found (no catalase-peroxidase or Mn-catalase). All presented a conserved catalytic domain and tertiary structure. Enzymatic activity of cloned chlamydial catalases was assessed by measuring hydrogen peroxide degradation. The catalases are enzymatically active with different efficiencies. The catalase of Parachlamydia acanthamoebae is the least efficient of all (its catalytic activity was 2 logs lower than that of Pseudomonas aeruginosa). Based on the phylogenetic analysis, we hypothesize that an ancestral class 2 catalase probably was present in the common ancestor of all current Chlamydiales but was retained only in Criblamydia sequanensis and Neochlamydia hartmannellae. The catalases of class 3, present in Estrella lausannensis and Parachlamydia acanthamoebae, probably were acquired by lateral gene transfer from Rhizobiales, whereas for Waddlia chondrophila they likely originated from Legionellales or Actinomycetales. The acquisition of catalases on several occasions in the Chlamydiales suggests the importance of this enzyme for the bacteria in their host environment.

Experimental bacterial endocarditis after dental extractions in rats with periodontitis.

The development of bacterial endocarditis was analyzed after dental extractions in rats with or without periodontal disease. Periodontal disease was produced in rats by tying silk ligatures around the two maxillary first molars and placing the animals on a high sucrose diet for 14 weeks. Sterile aortic valve vegetations were produced by means of a transaortic catheter, and 24 hr later the maxillary first molars were extracted. The animals were killed 72 hr after the extractions. In rats with periodontal disease, extractions resulted in a 48% (14 of 29) incidence of bacterial endocarditis, most cases of which were due to Streptococcus spp. (one was caused by Staphylococcus aureus). In contrast, when the teeth with a healthy periodontium were extracted, only 6% (one of 15) of the rats developed endocarditis. When catheters were placed in animals with periodontal disease but no extractions were performed, no endocarditis occurred.

Carotid artery rupture and cervicofacial actinomycosis.

Cervicofacial actinomycosis is an uncommon, progressive infection caused by bacilli of the Actinomyces genus. Actinomyces are common commensal saprophytes in the oral cavity which may have medical importance as facultative pathogens. Subsequent to local injuries to the oral mucosa, they may penetrate the deep tissues and be responsible for suppurative or granulomatous infections. We herein report a case of a 65-year-old man who underwent surgery followed by chemotherapy and radiotherapy for a tonsillar carcinoma. An ulcerous lesion in the base of the tongue developed and spread to the carotid artery wall. The man died of a massive hemorrhage due to left carotid artery rupture. Postmortem computed tomography angiography performed prior to autopsy allowed the precise localization of the source of bleeding to be detected. Postmortem biochemical investigations confirmed the presence of inflammation associated with local bacterial infection. Histological investigations revealed the rupture of the left carotid artery surrounded by numerous colonies of Actinomyces. Acute and chronic inflammation with tissue necrosis as well as post-actinic, fibrotic changes were also found in the tissues surrounding the ruptured artery wall.

The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models.

Gene expression data from microarrays are being applied to predict preclinical and clinical endpoints, but the reliability of these predictions has not been established. In the MAQC-II project, 36 independent teams analyzed six microarray data sets to generate predictive models for classifying a sample with respect to one of 13 endpoints indicative of lung or liver toxicity in rodents, or of breast cancer, multiple myeloma or neuroblastoma in humans. In total, >30,000 models were built using many combinations of analytical methods. The teams generated predictive models without knowing the biological meaning of some of the endpoints and, to mimic clinical reality, tested the models on data that had not been used for training. We found that model performance depended largely on the endpoint and team proficiency and that different approaches generated models of similar performance. The conclusions and recommendations from MAQC-II should be useful for regulatory agencies, study committees and independent investigators that evaluate methods for global gene expression analysis.