MHC class I expression dependent on bacterial infection and parental factors in whitefish embryos (Salmonidae).

Ecological conditions can influence not only the expression of a phenotype, but also the heritability of a trait. As such, heritable variation for a trait needs to be studied across environments. We have investigated how pathogen challenge affects the expression of MHC genes in embryos of the lake whitefish Coregonus palaea. In order to experimentally separate paternal (i.e. genetic) from maternal and environmental effects, and determine whether and how stress affects the heritable variation for MHC expression, embryos were produced in full-factorial in vitro fertilizations, reared singly, and exposed at 208 degree days (late-eyed stage) to either one of two strains of Pseudomonas fluorescens that differ in their virulence characteristics (one increased mortality, while both delayed hatching time). Gene expression was assessed 48 h postinoculation, and virulence effects of the bacterial infection were monitored until hatching. We found no evidence of MHC class II expression at this stage of development. MHC class I expression was markedly down-regulated in reaction to both pseudomonads. While MHC expression could not be linked to embryo survival, the less the gene was expressed, the earlier the embryos hatched within each treatment group, possibly due to trade-offs between immune function and developmental rate or further factors that affect both hatching timing and MHC expression. We found significant additive genetic variance for MHC class I expression in some treatments. That is, changes in pathogen pressures could induce rapid evolution in MHC class I expression. However, we found no additive genetic variance in reaction norms in our study population.

The influence of bacterial activity on phosphorite formation in the Miocene Monterey Formation, California

Authigenic phosphorites from the Miocene Monterey Formation (California) including an autochthonous phosphatic laminite were analyzed for molecular biomarkers, element content, and sulfur isotopic composition of associated pyrite and sulfate to evaluate the role of bacterial activity in the precipitation of phosphate minerals. The phosphorites formed in a depositional environment typified by upwelling with dynamic bottom currents and hardground formation. Pyrite enclosed in the phosphorites shows delta S-34 values as low as -36.5 parts per thousand VCDT, which is consistent with bacterial sulfate reduction. In a three-step extraction phosphorite dissolution extraction procedure, molecular fossils of sulfate-reducing bacteria (di-O-alkyl glycerol ethers and short-chain branched fatty acids i- and ai-C-15:0, i- and ai-C-17:0, and 10MeC(16:0)) were preferentially released from the mineral lattice. This suggests that the molecular fossils were tightly bound to carbonate fluorapatite, indicating that sulfate-reducing bacteria were involved in mineral formation. A close association of sulfate-reducing bacteria with large sulfide-oxidizing bacteria, which was previously suggested to favor carbonate fluorapatite precipitation, could neither be confirmed nor excluded for the Miocene Monterey Formation phosphorites. (C) 2012 Elsevier B.V. All rights reserved.

Structural investigations into the interaction of hemoglobin and part structures with bacterial endotoxins.

An understanding of details of the interaction mechanisms of bacterial endotoxins (lipopolysaccharide, LPS) with the oxygen transport protein hemoglobin is still lacking, despite its high biological relevance. Here, a biophysical investigation into the endotoxin:hemoglobin interaction is presented which comprises the use of various rough mutant LPS as well as free lipid A; in addition to the complete hemoglobin molecule from fetal sheep extract, also the partial structure alpha-chain and the heme-free sample are studied. The investigations comprise the determination of the gel-to-liquid crystalline phase behaviour of the acyl chains of LPS, the ultrastructure (type of aggregate structure and morphology) of the endotoxins, and the incorporation of the hemoglobins into artificial immune cell membranes and into LPS. Our data suggest a model for the interaction between Hb and LPS in which hemoglobins do not react strongly with the hydrophilic or with the hydrophobic moiety of LPS, but with the complete endotoxin aggregate. Hb is able to incorporate into LPS with the longitudinal direction parallel to the lipid A double-layer. Although this does not lead to a strong disturbance of the LPS acyl chain packing, the change of the curvature leads to a slightly conical molecular shape with a change of the three-dimensional arrangement from unilamellar into cubic LPS aggregates. Our previous results show that cubic LPS structures exhibit strong endotoxic activity. The property of Hb on the physical state of LPS described here may explain the observation of an increase in LPS-mediating endotoxicity due to the action of Hb.

Reprogramming plant cells for endosymbiosis.

The establishment of arbuscular mycorrhizal (AM) symbioses, formed by most flowering plants in association with glomeromycotan fungi, and the root-nodule (RN) symbiosis, formed by legume plants and rhizobial bacteria, requires an ongoing molecular dialogue that underpins the reprogramming of root cells for compatibility. In both endosymbioses, there are distinct phases to the interaction, including a presymbiotic anticipation phase and, subsequently, an intraradical accommodation of the microsymbiont. Maintenance of the endosymbiosis then depends on reciprocal nutrient exchange with the microsymbiont-obtaining plant photosynthates in exchange for mineral nutrients: enhanced phosphate and nitrogen uptake from AM fungi and fixed nitrogen from rhizobia. Despite the taxonomically distinct groups of symbionts, commonalities are observed in the signaling components and the modulation of host cell responses in both AM and RN symbioses, reflecting common mechanisms for plant cell reprogramming during endosymbiosis.

Impact of relative size and language on the attitudes between nations and linguistic groups : The case of Switzerland

This study explores the impact of relative size on the intra- and intergroup attitudes of groups who either share a language or have a different language. For that purpose, we examined international attitudes, comparing a small nation, Switzerland, and two larger nations, Germany and France. We found support for the assumption that large neighbouring nations pose a threat to the smaller nation's identity, especially when they are linguistically similar. Consequently, in line with Tajfel's Social Identity Theory (1978), the smaller nation's inhabitants evaluate those of the larger nation less positively, liking them less and perceiving them to be more arrogant than vice versa. By investigating the special case of the French-speaking and the German-speaking Swiss as linguistic groups within their own nation we were able to demonstrate that these groups seek support with the larger-linguistically-similar nation to defend themselves against the more direct in-country threat to their identity. They acknowledge the similarity with the larger nation, yet keep defending their social identity by expressing a dislike for this perceived similarity.

Exposure to bioaerosols in poultry houses at different stages of fattening; use of real-time PCR for airborne bacterial quantification

Previous studies have demonstrated that poultry house workers are exposed to very high levels of organic dust and consequently have an increased prevalence of adverse respiratory symptoms. However, the influence of the age of broilers on bioaerosol concentrations has not been investigated. To evaluate the evolution of bioaerosol concentration during the fattening period, bioaerosol parameters (inhalable dust, endotoxin and bacteria) were measured in 12 poultry confinement buildings in Switzerland, at three different stages of the birds' growth; samples of air taken from within the breathing zones of individual poultry house employees as they caught the chickens ready to be transported for slaughter were also analysed. Quantitative polymerase chain reaction (Q-PCR) was used to assess the quantity of total airborne bacteria and total airborne Staphylococcus species. Bioaerosol levels increased significantly during the fattening period of the chickens. During the task of catching mature birds, the mean inhalable dust concentration for a worker was 26 +/- 1.9 mg m(-3) and endotoxin concentration was 6198 +/- 2.3 EU m(-3) air, >6-fold higher than the Swiss occupational recommended value (1000 EU m(-3)). The mean exposure level of bird catchers to total bacteria and Staphylococcus species measured by Q-PCR is also very high, respectively, reaching values of 53 (+/-2.6) x 10(7) cells m(-3) air and 62 (+/-1.9) x 10(6) m(-3) air. It was concluded that in the absence of wearing protective breathing apparatus, chicken catchers in Switzerland risk exposure beyond recommended limits for all measured bioaerosol parameters. Moreover, the use of Q-PCR to estimate total and specific numbers of airborne bacteria is a promising tool for evaluating any modifications intended to improve the safety of current working practices

Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4

Rapport de synthèse : L'immunité innée regroupe les mécanismes moléculaires et cellulaires formant la première ligne de défense contre les infections microbiennes. La détection des micro-organismes pathogènes est assurée par des cellules sentinelles (cellules dendritiques et macrophages) qui jouent un rôle fondamental dans l'initiation des mécanismes de défense de l'hôte. Au contact de produits microbiens, ces cellules produisent un large échantillonnage de molécules, dont des cytokines, impliquées dans le développement de la réponse inflammatoire. La régulation de cette réponse relève d'un équilibre délicat, son insuffisance tant que son excès pouvant compromettre le devenir des patients infectés. La sepsis sévère et le choc septique représentent les formes les plus sévères d'infection, et leur mortalité demeure élevée (25 à 30% pour la sepsis sévère et 50 à 60% pour le choc septique). De plus, l'incidence de la sepsis tend à augmenter, atteignant en 2000 plus de 240 cas pour 100'000 personnes en Grande-Bretagne. La sepsis est caractérisée dans sa phase aiguë par une réponse inflammatoire exubérante. La plupart des thérapies visant à la bloquer ont toutefois montré des bénéfices incertains lors de leur application clinique. Il est donc impératif d'identifier de nouvelles cibles thérapeutiques. Les "Toll-like receptors" (TLRs) sont une famille de récepteurs qui jouent un rôle fondamental dans la détection des micro-organismes par les cellules du système immunitaire inné. Parmi eux, TLR4 est indispensable à la reconnaissance du lipopolysaccharide (LPS) des bactéries Gram-négatives. L'interaction entre TLR4 et le LPS représentant un élément précoce de la réponse de l'hôte à l'infection, nous avons émit l'hypothèse que TLR4 pourrait représenter une cible de choix en vue du développement de nouvelles thérapies contre la sepsis. Dans l'objectif de valider ce concept, nous avons, dans un premier temps, démontré que des souris génétiquement déficientes en TLR4 étaient totalement résistantes au choc septique induit par Escherichia coli (E. coli), une bactérie Gram-négative fréquemment responsable de sepsis. Forts de cette observation, nous avons développé une molécule recombinante composée du domaine extracellulaire de TLR4 fusionné à la partie IgGi-Fc. Cette molécule soluble, qui inhibait la réponse des macrophages au LPS in vitro, a été utilisée pour générer des anticorps anti-TLR4 chez le lapin. La spécificité et l'efficacité de ces anticorps ont été prouvées en démontrant que les anti-TLR4 bloquaient les signaux d'activation intracellulaire et la production de TNF et d'IL-6 en réponse au LPS et aux bactéries Gram-négatives in vitro et in vivo. Enfin, l'efficacité des ces anticorps a été testée dans des modèles de sepsis chez la souris. Ainsi, l'injection prophylactique (-lh) ou thérapeutique (+3h) d'anticorps anti-TLR4 réduisait la production de TNF et protégeait les animaux de la mort. De manière spectaculaire, ces anticorps réduisaient également la production de TNF et protégeaient de la sepsis à E. coli lorsqu'ils étaient administrés de manière prophylactique (-4h) et thérapeutique, jusqu'à 13 heures après l'initiation de l'infection. Ces résultats indiquent donc qu'il est possible de bloquer le développement de la réponse inflammatoire et de protéger du choc septique à bactéries Gram-négatives en utilisant des thérapies ciblant TLR4. Par ailleurs, ils suggèrent qu'une fenêtre d'opportunité de plusieurs heures pourrait être mise à profit pour initier un traitement chez les patients septiques. Ces résultats devraient encourager la poursuite des essais cliniques en cours qui visent à tester l'efficacité de thérapies dirigées contre TLR4 comme traitement complémentaire de la sepsis.

Epigenetic regulation of the bacterial cell cycle.

N(6)-methyl-adenines can serve as epigenetic signals for interactions between regulatory DNA sequences and regulatory proteins that control cellular functions, such as the initiation of chromosome replication or the expression of specific genes. Several of these genes encode master regulators of the bacterial cell cycle. DNA adenine methylation is mediated by Dam in gamma-proteobacteria and by CcrM in alpha-proteobacteria. A major difference between them is that CcrM is cell cycle regulated, while Dam is active throughout the cell cycle. In alpha-proteobacteria, GANTC sites can remain hemi-methylated for a significant period of the cell cycle, depending on their location on the chromosome. In gamma-proteobacteria, most GATC sites are only transiently hemi-methylated, except regulatory GATC sites that are protected from Dam methylation by specific DNA-binding proteins.

Release of vancomycin and teicoplanin from a plasticized and resorbable gelatin sponge: in vitro investigation of a new antibiotic delivery system with glycopeptides.

BACKGROUND: The aim of this study was to evaluate the efficacy of sustained release of vancomycin and teicoplanin from a resorbable gelatin glycerol sponge, in order to establish a new delivery system for local anti-infective therapy. MATERIALS AND METHODS: 60 plasticized glycerol gelatin sponges containing either 10 or 20% gelatin (w/v) were incubated in vancomycin or teicoplanin solution at 20 degrees C for either 1 or 24 h. In vitro release properties of the sponges were investigated over a period of 1 week by determining the levels of vancomycin and teicoplanin eluted in plasma using fluorescent polarization immunoassay. The rate constant and the half-life for the antibiotic release of each group were calculated by linear regression assuming first order kinetics. RESULTS: Presoaking for 24 h was associated with a significant increase in the total antibiotic release in all groups opposed to 1 h of incubation, except for the 10% sponges presoaked in teicoplanin. Doubling the gelatin content of the sponges from 10 to 20% significantly increased the total release of antibiotic load only in teicoplanin-containing sponges after 24 h incubation. In all corresponding groups investigated, release of vancomycin was more prolonged compared to teicoplanin, which allowed a gradual release beyond 5 days. The half-life (h +/- SEM) of both types of vancomycin-containing sponges was significantly prolonged by 24 h incubation in comparison to 1 h incubation (29.1 +/- 5.9 vs 5.9 +/- 1.0; p < 0.001, 30.0 +/- 2.1 vs 11.1 +/- 1.9; p < 0.001). However, neither doubling the gelatin content of the sponges nor a prolonged incubation was associated with a significantly prolonged delivery of teicoplanin. CONCLUSION: This study demonstrated a better diffusion-controlled release of vancomycin-impregnated glycerol gelatin sponges compared to those pretreated with teicoplanin. The plasticized glycerol gelatin sponge may be a promising carrier for the application of vancomycin to infected wounds for local anti-infective therapy.

Citrobacter rodentium Requires Intestinal Neural Wiskott-Aldrich Syndrome Protein (N-WASp) for Actin Pedestal Formation, Colonization and Epithelial Barrier Disruption In Vivo

Background: Citrobacter rodentium is a natural mouse pathogen that is genetically closelyrelated to the human enteric pathogens enteropathogenic and enterohemorrhagic E. coli.Among the repertoire of conserved virulence factors that these pathogens deliver via typeIII secretion, Tir and EspF are responsible for the formation of characteristic actin-richpedestals and disruption of tight junction integrity, respectively. There is evidence In Vitrothese effectors accomplish this, at least in part, by subverting the normal host cellularfunctions of N-WASP, a critical regulator of branched chain actin assembly. Although NWASPhas been shown to be involved in pedestal formation In Vitro, the requirements ofN-WASP-mediated actin pedestals for intestinal colonization by attaching/effacing (A/E)pathogens In Vivo is not known. Furthermore, it is not known whether N-WASP is requiredfor EspF-mediated tight junction disruption. Methods: To investigate the role of N-WASPin the gut epithelium, we generated mice with intestine-specific deletion of N-WASP(iNWKO), by mating mice homozygous for a floxed N-WASP allele (N-WASPL2L/L2L) tomice expressing Cre recombinase under the villin promoter. Separately housed groups ofWT and iNWKO mice were inoculated with 5x108 GFP-expressing C. rodentium by intragastriclavage. Stool was collected 2, 4, 7, and 12 days after infection, and recoverablecolony forming units (CFUs) of C. rodentium were quantified by plating serial dilutions ofhomogenized stool on MacConkey's agar. GFP+ colonies were counted after 24 hoursincubation at 37°C. The presence of actin pedestals was investigated by electron microscopy(EM), and tight junction morphology was assessed by immunofluorescence staining ofoccludin, ZO-1 and claudin-2. Results: C. rodentium infection did not result in mortalityin WT or iNWKO mice. Compared to controls, iNWKO mice exhibited higher levels ofbacterial shedding during the first 4 days of infection (day 4 average: WT 5.2x104 CFU/gvs. iNWKO 4.7x105 CFU/g, p=0.08), followed by a more rapid clearance of C. rodentium, (day7-12 average: WT 2x106 CFU/g vs. iNWKO 2.7x105, p=0.01). EM and immunofluorescencerevealed the complete lack of actin pedestals in iNWKO mice and no mucosa-associatedGFP+ C. rodentium by day 7. WT controls exhibited tight junction disruption, reflected byaltered distribution of ZO-1, whereas iNWKO mice had no change in the pattern of ZO-1.Conclusion: Intestinal N-WASP is required for actin pedestal formation by C. rodentium InVivo, and ablation of N-WASP is associated with more rapid bacterial clearance and decreasedability of C. rodentium to disrupt intercellular junctions.

Cutting edge: cell autonomous rather than environmental factors control bacterial superantigen-induced T cell anergy in vivo.

Anergic T cells display a marked decrease in their ability to produce IL-2 and to proliferate in the presence of an appropriate antigenic signal. Two nonmutually exclusive classes of models have been proposed to explain the persistence of T cell anergy in vivo. While some reports indicate that anergic T cells have intrinsic defects in signaling pathways or transcriptional activities, other studies suggest that anergy is maintained by environmental "suppressor" factors such as cytokines or Abs. To distinguish between these conflicting hypotheses, we employed the well-characterized bacterial superantigen model system to evaluate in vivo the ability of a trace population of adoptively transferred naive or anergized T cells to proliferate in a naive vs anergic environment upon subsequent challenge. Our data clearly demonstrate that bacterial superantigen-induced T cell anergy is cell autonomous and independent of environmental factors.

A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes.

The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor--Burkholderia sp. strain RP037--produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed.

A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system.

BACKGROUND: The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs). RESULTS: On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit. CONCLUSIONS: A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.