The biosynthesis and biological role of lipopolysaccharide O-antigens of pathogenic Yersiniae

Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria. The LPS molecule is composed of two biosynthetic entities: the lipid A--core and the O-polysaccharide (O-antigen). Most biological effects of LPS are due to the lipid A part, however, there is an increasing body of evidence indicating that O-antigen (O-ag) plays an important role in effective colonization of host tissues, resistance to complement-mediated killing and in the resistance to cationic antimicrobial peptides that are key elements of the innate immune system. In this review, we will discuss: (i) the work done on the genetics and biosynthesis of the O-ags in the genus Yersinia; (ii) the role of O-ag in virulence of these bacteria; (iii) the work done on regulation of the O-ag gene cluster expression and; (iv) the impact that the O-ag expression has on other bacterial surface and membrane components.

Evaluation of in Situ DGT Measurements for Predicting the Concentration of Cd in Chinese Field-Cultivated Rice: Impact of Soil Cd:Zn Ratios

DGT (diffusive gradients in thin-films) has been proposed as a tool for predicting Cd concentrations in rice grain, but there is a lack of authenticating data. To further explore the relationship between DGT measured Cd and concentrations in rice cultivated in challenging, metal degraded, field locations with different heavy metal pollutant sources, 77 paired soil and grain samples were collected in Southern China from industrial zones, a "cancer village" impacted by mining waste and an organic farm. In situ deployments of DGT in flooded paddy rice rhizospheres were compared with a laboratory DGT assay on dried and rewetted soil. Total soil concentrations were a very poor predictor of plant uptake. Laboratory and field deployed DGT assays and porewater measurements were linearly related to grain concentrations in all but the most contaminated samples where plant toxicity occurred. The laboratory DGT assay was the best predictor of grain Cd concentrations, accommodating differences in soil Cd, pollutant source, and Cd:Zn ratios. Field DGT measurements showed that Zn availability in the flooded rice rhizospheres was greatly diminished compared to that of Cd, resulting in very high Cd:Zn ratios (0.1) compared to commonly observed values (0.005). These results demonstrate the potential of the DGT technique to predict Cd concentrations in field cultivated rice and demonstrate its robustness in a range of environments. Although, field deployments provided important details about in situ element stoichiometry, due to the inherent heterogeneity of the rice rhizosphere soils, deployment of DGT in dried and homogenized soils offers the best possibility of a soil screening tool.

Use of immunosorbent-purified antigens of Fasciola hepatica in enzyme immunoassays

A monoclonal antibody specific for the T1 tegumental antigen of Fasciola hepatica was used as a solid-phase immunosorbent for the purification of T1 antigen from homogenised mature F hepatica. Material fractionated by this technique was successfully used in enzyme-linked immunoassays to detect antibodies to F hepatica in sera from sheep and cattle. Species differences in response to infection by F hepatica were demonstrated.

Fasciola hepatica:development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.

Langerhans cells promote early germinal center formation in response to Leishmania-derived cutaneous antigens

Efficient formation of early GCs depends on the close interaction between GC B cells and antigen-primed CD4+ follicular helper T cells (TFH). A tight and stable formation of TFH/B cell conjugates is required for cytokine-driven immunoglobulin class switching and somatic hypermutation of GC B cells. Recently, it has been shown that the formation of TFH/B cell conjugates is crucial for B-cell differentiation and class switch following infection with Leishmania major parasites. However, the subtype of DCs responsible for TFH-cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin+ DC subsets in vivo, we show that the functionality of TFH/B cell conjugates is disturbed after depletion of Langerhans cells (LCs): LC-depleted mice show a reduction in somatic hypermutation in B cells isolated from TFH/B cell conjugates and markedly reduced GC reactions within skin-draining lymph nodes. In conclusion, this study reveals an indispensable role for LCs in promoting GC B-cell differentiation following cutaneous infection with Leishmania major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.

Fragmentation of CD+ induced by intense ultrashort laser pulses

The fragmentation of CD+ in intense ultrashort laser pulses was investigated using a coincidence three-dimensional momentum imaging technique improved by employing both transverse and longitudinal electric fields. This allowed clear separation of all fragmentation channels and the determination of the kinetic energy release down to nearly zero, for a molecule with significant mass asymmetry. The most probable dissociation pathways for the two lowest dissociation limits, C+ + D and C+ D+, were identified for both 22-fs, 798-nm and 50-fs, 392-nm pulses. Curiously, the charge asymmetric dissociation of CD2+ was not observed for 392-nm photons, even though it was clearly visible for the fundamental 798 nm at the same peak intensity.

Sensitivity to Cd or Zn of host and symbiont of ectomycorrhizal Pinus sylvestris L. (Scots pine) seedlings

Pinus sylvestris seedlings infected with either the ectomycorrhizal (ECM) fungus Paxillus involutus or Suillus variegatus were exposed to a range of Cd or Zn concentrations. This was done to investigate the relationship between the sensitivity of ECM fungi and their host plants over a wide range of concentrations. P involutus ameliorated the toxicity of Cd and Zn to P. sylvestris with respect to root length, despite significant inhibition of ECM infection levels by Cd (Cd EC₅₀ [effective concentration which inhibits ECM infection by 50%] values were: P. involutus 3.7 μg g^-1 Cd; S. variegatus 2.3 μg g^-1 Cd). ECM infection by P. involutus also decreased Cd and Zn transport to the plant shoots at potentially toxic concentrations and also influenced the proportion of Zn transported to the roots and shoots, with a higher proportion retained in the roots of the seedlings. ECM infection did increase host biomass production, but this was not affected by the presence of Cd or Zn. Root and shoot biomass production by P. sylvestris, in both the presence and absence of ECM fungi, was unaffected by Cd and Zn at all concentrations tested.

Cross-colonization of scots pine (Pinus sylvestris) seedlings by the ectomycorrhizal fungus Paxillus involutus in the presence of inhibitory levels of Cd and Zn

The effects of Cd and Zn on cross-colonization by Paxillus involutus of Scots pine seedlings was examined by using pairs of ectomycorrhizal (ECM) and non-mycorrhizal (NM) seedlings grown in the same vessel. This was done to assess, first, the ability of P. involutus to colonize NM Scots pine seedlings by growth from colonized roots of other Scots pine seedlings in the presence of Cd or Zn, and, second whether ECM colonization of Scots pine by P. involutus provided a competitive advantage over NM seedlings. Ectomycorrhizal colonization of Scots pine was shown to be more sensitive than Scots pine itself to Cd and Zn, but prior colonization did provide a competitive advantage with respect to biomass production. This beneficial effect over NM seedlings was, however, equal in the control, Cd and Zn treatments, and was due simply to growth stimulation in the presence of ECM colonization. Cross-colonization from an ECM to a NM seedling was reduced but not prevented by Cd and Zn. Cd had a more negative effect on cross-colonization than on initial colonization of seedlings, whereas Zn had an equally inhibitory effect on both parameters. These results have important implications for plant establishment on metal-contaminated sites. If cross-colonization between plants is reduced by toxic metals, plant establishment on contaminated sites might be retarded.

Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

A commercial Bacillus anthracis (Anthrax) whole genome protein microarray has been used to identify immunogenic Anthrax proteins (IAP) using sera from groups of donors with (a) confirmed B. anthracis naturally acquired cutaneous infection, (b) confirmed B. anthracis intravenous drug use-acquired infection, (c) occupational exposure in a wool-sorters factory, (d) humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups. Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However, a number of other chromosomally-located and plasmid encoded open reading frame proteins were also recognized by infected or exposed groups in comparison to controls. Some of these antigens e.g., BA4182 are not recognized by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo that are not currently found in the UK licensed Anthrax Vaccine (AVP). These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis "infectome." These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesized, tested in mouse immunogenicity studies and validated in parallel using human sera from the same study.

Availability and transfer to grain of As, Cd, Cu, Ni, Pb and Zn in a barley agri-system: Impact of biochar, organic and mineral fertilizers

With biochar becoming an emerging soil amendment and a tool to mitigate climate change, there are only a few studies documenting its effects on trace element cycling in agriculture. Zn and Cu are deficient in many human diets, whilst exposures to As, Pb and Cd need to be decreased. Biochar has been shown to affect many of them mainly at a bench or greenhouse scale, but field research is not available. In our experiment we studied the impact of biochar, as well as its interactions with organic (compost and sewage sludge) and mineral fertilisers (NPK and nitrosulfate), on trace element mobility in a Mediterranean agricultural field (east of Madrid, Spain) cropped with barley. At harvesting time, we analysed the soluble fraction, the available fraction (assessed with the diffusive gradients in thin gels technique, DGT) and the concentration of trace elements in barley grain. No treatment was able to significantly increase Zn, Cu or Ni concentration in barley grain, limiting the application for cereal fortification. Biochar helped to reduce Cd and Pb in grain, whereas As concentration slightly increased. Overall biochar amendments demonstrated a potential to decrease Cd uptake in cereals, a substantial pathway of exposure in the Spanish population, whereas mineral fertilisation and sewage sludge increased grain Cd and Pb. In the soil, biochar helped to stabilise Pb and Cd, while marginally increasing As release/mobilisation. Some of the fertilisation practises or treatments increased toxic metals and As solubility in soil, but never to an extent high enough to be considered an environmental risk. Future research may try to fortify Zn, Cu and Ni using other combinations of organic amendments and different parent biomass to produce enriched biochars.