Recombinant Lactococcus lactis expressing haemagglutinin from a polish avian H5N1 isolate and its immunological effect in preliminary animal trials.

Lactic acid bacteria (LAB) are Gram-positive, non-pathogenic microorganisms that are gaining much interest as antigen producers for development of live vaccine vectors. Heterologous proteins of different origin have been successfully expressed in such LAB species as Lactococcus lactis. Recombinant L. lactis strains have been shown to induce specific local and systemic immune responses against various antigens. Our study aimed at constructing a L. lactis strain expressing haemagglutinin of the Polish avian H5H1 influenza isolate and examining its effect on animals. Expression of the cloned H5 gene was achieved using the nisin-controlled gene expression system. Detection of the intracellular H5 antigen produced in L. lactis was performed by Western blot analysis and confirmed using Mass Spectrometry. The potential of L. lactis recombinant cells to induce an immune response was examined by setting up preliminary immunization trials on mice and chicken. Obtained sera were tested for specific antibodies by ELISA assays. The results of these preliminary studies are a promising step toward developing a vaccine against the avian bird flu using Lactococcus lactis cells as bioreactors for efficient antigen production and delivery to the mucosal surface

Microbial community changes along the Ecology Glacier ablation zone (King George Island, Antarctica)

In recent years glacial surfaces have received much attention as microbial habitats of diverse photoautotrophic and heterotrophic cells. Supraglacial ecosystems are annually covered and uncovered by snow. The aim of this study was to investigate the microbial community response to changing environmental conditions in a transect following the receding snow line on the surface of Ecology Glacier (King George Island, Antarctica). Parameters of surface ice and cryoconite holes included chemical composition of ice and sediment, Bacteria diversity by denaturating gradient gel electrophoresis (DGGE), microbial functional diversity (Biolog Ecoplates), and microbial counts (epifluorescence microscopy, colony forming units - CFU). Data demonstrated profound differences between surface ice and cryoconite holes. Changing environmental factors along the transect influenced composition and abundance of the microbiocenosis in both habitat types. Several parameters correlated positively with distance from the glacier edge, including the cell morphotype Shannon Index, chlorophyll a, nitrogen and seston concentrations. Suspended solids content positively correlated with microbial 2 abundance and diversity. Nitrogen and phosphorus were limiting factors of microbial growth as amounts of organic nitrogen and phosphorus positively correlated with the cell numbers, fission rates and photoautotroph contribution. Our findings indicate that microbial community shows a response in terms of abundance and diversity to exposure of the glacial surface as snow-cover melts. To our knowledge this is the first study to recognize a microbial development pattern on a glacier surface in connection with the receding snow line. This may help better understand variability within supraglacial habitats, correct sampling procedures and inform biocenotic development models

Carbon assimilation profiles of mucoralean fungi show their metabolic versatility

Most mucoralean fungi are common soil saprotrophs and were probably among the first land colonisers. Although Mucoromycotina representatives grow well on simple sugar media and are thought to be unable to assimilate more complex organic compounds, they are often isolated from plant substrates. The main goal of the study was to explore the effects of isolation origin and phylogenetic placement on the carbon assimilation capacities of a large group of saprotrophic Mucoromycotina representatives (i.e. Umbelopsidales and Mucorales). Fifty two strains representing different Mucoromycotina families and isolated from different substrates were tested for their capacity to grow on 99 different carbon sources using the Biolog phenotypic microarray system and agar plates containing selected biopolymers (i.e. cellulose, xylan, pectin, and starch) as a sole carbon source. Although our results did not reveal a correlation between phylogenetic distance and carbon assimilation capacities, we observed 20 significant differences in growth capacity on specific carbon sources between representatives of different families. Our results also suggest that isolation origin cannot be considered as a main predictor of the carbon assimilation capacities of a particular strain. We conclude that saprotrophic Mucoromycotina representatives are, contrary to common belief, metabolically versatile and able to use a wide variety of carbon sources

Metabolic Potential, Ecology and Presence of Associated Bacteria Is Reflected in Genomic Diversity of Mucoromycotina

Mucoromycotina are often considered mainly in pathogenic context but their biology remains understudied. We describe the genomes of six Mucoromycotina fungi representing distant saprotrophic lineages within the subphylum (i.e., Umbelopsidales and Mucorales). We selected two Umbelopsis isolates from soil (i.e., U. isabellina, U. vinacea), two soil-derived Mucor isolates (i.e., M. circinatus, M. plumbeus), and two Mucorales representatives with extended proteolytic activity (i.e., Thamnidium elegans and Mucor saturninus). We complement computational genome annotation with experimental characteristics of their digestive capabilities, cell wall carbohydrate composition, and extensive total lipid profiles. These traits inferred from genome composition, e.g., in terms of identified encoded enzymes, are in accordance with experimental results. Finally, we link the presence of associated bacteria with observed characteristics. Thamnidium elegans genome harbors an additional, complete genome of an associated bacterium classified to Paenibacillus sp. This fungus displays multiple altered traits compared to the remaining isolates, regardless of their evolutionary distance. For instance, it has expanded carbon assimilation capabilities, e.g., efficiently degrades carboxylic acids, and has a higher diacylglycerol:triacylglycerol ratio and skewed phospholipid composition which suggests a more rigid cellular membrane. The bacterium can complement the host enzymatic capabilities, alter the fungal metabolism, cell membrane composition but does not change the composition of the cell wall of the fungus. Comparison of early-diverging Umbelopsidales with evolutionary younger Mucorales points at several subtle differences particularly in their carbon source preferences and encoded carbohydrate repertoire. Nevertheless, all tested Mucoromycotina share features including the ability to produce 18:3 gamma-linoleic acid, use TAG as the storage lipid and have fucose as a cell wall component

Microbial community development on the surface of Hans and Werenskiold Glaciers (Svalbard, Arctic) – a comparison.

Surface ice and cryoconite holes of two types of polythermal Svalbard Glaciers (Hans Glacier - grounded tidewater glacier and Werenskiold Glacier - land-based valley glacier) were investigated in terms of chemical composition, microbial abundance and diversity. Gathered data served to describe supraglacial habitats and to compare microbe-environment interactions on those different type glaciers. Hans Glacier samples displayed elevated nutrient levels (DOC, nitrogen and seston) compared to Werenskiold Glacier. Adjacent tundra formations, bird nesting sites and marine aerosol were candidates for allochtonic enrichment sources. Microbial numbers were comparable on both glaciers, with surface ice containing cells in the range of 104 mL-1 and cryoconite sediment 108 g-1 dry weight. Denaturating gradient gel electrophoresis band-based clustering revealed differences between glaciers in terms of dominant bacterial taxa structure. Microbial community on Werenskiold Glacier benefited from the snow-released substances. On Hans Glacier this effect was not as pronounced, affecting mainly the photoautotrophs. Over-fertilization of Hans Glacier surface was proposed as the major factor, desensitizing the microbial community to the snow melt event. Nitrogen emerged as a limiting factor in surface ice habitats, especially to Eukaryotic algae

Lactic acid bacteria — promising vaccine vectors: possibilities, limitations, doubts

Gram-positive and nonpathogenic lactic acid bacteria (LAB) are considered to be promising candidates for the development of new, safe systems of heterologous protein expression. Additionally, many experiments have shown that specific systemic and mucosal immune responses against selected pathogens can be elicited using modified LAB strains. For that reason they could be a good replacement of classical, often pathogenic, attenuated carriers. This solution offers many advantages in comparison to systemic inoculation, as well as, from immunological and practical points of view. The development of efficient mucosal vaccines is nowadays a priority in modern vaccinology, together with improvement of immunization efficiency, monitoring of antigen production in vivo, determination of optimal dose for vaccination, strain selection and characterization

Expression of avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) under control of the ptcB promoter in Lactococcus lactis.

Gram-positive and nonpathogenic lactic acid bacteria (LAB) are considered to be promising candidates for the development of new, safe systems of heterologous protein expression. Recombinant LAB has been shown to induce specific local and systemic immune response against selected pathogens, and could be a good alternative to classical attenuated carriers. The main goal of our study was to express the avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) in Lactococcus lactis. Results of this study were anticipated to lead to construction of lactococcal strain(s) with potential vaccine properties against the avian influenza A (H5N1) virus. Expression of the cloned H5 gene, its His-tagged variant and chIL-2 gene, under the control of the ptcB gene promoter was attested by RT-PCR on transcriptional level and Western or dot blot analysis on translational level, demonstrating that system can be an attractive solution for production of heterologous proteins. The results of the preliminary animal trial conducted in mice are a promising step toward development of a vaccine against avian bird flu using Lactococcus lactis cells as antigen carriers

Recombinant Lactococcus lactis Expressing Haemagglutinin from a Polish Avian H5N1 Isolate and Its Immunological Effect in Preliminary Animal Trials

Lactic acid bacteria (LAB) are Gram-positive, nonpathogenic microorganisms that are gaining much interest as antigen producers for development of live vaccine vectors. Heterologous proteins of different origin have been successfully expressed in various LAB species, including Lactococcus lactis. Recombinant L. lactis strains have been shown to induce specific local and systemic immune responses against various antigens. Our study aimed at constructing a L. lactis strain expressing haemagglutinin of a Polish avian H5H1 influenza isolate and examining its effect on animals. Expression of the cloned H5 gene was achieved using the nisin-controlled gene expression system. Detection of the intracellular H5 antigen produced in L. lactis was performed by Western blot analysis and confirmed using mass spectrometry. The potential of L. lactis recombinant cells to induce an immune response was examined by setting up preliminary immunization trials on chickens and mice. Obtained sera were tested for specific antibodies by ELISA assays. The results of these studies are a promising step toward developing a vaccine against the bird flu using Lactococcus lactis cells as bioreactors for efficient antigen production and delivery to the mucosal surface