Delivery of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> HpaA to gastrointestinal mucosal immune sites using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and its immune efficacy in mice

Objective

To develop a safe and effective oral vaccine against Helicobacter pylori using its HpaA protein expressed in Lactococcus lactis.

Results

The gene encoding HpaA was obtained by PCR and ligated to pNZ8110-lysM following digestion with NaeI + SphI. The recombinant plasmid was transferred into E. coli for multiplication, and then into L. lactis. The recombinant L. lactis was induced to express HpaA, resulting in two products of 29 and 25 kDa, both of which yielded positive immunoreaction with mouse antisera against H. pylori, as confirmed by immunoblot assays. The 29 kDa product constituted 12% of the cell lysates. Oral inoculation with the engineered L. lactis evoked significantly elevated serum IgG level in mice (P < 0.05).

Conclusions

A novel engineered L. lactis strain was developed that efficiently produces whole HpaA protein with desired antigenicity and potent immunogenicity. It provides a basis for approaches to L. lactis-delivered anti-H. pylori vaccination.

     

Construction of a recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strain expressing a fusion protein of Omp22 and HpaA from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> for oral vaccine development

Objectives

To develop orally administrated anti-Helicobacter pylori vaccination, a Lactococcus lactis strain was genetically constructed for fusion expression of H. pylori protective antigens HpaA and Omp22.

Results

The fusion gene of omp22 and hpaA with an adapter encoding three glycines was cloned from a plasmid pMAL-c2x-omp22-hpaA into Escherichia coli MC1061 and L. lactis NZ3900 successively using a shutter vector pNZ8110. Expression of the fusion gene in L. lactis was induced with nisin resulting in production of proteins with molecular weights of 50 and 28 kDa. Both of them were immunoreactive with mouse anti-H. pylori sera as determined via western blotting. Oral vaccination of BALB/c mice using the L. lactis strain carrying pNZ8110-omp22-hpaA elicited significant systematic humoral immune response (P < 0.05).

Conclusions

This is the first report showing that a fusion protein of two H. pylori antigens was efficiently expressed in L. lactis with immunogenicity. This is a considerable step towards H. pylori vaccines.

     

Cytoplasmic expression of a thermostable invertase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis> in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Objectives

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis.

Results

The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively.

Conclusions

Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

     

Bile salt tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> is enhanced by expression of bile salt hydrolase thereby producing less bile acid in the cells

Objectives

Changes of bile salt tolerance, morphology and amount of bile acid within cells were studied to evaluate the exact effects of bile salt hydrolase (BSH) on bile salt tolerance of microorganism.

Results

The effect of BSHs on the bile salt tolerance of Lactococcus lactis was examined by expressing two BSHs (BSH1 and BSH2). Growth of L. lactis expressing BSH1 or BSH2 was better under bile salt stress compared to wild-type L. lactis. As indicated by transmission electron microscopy, bile acids released by the action of BSH induced the formation of micelles around the membrane surface of cells subject to conjugated bile salt stress. A similar micelle containing bile acid was observed in the cytoplasm by liquid chromatography-mass spectrometry. BSH1 produced fewer bile acid micelles in the cytoplasm and achieved better cell growth of L. lactis compared to BSH2.

Conclusions

Expression of BSH improved bile salt tolerance of L. lactis but excessive production by BSH of bile acid micelles in the cytoplasm inhibited cell growth.

     

Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.

     

Expression of lycopene biosynthesis genes fused in line with Shine-Dalgarno sequences improves the stress-tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Objectives

Lycopene biosynthetic genes from Deinococcus radiodurans were co-expressed in Lactococcus lactis to produce lycopene and improve its tolerance to stress.

Results

Lycopene-related genes from D. radiodurans, DR1395 (crtE), DR0862 (crtB), and DR0861 (crtI), were fused in line with S hine-Dalgarno (SD) sequences and co-expressed in L. lactis. The recombinant strain produced 0.36 mg lycopene g^-1 dry cell wt after 48 h fermentation. The survival rate to UV irradiation of the recombinant strain was higher than that of the non-transformed strain.

Conclusion

The L. lactis with co-expressed genes responsible for lycopene biosynthesis from D. radiodurans produced lycopene and exhibited increased resistance to UV stress, suggesting that the recombinant strain has important application potential in food industry.

     

Improvement of the respiration efficiency of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> by decreasing the culture pH

Objective

The growth characteristics and intracellular hemin concentrations of Lactococcus lactis grown under different culture pH and aeration conditions were examined to investigate the effect of culture pH on the respiration efficiency of L. lactis NZ9000 (pZN8148).

Results

Cell biomass and biomass yield of L. lactis grown with 4 μg hemin/ml and O₂ were higher than those without aeration when the culture pH was controlled at 5–6.5. The culture pH affected the respiratory efficiency in the following order of pH: 5 > 5.5 > 6 > 6.5; the lag phase increased as the culture pH decreased. Hemin accumulation was sensitive to culture pH. Among the four pH conditions, pH 5.5 was optimal for hemin accumulation in the cells. The highest intracellular hemin level in L. lactis resting cells incubated at different pH saline levels (5–6.5) was at pH 5.5.

Conclusion

The respiration efficiency of L. lactis under respiration-permissive conditions increases markedly as the culture pH decreases. These results may help develop high cell-density L. lactis cultures. Thus, this microorganism may be used for industrial applications.

     

Oral vaccine of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice

Objective

An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus.

Results

Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group.

Conclusion

Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

<Emphasis Type="Italic">massPix</Emphasis>: an R package for annotation and interpretation of mass spectrometry imaging data for lipidomics

Introduction

Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools.

Objectives

We have developed massPix—an R package for analysing and interpreting data from MSI of lipids in tissue.

Methods

massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries.

Results

Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering.

Conclusion

massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications.

     

Improvement of 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> by moderate expression of <Emphasis Type="Italic">puuC</Emphasis> (encoding an aldehyde dehydrogenase)

Objective

To improve 1,3-propanediol production in Klebsiella pneumoniae, the effects of puuC expression in lactate- and lactate/2,3-butanediol-deficient strains were assessed.

Results

Overexpression of puuC (encoding an aldehyde dehydrogenase) inhibited 1,3-propanediol production and increased 3-hydroxypropionic acid formation in both lactate- and lactate/2,3-butanediol-deficient strains. An improvement in 1,3-propanediol production was only achieved in a lactate-deficient strain via moderate expression of puuC; at the end of the fermentation, 1,3-propanediol productivity increased by 14 % compared with the control. Further comparative analysis of the metabolic flux distributions in different strains indicated that 3-hydroxypropionic acid formation could play a considerable role in cell metabolism in K. pneumoniae.

Conclusion

An improvement in 3-hydroxypropionic acid formation would be beneficial for cell metabolism, which can be accomplished by enhancing 1,3-propanediol productivity in a lactate-deficient strain via moderate expression of puuC.

     

Metabolic profiling identifies trehalose as an abundant and diurnally fluctuating metabolite in the microalga <Emphasis Type="Italic">Ostreococcus tauri</Emphasis>

Introduction

The picoeukaryotic alga Ostreococcus tauri (Chlorophyta) belongs to the widespread group of marine prasinophytes. Despite its ecological importance, little is known about the metabolism of this alga.

Objectives

In this work, changes in the metabolome were quantified when O. tauri was grown under alternating cycles of 12 h light and 12 h darkness.

Methods

Algal metabolism was analyzed by gas chromatography-mass spectrometry. Using fluorescence-activated cell sorting, the bacteria associated with O. tauri were depleted to below 0.1% of total cells at the time of metabolic profiling.

Results

Of 111 metabolites quantified over light–dark cycles, 20 (18%) showed clear diurnal variations. The strongest fluctuations were found for trehalose. With an intracellular concentration of 1.6 mM in the dark, this disaccharide was six times more abundant at night than during the day. This fluctuation pattern of trehalose may be a consequence of starch degradation or of the synchronized cell cycle. On the other hand, maltose (and also sucrose) was below the detection limit (~10 μM). Accumulation of glycine in the light is in agreement with the presence of a classical glycolate pathway of photorespiration. We also provide evidence for the presence of fatty acid methyl and ethyl esters in O. tauri.

Conclusions

This study shows how the metabolism of O. tauri adapts to day and night and gives new insights into the configuration of the carbon metabolism. In addition, several less common metabolites were identified.

     

Global proteomic mapping of alkali stress regulated molecular networks in <Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L.

Background and aims

Soil salinization with high pH condition is a major abiotic stress to plant growth and crop productivity. Helianthus tuberosus L. is an important stress tolerant plant and can survive in the saline-alkali soil and semiarid areas. The aim of this study is to identify the effect of alkali stress on H. tuberosus through global proteomics analysis and improve understanding of the alkalinity resistance of plants.

Methods

H. tuberosus seedlings were exposed to different level alkali stress for 7 days. Protein profiling was quantified by conducting MS-based comparative proteomics analysis. RT-PCR study was carried out to analyze the mRNA expression levels of candidate alkali stress response proteins.

Results

The response of H. tuberosus to alkali stress was detected at both physiological and molecular levels. 104 differentially expressed proteins from H. tuberosus leaves response to Na₂CO₃ treatment were successfully identified. Functional categorization of these identified proteins showed that the accumulation level of proteins involved in glycolysis, TCA cycle, PSI system, ROS scavenging and signal transduction increased under alkali stress.

Conclusions

Based on the observation of plant growth and the investigation of molecular regulation, H.tuberosus could resist certain alkali stress by modulating carbohydrate metabolism and redox homeostasis. These findings provide a new sight into the underlying molecular mechanisms of alkali resistance in plant.

     

Profiling,isolation and structure elucidation of specialized acylsucrose metabolites accumulating in trichomes of <Emphasis Type="Italic">Petunia</Emphasis> species

Introduction

Acylsugar specialized metabolites function as defenses against insect herbivores, and are the most abundant specialized metabolites produced in Solanaceous trichomes. Metabolite profiling provides the foundation for determining the genetic basis of specialized metabolism and its evolution.

Objectives

To profile and identify acylsugar specialized metabolites in three Petunia species: P. axillaris, P. integrifolia and P. exserta.

Methods

Metabolites were profiled using ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOF MS). Metabolites were purified using solid phase extraction and HPLC, and structures were established using NMR spectroscopy.

Results

Twenty-eight distinct acylsucrose formulas, representing a sampling of more than 100 different detected chemical forms, were purified from three Petunia species and structures have been proposed based on one- and two-dimensional NMR data. 15 of the 28 purified acylsugars were sucrose pentaesters that possess a malonyl group on the fructose ring. These malonate esters can be readily distinguished from other acylsugars based on distinct masses of pseudomolecular ions and fragment ions generated using multiplexed collision-induced dissociation. Chemical diversity of acylsugars was observed between Petunia species, particularly with respect to the lengths of acyl chains and specific acylation positions.

Conclusions

These findings suggest substrate selectivity of various acyltransferases in Petunia species.