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Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.

Lactobacillus acidophilus NCFM (NCFM) is a well-documented probiotic bacterium isolated from human gut. Detailed 2D gel-based NCFM proteomics addressed the so-called alkaline range, i.e., pH 6-11. Proteins were identified in 150 of the 202 spots picked from the Coomassie Brilliant Blue stained 2D gel using MALDI-TOF-MS. The 102 unique gene products among the 150 protein identifications were assigned to different functional categories, and evaluated by considering a calculated distribution of abundance as well as grand average of hydrophobicity values. None of the very few available lactic acid bacteria proteome reference maps included the range of pI >7.0. The present report of such data on the proteome of NCFM fundamentally complements current knowledge on protein profiles limited to the acid and neutral pH range.

Lactobacillus acidophilus NCFM is a probiotic bacterium adapted to survive in the gastrointestinal tract and with potential health benefits to the host. Lactitol is a synthetic sugar alcohol used as a sugar replacement in low calorie foods and selectively stimulating growth of L. acidophilus NCFM. In the present study the whole-cell extract proteome of L. acidophilus NCFM grown on glucose until late exponential phase was resolved by 2-DE (pH 3-7). A total of 275 unique proteins assigned to various physiological processes were identified from 650 spots. Differential 2-DE (DIGE) (pH 4-7) of L. acidophilus NCFM grown on glucose and lactitol, revealed 68 spots with modified relative intensity. Thirty-two unique proteins were identified in 41 of these spots changing 1.6-12.7-fold in relative abundance by adaptation of L. acidophilus NCFM to growth on lactitol. These proteins included β-galactosidase small subunit, galactokinase, galactose-1-phosphate uridylyltransferase and UDP-glucose-4-epimerase, which all are potentially involved in lactitol metabolism. This first comprehensive proteome analysis of L. acidophilus NCFM provides insights into protein abundance changes elicited by the prebiotic lactitol.

Probiotics are live microorganisms that exert health-promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well-studied probiotic strain Bifidobacterium animalis subsp. lactis BB-12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2-DE coupled with MALDI-TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either outside the cell or on its surface. These proteins include solute-binding proteins for oligosaccharides, amino acids and manganese, cell wall-metabolizing proteins, and 18 proteins that have been described to interact with human host epithelial cells or extracellular matrix proteins. The potential functions include binding of plasminogen, formation of fimbriae, adhesion to collagen, attachment to mucin and intestinal cells as well as induction of immunomodulative response. These findings suggest a role of the proteins in colonization of the gastrointestinal tract, adhesion to host tissues, or immunomodulation of the host immune system. The identification of proteins predicted to be involved in such interactions can pave the way towards well targeted studies of the protein-mediated contacts between bacteria and the host, with the goal to enhance the understanding of the mode of action of probiotic bacteria.

Increased climatic variability is resulting in an increase of both the frequency and the magnitude of extreme climate events. Therefore, cereals may be exposed to more than one stress event in the growing season, which may ultimately affect crop yield and quality. Here, effects are reported of interaction of water deficits and/or a high-temperature event (32°C) during vegetative growth (terminal spikelet) with either of these stress events applied during generative growth (anthesis) in wheat. Influence of combinations of stress on protein fractions (albumins, globulins, gliadins and glutenins) in grains and stress-induced changes on the albumin and gliadin proteomes were investigated by 2-DE and MS. The synthesis of individual protein fractions was shown to be affected by both the type and time of the applied stresses. Identified drought or high-temperature-responsive proteins included proteins involved in primary metabolism, storage and stress response such as late embryogenesis abundant proteins, peroxiredoxins and α-amylase/trypsin inhibitors. Several proteins, e.g. heat shock protein and 14-3-3 protein changed in abundance only under multiple high temperatures. Increased climatic variability is resulting in an increase of both the frequency and the magnitude of extreme climate events. Therefore, cereals may be exposed to more than one stress event in the growing season, which may ultimately affect crop yield and quality. Here, effects are reported of interaction of water deficits and/or a high-temperature event (32°C) during vegetative growth (terminal spikelet) with either of these stress events applied during generative growth (anthesis) in wheat. Influence of combinations of stress on protein fractions (albumins, globulins, gliadins and glutenins) in grains and stress-induced changes on the albumin and gliadin proteomes were investigated by 2-DE and MS. The synthesis of individual protein fractions was shown to be affected by both the type and time of the applied stresses. Identified drought or high-temperature-responsive proteins included proteins involved in primary metabolism, storage and stress response such as late embryogenesis abundant proteins, peroxiredoxins and α-amylase/trypsin inhibitors. Several proteins, e.g. heat shock protein and 14-3-3 protein changed in abundance only under multiple high temperatures.

The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms.

Udgivelsesdato: Jul Cereal seed germination involves a complex coordination between different seed tissues. Plasma membranes must play crucial roles in coordination and execution of germination; however, very little is known about seed plasma membrane proteomes due to limited tissue amounts combined with amphiphilicity and low abundance of membrane proteins. A fraction enriched in plasma membranes was prepared from embryos dissected from 18 h germinated barley seeds using aqueous two-phase partitioning. Reversed-phase chromatography on C(4) resin performed in micro-spin columns with stepwise elution by 2-propanol was used to reduce soluble protein contamination and enrich for hydrophobic proteins. Sixty-one proteins in 14 SDS-PAGE bands were identified by LC-MS/MS and database searches. The identifications provide new insight into the plasma membrane functions in seed germination.

Udgivelsesdato: 2007-Dec Germination of barley is accompanied by changes in water-soluble seed proteins. 2-DE was used to describe spatio-temporal proteome differences in dissected seed tissues associated with germination and the subsequent radicle elongation. Protein identification by MS enabled assignment of proteins and functions to the seed embryo, aleurone, and endosperm. Abundance in 2-DE patterns was monitored for 48 different proteins appearing in 79 gel spots at 8 time-points up to 72 h post imbibition (PI). In embryo, a beta-type proteasome subunit and a heat shock protein 70 fragment were among the earliest proteins to appear (at 4 h PI). Other early changes were observed that affected spots containing desiccation stress-associated late embryogenesis abundant and abscisic acid (ABA)-induced proteins. From 12 h PI proteins characteristic for desiccation stress disappeared rapidly, as did a putative embryonic protein and an ABA-induced protein, suggesting that these proteins are also involved in desiccation stress. Several redox-related proteins differed in spatio-temporal patterns at the end of germination and onset of radicle elongation. Notably, ascorbate peroxidase that was observed only in the embryo, increased in abundance at 36 h PI. The surprisingly early changes seen in the protein profiles already 4 h after imbibition indicate that germination is programmed during seed maturation

Using thiol-specific fluorescence labelling, over 30 putative target proteins of thioredoxin h with diverse structures and functions have been identified in seeds of barley and other plants. To gain insight at the structural level into the specificity of target protein reduction by thioredoxin h, thioredoxin h-reducible disulphide bonds in individual target proteins are identified using a novel strategy based on differential alkylation of cysteine thiol groups by iodoacetamide and 4-vinylpyridine. This method enables the accessible cysteine side chains in the thiol form (carbamidomethylated) to be distinguished from those inaccessible or disulphide bound form (pyridylethylated) according to the mass difference in the peptide mass maps obtained by matrix-assistend laser desorption/ionisation-time of flight mass spectrometry. Using this approach, in vitro reduction of disulphides in recombinant barley alpha-amylase/subtilisin inhibitor (BASI) by barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with two-dimensional electrophoresis for convenient thioredoxin h-reducible disulphide identification in barley seed extracts without the need for protein purification or production of recombinant proteins. Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four alpha-amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located structurally close to the alpha-amylase binding surfaces of BASI and alpha-amylase inhibitor BMAI-1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects of the recognition mechanism and regulation of target proteins.

Probiotic cultures encounter oxidative conditions during manufacturing, yet protein abundance changes induced by such stress have not been characterized for some of the most common probiotics and starters. This comparative proteomics investigation focuses on the response by Lactobacillus acidophilus NCFM to H2 O2, simulating an oxidative environment. Bacterial growth was monitored by BioScreen and batch cultures were harvested at exponential phase for protein profiling of stress responses by 2D gel based comparative proteomics. Proteins identified in 19 of 21 spots changing in abundance due to H2 O2 were typically related to carbohydrate and energy metabolism, cysteine biosynthesis, and stress. In particular, increased cysteine synthase activity may accumulate a cysteine pool relevant for protein stability, enzyme catalysis, and the disulfide-reducing pathway. The stress response further included elevated abundance of biomolecules reducing damage such as enzymes from DNA repair pathways and metabolic enzymes with active site cysteine residues. By contrast, a protein-refolding chaperone showed reduced abundance, possibly reflecting severe oxidative protein destruction that was not overcome by refolding. The proteome analysis provides novel insight into resistance mechanisms in lactic acid bacteria against reactive oxygen species and constitutes a valuable starting point for improving industrial processes, food design, or strain engineering preserving microorganism viability.

A proteomic analysis was conducted to map the events during the initial stages of the interaction between the fungal pathogen Fusarium graminearum and the susceptible barley cultivar Scarlett. Quantification of fungal DNA demonstrated a sharp increase in fungal biomass in barley spikelets at 3 days after inoculation. This coincided with the appearance of discrete F. graminearum-induced proteolytic fragments of β-amylase. Based on these results, analysis of grain proteome changes prior to extensive proteolysis enabled identification of barley proteins responding early to infection by the fungus. In total, the intensity of 51 protein spots was significantly changed in F. graminearum-infected spikelets and all but one were identified. These included pathogenesis-related proteins, proteins involved in energy metabolism, secondary metabolism and protein synthesis. A single fungal protein of unknown function was identified. Quantitative real-time RT-PCR analysis of selected genes showed a correlation between high gene expression and detection of the corresponding proteins. Fungal genes encoding alkaline protease and endothiapepsin were expressed during 1-3 days after inoculation, making them candidates for generation of the observed β-amylase fragments. These fragments have potential to be developed as proteome-level markers for fungal infection that are also informative about grain protein quality.

In Central and Southern Italy, where durum wheat represents one of the most widely cultivated crops, grain filling occurs during Spring, a period characterised by sudden increases in temperature. Wheat grain proteins are classified into albumins, globulins, and prolamins. The non-prolamin fractions include proteins with metabolic activity or structural function. In order to investigate the consequences of heat stress on the accumulation of non-prolamin proteins in mature durum wheat kernels, the Italian cultivar Svevo was subjected to two thermal regimes (heat stress vs. control) during grain filling. The 2D patterns of non-prolamin proteins were monitored to identify polypeptides affected by heat stress. This study shows that heat stress alters significantly the durum wheat seed proteome, although the fold changes range only between 1.2 and 2.2. This analysis revealed 132 differentially expressed polypeptides, 47 of which were identified by MALDI TOF and MALDI-TOF-TOF MS and included heat shock proteins, proteins involved in the glycolysis and carbohydrate metabolism, as well as stress related proteins. Many of the heat induced polypeptides are considered to be allergenic for sensitive individuals. The differences observed with previously reported data regarding bread wheat may be explained by the absence of the D genome in durum wheat L'articolo è disponibile sul sito dell'editore: www.interscience.wiley.com Project “Proteine e geni per la protezione delle piante dagli stress biotici e abiotici (PROTEO-STRESS), MIPAF, Italy - MIUR, "Fondo per il sostegno dei giovani e per favorire la mobilità degli studenti" in the framework “Valorizzazione dei prodotti tipici dell’agroalimentare e sicurezza alimentare attraverso nuovi sistemi di caratterizzazione e garanzia di qualità” - COST Action FA0603 Plant Proteomics.

Several barley (Hordeum vulgare) cultivars are used in the production of malt for brewing. The malt quality depends on the cultivar, its growth and storage conditions, and the industrial process. To enhance studies on malt quality, we embarked on a proteome analysis approach for barley seeds and malt. The proteome analysis includes two-dimensional (2-D) gel electrophoresis, mass spectrometry, and bioinformatics for identification of selected proteins. This project initially focused on proteins in major spots in the neutral isoelectric point range (pI 4-7) including selected spots that differ between four barley cultivars. The excellent malting barley cultivar Barke was used as reference. Cultivar differences in the 2-D gel spot patterns are observed both at the seed and the malt level. In seed extracts one of the proteins causing variations has been identified as an alpha-amylase/trypsin inhibitor. In malt extracts multiple forms of the alpha-amylase isozyme 2 have been identified in varying cultivar characteristic spot patterns. The present identification of proteins in major spots from 2-D gels includes 27 different proteins from 42 spots from mature seed extract, while only three specific proteins were identified by analysing 13 different spots from the corresponding malt extract. It is suggested that post-translational processing causes the same protein to occur in different spots.