Cite this: DOI: 10.1039/c3mb70557a
Selenium effects on the metabolism of a
Se-metabolizing Lactobacillus reuteri: analysis of
envelope-enriched and extracellular proteomes†
E. Mangiapane,*aC. Lamberti,b A. Pessione,aE. Galano,cA. Amoresanocand E. PessioneaSelenium (Se) has received great attention in the last few years, as it is considered to be essential for human health (prevention of viral infections, heart diseases and ageing-related diseases). Se deficiency can be counteracted by the administration of selenium-enriched probiotics that are able to convert inorganic selenium into less toxic and more bio-available organic forms. This study was performed on Lactobacillus reuteri Lb2 BM DSM 16143, a probiotic LAB previously demonstrated to be able to fix Se into selenocysteines. The aim was to assess Se influence on its metabolism, by a 2-DE proteomic approach, on two different cellular districts: envelope-enriched and extracellular proteomes. While in the envelope-enriched fraction 15 differentially expressed proteins were identified, in the extracellular proteome no quantitative difference was detected. However, at a molecular level, we observed the insertion of Se into selenocysteine, exclusively under the stimulated conditions. The obtained results confirmed the possibility to use L. reuteri Lb2 BM DSM 16143 as a carrier of organic Se that can be easily released in the gut becoming available for the human host.
Introduction
In the post genomic era, the availability of more and more improved methods for analyzing bacterial sub-proteomes has shed light on complex physiological phenomena such as the protein secretion mechanisms,1energy metabolism,2quorum
sensing,3 moonlighting proteins,4,5 parallel catabolic path-ways,6 environmental adaptation7and detoxification mechan-isms related to stress.8,9 The analysis of extracellular and envelope-enriched proteomes can also supply information about exported virulence factors10,11 useful for developing suitable diagnostics and therapeutics12 and for discovering new interesting targets for vaccine development and anti-microbial chemotherapy.13
As far as commensal and/or probiotic bacteria are con-cerned, studying extracellular-surface proteomes allows us to discover proteins involved in bacterial–host cross-talk such as chaperones and moonlighting glycolytic enzymes,14,15immune
system modulators16 and proteins involved in adhesion to biotic surfaces.17–19
In recent years, one of the applications of probiotics is represented by their use as nutraceutical supplements; they can be supplemented with zinc, selenium, vitamins and o-3-fatty acids, in order to enhance beneficial effects on human health.20,21 Selenium (Se) is a fundamental trace element for humans, since its deficiency has been correlated to several diseases (i.e. viral infections, thyroid dysfunctions, different types of cancer and ageing).22,23 Indeed, Se is essential for several functions within our cells: it is present as selenocysteine in 25 selenoproteins that possess antioxidant activity (GPx), and are involved in redox signaling (TrxR) and thyroid hormone metabolism (DIO) or in Se transport and storage (SelP).24,25 Furthermore, it has been widely described as a powerful anti-oxidant and anti-carcinogenic element.26Therefore, the use of probiotics as a vehicle of organic selenium forms in human body represents an interesting perspective in the nutraceutical field. For this reason, the effects of selenium on probiotic cells must be deeply studied, in order to elucidate the mechanisms involved in Se metabolism and to exclude possible harmful effects.
In a previous paper27the up-regulation of a selenocysteine lyase, involved in the selenium-fixing process, has been demonstrated by in toto proteome analyses in a probiotic LAB: Lactobacillus reuteri Lb2 BM DSM 16143 grown in a sodium selenite medium.
aDepartment of Life Sciences and Systems Biology, University of Turin,
Via Accademia Albertina 13, 10123, Torino, Italy. E-mail: erika.mangiapane@unito.it
bCNR ISPA, c/o Bioindustry Park S. Fumero, Via Ribes 5, 10010, Colleretto Giacosa,
Italy
cDepartment of Chemical Sciences, University of Naples ‘‘Federico II’’,
Via Cinthia 4, 80125, Napoli, Italy
†Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3mb70557a Received 6th December 2013, Accepted 16th January 2014 DOI: 10.1039/c3mb70557a www.rsc.org/molecularbiosystems
BioSystems
PAPER
Published on 30 January 2014. Downloaded by UNIVERSITÀ DEGLI STUDI DI TORINO on 26/02/2014 11:19:57.
View Article Online View Journal
The excess of selenite, which could be toxic, undergoes a reduction to elemental selenium that aggregates generating particles on the bacterial cell surface.27 More recent results
revealed that the same strain has the capability to specifically fix inorganic selenium into the selenocysteines of 7 enzymes involved in different pathways within the cell: glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase, phosphoketo-lase, 6-phosphogluconate dehydrogenase, arginine deiminase, ornithine carbamoyltransferase and ribonucleoside hydrolase RihC.28
The aim of the present study was to investigate L. reuteri Lb2 BM DSM 16143 sub-proteomes, such as the envelope-enriched fraction and extracellular compartment, in order to complete an overall study dealing with the influence of selenium on the metabolism of this probiotic confirming its potential as a nutraceutical supplement. For this purpose a 2-DE comparative proteomic approach was used on cells grown in selenite-enriched medium and under control conditions without selenium. To corroborate the proteomic results, a phenotypic test confirming the role of proteins whose expression was influenced by selenium has been performed as well.
Experimental
Culture conditions
Lactobacillus reuteri Lb2 BM DSM 16143, belonging to the collection of BioMan life science S.r.l., was maintained in a modified MRS (de Man, Rogosa and Sharpe) medium (10 g L 1
tryptone enzymatic digest from casein, 8 g L 1 peptone from soybean, 10 g L 1 yeast extract, 10 g L 1 sucrose, 1 mL L 1 tween80, 2 g L 1potassium phosphate dibasic, 5 g L 1sodium acetate, 2 g L 1ammonium citrate tribasic anhydrous, 0.2 g L 1 magnesium sulfate, 0.05 g L 1manganese sulfate) at 24 1C in 0.5 mL aliquots with 0.5 mL of 40% glycerol.
The cultures were grown in closed screw cap bottles, at 37 1C, without shaking. The pH of the medium was adjusted at 6.4 before the inoculum. The bacterial growth was monitored by 600 nm optical density (OD600) measurement. L. reuteri Lb2 BM
DSM 16143 was grown under control condition (MRS modified medium) and under stimulated condition (the same medium supplemented with 4.38 mg L 1of sodium selenite). A sodium
selenite stock solution (2.19 g L 1corresponding to 1 g L 1of Se)
was prepared and sterilized by filtration (single use syringe filter, 0.20 mm, Sartorius Stedim Biotech) and added to the cold autoclaved medium.
For all the cultures three biological replicates were made.
Preparation of envelope-enriched protein extracts
Equivalent amounts of cells (150 mg dry weight for each experiment) were treated in each protein preparation. The cells were harvested in the exponential growth phase (15 h after the inoculum). The biomass was collected by centrifugation and washed in 50 mL 0.85% NaCl. The obtained pellets were resuspended in 3 mL 50 mM Tris-HCl, pH 7.3, EDTA 1 mM, sonicated and clarified as previously described.29To recover the
highest amount of proteins, the pellet was resuspended, soni-cated and clarified again as already described and the two supernatants were put together.6To obtain a fraction enriched
in envelope proteins, the samples were centrifuged (100 000 g, 8 h, 4 1C) to pelletize the membrane fragments. Pellets were solubilized in 20 mM Tris, pH 6.8, 1 mM MgCl2, centrifuged
(45 000 g, 5 h, 4 1C) and resuspended in 200 mL of 50 mM ammonium bicarbonate. The sample was supplemented with 1 mL of 2 : 1 v/v trifluoroethanol/chloroform and incubated at 0 1C for 1 h, vortexing it for 10 s every 5 min. By centrifuging (10 000 g, 5 min, 4 1C) this suspension, an upper aqueous phase, an insoluble interphase and a lower chloroformic phase were obtained. The upper phase was recovered and dried using a vacuum centrifuge.6,30,31The residue was solubilized in rehydration solution (6.5 M urea, 2.2 M thiourea, 1% w/v aminosulfobetaine-14, 5 mM Tris-HCl, pH 8.8, 0.5% IPG buffer (GE Healthcare), 100 mM DTT) and proteins were quantified by the 2-D quant kit (GE Healthcare) and Bradford assay.
Preparation of extracellular protein fraction
The samples were prepared as previously described by Mangia-pane et al.32 Briefly, middle exponential phase cultures were centrifuged (4000 g, 20 minutes, 4 1C, Thermo Scientific SL 16R) and the obtained supernatants were filtered using Steri-cup filters (Millipore), in order to eliminate the biomass. Trichloroacetic acid 16% w/v was added to promote protein precipitation under shaking over night at 4 1C. The obtained suspensions were then ultracentrifuged (35 000 g, 90 min, 4 1C). Pellets were dried, pulverized and resuspended in 50 mM Tris-HCl pH 7.3. 1 mL aliquots of the obtained sample were subjected to phenol extraction as described before.11One mL phenol was added and the mixtures were incubated for 10 min at 70 1C and for 5 min at 0 1C and centrifuged (7000 g, 10 min, room temperature). The upper phase was discarded and 1 mL of MilliQ water was added to the lower phase, which was then incubated for 10 min at 70 1C and for 5 min at 0 1C and centrifuged (7000 g, 10 min, room temperature) again. The upper phase was discarded and 1 mL of ice cold acetone was added to the lower phase before incubating over night at 20 1C. Precipitated proteins were recovered by centrifuging (15 000 g, 20 min, 4 1C) and washed with ice cold acetone (15 000 g, 20 min, 4 1C). Pellets were pulverized and resus-pended in rehydration solution and proteins were quantified by the 2-D quant kit (GE Healthcare) and Bradford assay. 2-DE
IEF (Isoelectrofocusing) was performed as previously described.29 The proteins were separated into 13 cm IPG strips (GE Healthcare) with a linear gradient ranging from 4 to 7: 235 mg of proteins were loaded for the envelope-enriched protein fraction, while 275 mg for the extracellular proteome. IEF was performed using IPGphor (GE Healthcare) at 20 1C, with 66 000 Vh (envelope-enriched protein fraction) and 83 000 Vh (extracellular fraction). After IEF, the strips were incubated at room temperature in 6 M urea, 30% v/v glycerol, 2% w/v SDS, 50 mM Tris-HCl, pH 8.6, enriched at first with 2% w/v DTT for 15 min and afterwards with 4.5% w/v iodoacetamide
for 15 min. They were then sealed at the top of the 1.0 mm vertical second dimension gels. For each sample, SDS-PAGE was carried out on 11.5% T and 3.3% C acrylamide (Biorad Acrylamide) homogeneous gels. The running buffer was 25 mM Tris, 192 mM glycine, 0.1% SDS. The running conditions were 11 1C, 600 V constant voltage, 20 mA per gel, 60 W for 15 min and 11 1C, 600 V constant voltage, 40 mA per gel, 80 W for about 2.5 h. The molecular weight markers were from the Low Mr Electrophoresis Calibration Kit (GE-Healthcare). The gels were automatically stained using Processor Plus (Amersham Biosciences) with freshly prepared Neuhoff stain (Colloidal Coomassie Blue)33and, after image acquisition, they were dried in a GD 2000 Vacuum Gel Drier System (GE Healthcare). Image analysis and statistical analysis
2-DE gels were digitized using Personal Densitometer SI (Amersham Biosciences). Image analysis and spot detection were performed with Progenesis PG200 software (Non Linear Dynamics). Spot detection was automatically performed by using the algorithm named ‘‘2005 detection’’ and manually checked. After the establishment of some user seeds, matching was automatically performed and manually verified. For both the envelope-enriched and the extracellular fractions, two analytical replicates for each of the three biological replicates were used. Spot intensities were measured via normalized spot volumes and were statistically analyzed by means of the t-test: the mean values were considered significantly different when po 0.05. All the spots with po 0.05 and present in at least five out of six replicates identified were selected for MS identification.
Protein identification
Enzymatic digestion was carried out with 200 ng of trypsin in 50 mL of 10 mM NH4HCO3 buffer, pH 7.8. Gel pieces were
incubated at 37 1C overnight. Peptides were then extracted by washing the gel particles with 10 mM NH4HCO3and 1% formic
acid in 50% ACN at room temperature. The resulting peptide mixtures were filtered using a 0.22 PVDF filter from Millipore. The peptide mixtures were analysed by nanoLC-chip MS/MS, using a CHIP MS 6520 QTOF equipped with a capillary 1200 HPLC system and a chip cube (Agilent Technologies). After loading, the peptide mixture (8 mL in 0.1% formic acid) was first concentrated and washed at 4 mL min 1in 40 nL enrichment column (Agilent Technologies chip), with 0.1% formic acid as an eluent. The sample was then fractionated on a C18 reverse-phase capillary column (75 mm 43 mm in the Agilent Technologies chip) at a flow rate of 400 nL min 1with a linear gradient of eluent B (0.1% formic acid in 95% ACN) in A (0.1% formic acid in 2% ACN) from 7 to 60% for 50 min. Doubly and triply charged peptides were selected and analyzed using data-dependent acquisition of one MS scan (mass range from 300 to 2000 m/z) followed by MS/MS scans of the three most abundant ions in each MS scan. Collision energy (CE) applied during peptide fragmentation is calculated by the subsequent empirical equations: CE = 4 V/100 Da 2.4 V. Raw data from nanoLC-MS/ MS were analyzed and converted in common spectral file formats (.mgf mascot generic file), using Qualitative Analysis software
(Agilent MassHunter Workstation Software, version B.02.00). MASCOT software (www.matrixscience.com) version: 2.4.0 was used for the protein identification against the NCBInr database (NCBInr_20120920.fasta; 21582400 sequences; 7401135489 resi-dues), with the taxonomy restriction to Other Firmicutes (2926062 sequences). The MASCOT search parameters were: ‘‘trypsin’’ as enzyme allowing up to 3 missed cleavages, carbamidomethyl on cysteine residues as fixed modification, oxidation of methionine and formation of pyroGlu N-term on glutamine were selected as variable modifications, 10 ppm MS/MS tolerance and 0.6 Da peptide tolerance. By data analysis, the threshold provided to evaluate quality of matches for MS/MS data was found to be 41.
Bile resistance experiment
Four different cultures were set up: L. reuteri Lb2 BM DSM 16143 was grown in MRS medium, MRS medium supplemented with 4.38 mg L 1sodium selenite, MRS medium supplemented with 0.5% bile salts (Sigma Aldrich) and MRS medium supple-mented with both sodium selenite and bile salts. An over-night pre-culture was used to perform the inoculation at 0.1% and after 5 hour growth the number of colony forming units per mL (CFU per mL) was determined by plating serial 10-fold dilutions onto MRS agar. For all the four cultures two different biological replicates were performed.
Results and discussion
Expression pattern of the envelope-enriched protein fraction under Se-fortified and Se-free conditions
Comparative proteomic analyses in the acidic (4–7) pI range were performed on the envelope-enriched protein fraction to detect the differentially expressed proteins between control (MRS modified medium) and stimulated (MRS + 4.38 mg L 1
Na2SeO3) conditions. Maps are shown in Fig. 1.
The image analysis revealed 15 differentially expressed spots: 10 were up-regulated by Se (phosphoketolase, spot 1; phosphoketolase, spot 2; 6-phosphogluconate dehydrogenase, spot 3; bifunctional acetaldehyde-CoA/alcohol dehydrogenase, spot 4; molybdopterin biosynthesis protein MoeA, spot 5; choloylglycine hydrolase, spot 6; F0F1 ATP synthase subunit
beta, spot 7; elongation factor Tu, spot 8; tRNA/rRNA methyl-transferase (SpoU), spot 9; ATP-dependent Clp protease proteo-lytic subunit, ClpP, spot 10) and 5 were down-regulated by Se (diol/glycerol dehydratase reactivating factor, large subunit, spot 11; dehydratase, medium subunit, spot 12; a-enolase, spots 13 and 14; ornithine carbamoyltransferase, spot 15). All of them were identified by nanoLC-chip MS/MS (Table 1; Table S1, ESI†). Fig. 2 shows the average normalized volumes and their variations under the two experimental conditions for each spot.
A very interesting enzyme up-regulated in selenium is molybdopterin biosynthesis protein MoeA (spot 5) that is involved in the synthesis of molybdenum cofactor (Moco) in all Mo-dependent enzymes in bacteria. Moco consists of a molybdopterin (MPT) compound that coordinates a Mo atom.34
MoeA is actually responsible for the insertion of Mo into MPT in order to obtain the activated form of the cofactor.35Mo-cofactor containing enzymes are named Mo hydrolases (MH): this family includes selenium-dependent molybdenum hydroxylases (SDMH) which contain a labile selenium atom, probably not contributed by SeCys, but through some other mechanisms. These enzymes are present in species also carrying the SelD protein (specifically involved in the production of selenophosphate, the activated form of Se), suggesting its involvement in the activation of Se for SDMH maturation.36In a study by Srivastava and coworkers,37 an eubacterial gene cluster that links selD with a gene encoding a molybdenum hydroxylase has been described. In other anaerobic and facultative microorganisms, other genes, probably involved in Se metabolism and Mo cofactor synthesis, co-localize with this group. This specific cluster is present in a lactic acid bacterium, Enterococcus faecalis,37 suggesting the possibility that the same scenario can occur in other LABs, such as L. reuteri. MoeA up-regulation is therefore probably linked to the expression in L. reuteri Lb2 BM of Se-dependent molybdenum hydroxylases, even if no SDMH has been identified in this study.
The up-regulation of F0F1ATP synthase subunit beta (spot 7)
is consistent with the results already obtained for the in toto proteome27in which the ATP synthase F1epsilon subunit has
been observed to be over-expressed in selenium. The beta subunit identified in this work is part of the F1catalytic core
and it is responsible for ATP synthesis.38The F0F1complex is
present at the membrane level, confirming the efficacy of the protocol used to recover proteins. Furthermore this result is in agreement with the observed stimulation of the energy-generating routes (glycolysis and pentose-phosphate pathway) resulting in enhanced ATP synthesis.27
Elongation factor Tu (spot 8) is up-regulated under the stimulated conditions. This protein, normally present in the cytosol and involved in protein synthesis, is described as a moonlighting protein, since it is responsible for adhesion when surface-exposed. EF–Tu has also been detected in the exopro-teome of Bifidobacterium animalis subsp. lactis as putative moonlighting protein with adhesion roles that contribute to the probiotic features of the strain.39In the literature EF–Tu involve-ment in adhesion processes is well docuinvolve-mented: in Mycoplasma
Table 1 List of the identified spots from the envelope-enriched protein fraction after nanoLC-chip MS/MS. For each protein number of peptides, score and percentage of sequence coverage are reported
Spot MW(Da) Identified protein NCBInr ID
No. of peptides Score Sequence coverage (%) 1 91 374 Phosphoketolase gi|194467185 70 1695 58 2 91 374 Phosphoketolase gi|194467185 33 798 47
3 53 498 6-Phosphogluconate dehydrogenase gi|184154309 19 394 32
4 97 640 Bifunctional acetaldehyde-CoA/alcohol dehydrogenase gi|148543558 15 451 15
5 44 850 Molybdopterin biosynthesis protein MoeA gi|184153597 8 276 20
6 36 082 Choloylglycine hydrolase gi|148543961 26 861 76
7 51 600 F0F1ATP synthase subunit beta gi|148543702 29 1100 51
8 43 405 Elongation factor Tu gi|148543883 24 700 47
9 28 292 tRNA/rRNA methyltransferase SpoU gi|148544445 35 945 86
10 21 420 ATP-dependent Clp protease proteolytic subunit gi|148543625 39 1227 85 11 65 655 Diol/glycerol dehydratase reactivating factor, large subunit gi|148544950 55 1857 72
12 25 849 Dehydratase, medium subunit gi|148544952 9 350 32
13 48 010 Phosphopyruvate hydratase gi|194468183 18 619 37
14 48 010 Phosphopyruvate hydratase gi|194468183 56 1856 73
15 37 536 Ornithine carbamoyltransferase gi|148543661 33 1006 83
Fig. 1 Envelope-enriched protein fraction maps of L. reuteri LB2 BM DSM 16143 grown under control and stimulated conditions in the acidic (4–7) pI range. Fifteen spots were differentially expressed between the two conditions.
pneumoniae EF–Tu binds to fibronectin,40 and in Lactobacillus johnsonii it is able to bind mucin and thus intestinal epithelial cells, also displaying immunomodulatory properties.17
Another interesting up-regulated enzyme is bifunctional acetaldehyde-CoA/alcohol dehydrogenase (spot 4). This protein is made up of two conserved domains (Aldh and Adh), it is NADH-dependent, and it is involved in the two-step sequential conversion of acetyl-CoA to ethanol as an end product of fermentation.41 A few bacteria are known to catalyze these two reactions sequentially among which Streptococcus bovis42 and Clostridium acetobutylicum.43 In acetic acid bacteria this enzyme is membrane-bound and it is composed of three sub-units with different molecular masses (72–80 kDa, 44–54 kDa, 8–20 kDa).44 On the basis of the protein spot position in the
obtained 2-DE maps of L. reuteri Lb2 BM DSM 16143, it is possible to assert that the identified subunit is probably the intermediate molecular mass component. The up-regulation of this enzyme in the heterofermenter L. reuteri is consistent with the over-expression of phosphoketolase (spot 1, 2) and of both glycolysis and pentose phosphate pathway previously demon-strated in selenium-fortified media,27 since bifunctional acetaldehyde-CoA/alcohol dehydrogenase is at the end of the fermentation process, allowing conversion of acetate into ethanol, the second main product of heterofermenters. The correlation between these two proteins has also been confirmed by an interactome bioinformatic analysis performed with STRING 9.05 with a score of 0.557 (medium confidence).
The over-expression of the ATP-dependent Clp protease proteolytic subunit, ClpP, (spot 10) suggests a stress-response in L. reuteri Lb2 BM grown in selenium. It has been previously demonstrated that the growth on selenium can cause a certain degree of stress in L. reuteri: GroEL and GrpE, two well known stress protein, were up-regulated in the presence of 4.38 mg L 1 sodium selenite.27ClpP is a serine protease that degrades small
peptides (o7 amino acids). In Lactococcus lactis ClpP is involved in the degradation of misfolded proteins originated after stress exposure.45 ClpP protease gene has been demon-strated to be induced under stress conditions (stationary phase and ethanol stress) also in the LAB Oenococcus oeni by real-time reverse transcription-PCR analysis46and to be regulated during heat stress in L. plantarum by proteomic studies.47
Among selenium up-regulated proteins, choloylglycine hydrolase (spot 6) represents an interesting enzyme since it catalyzes the initial ‘‘gateway’’ reaction in the bacterial meta-bolism of CBAs (conjugated bile acids): the reaction consists in a deconjugation of CBAs to liberate free primary bile acids (BAs; cholic acid or chenodeoxycholic acid) and amino acids.48CBAs
have been suggested to repress bacterial growth in the small intestine by means of direct antimicrobial effects, up-regulation of host mucosal defenses, or synergistic action of both mechan-isms.49Choloylglycine hydrolase has widely been reported to be exposed on the surface of several Gram positive bacteria, such as Clostridium perfringens50 and Bifidobacterium lactis BI07, where it is responsible for Plg-binding.14 The expression and up-regulation of this enzyme by L. reuteri Lb2 BM DSM 16143 have a great importance for its probiotic traits, suggesting both the ability of the strain to survive to CBAs action and also a positive role of selenium in enhancing bile-salt survival. In order to confirm this capacity and to understand the effect of selenium on this phenomenon, the resistance to bile salts has been tested under both conditions (with and without Se). As shown in Fig. 3, a real increment of the resistance ability of L. reuteri Lb2 BM to bile salts in the presence of selenium was observed: 30% of survival with Se versus 17% without Se. In the literature there is no evidence of a link between bile salt resistance and selenium; however, such a capacity can encourage the use of this strain, grown in the presence of selenium, as a good probiotic in general and especially to treat hypercholesterolemic
Fig. 2 For each identified protein average normalized volumes and their variations (SEM = standard error of the mean) under the two experimental conditions are shown.
subjects. Indeed, since deconjugated bile acids are not well absorbed by the gut mucosa and are excreted with feces, new cholesterol is driven from blood to liver for de novo CBA synthesis,51allowing a reduction of blood cholesterol levels.
The down-regulated ornithine carbamoyltransferase (OTC, spot 15) is a very interesting protein. This enzyme is involved in the energy- and ammonia-generating ADI pathway and catalyzes the conversion of citrulline into carbamoyl-phosphate and ornithine.
Its down-regulation is consistent with the previous finding of a Se-induced down-expression of other two enzymes belonging to ADI pathway, namely arginine deiminase and carbamate kinase.27
These results corroborate the suggestion of the need for the acidophilic L. reuteri to control the entire operon to attenuate NH3-induced toxicity, since it is meanwhile involved to cope with
stress induced by selenium. Interestingly OTC was found in the envelope-enriched fraction as well as on the surface of Clostridium perfringens,50and the opportunistic Staphylococcus epidermidis.52If we consider the physiological role of ADI, we find that this route is coupled with an antiport system ensuring new arginine supply inside the cell by exchanging the OTC-generated ornithine. The presence of this OTC enzyme in the proximity of the ornithine/ arginine antiporter (i.e. membrane) can constitute an advantage for L. reuteri Lb2 BM physiology, by facilitating substrate supply to the antiport system.
Expression pattern of the extracellular protein fraction under Se-fortified and Se-free conditions
The comparative proteomic analysis of the extracellular frac-tion of L. reuteri Lb2 BM DSM 16143 revealed that selenium does not induce quantitative differences in protein expression (Fig. 4). For this reason, we focused our attention exclusively on the map deriving from Se-grown cultures. In these maps, image analysis revealed 59 spots that were then identified by nano-LC-chip MS/MS as 21 different proteins (considering isoforms).
Fig. 3 Effects of selenium supplementation on bile salt resistance. CFU number without treatment of bile salts was considered as a positive control (100% growth) both in the presence and absence of selenium. The survival percentage after treatment of bile salts is reported under both the tested conditions. The percentages were calculated as the mean values between the two biological replicates.
Fig. 4 Extracellular protein fraction map of L. reuteri LB2 BM DSM 16143 grown in the presence of sodium selenite in the acidic (4–7) pI range. Spots corresponding to proteins in which selenocysteines were identified are in the thicker boxes (spots 8 and 19).
These proteins are the same as those already described in a recent paper of our group32 in which we considered only extracellular proteins derived from cultures without selenium. Identified proteins have been divided into three func-tional groups: (1) cell wall processing enzymes (spot 1, mannosyl-glycoprotein endo-beta-N-acetylglucosamidase and N-acetylmuramoyl-L-alanine amidase; spot 2,
binding LysM; spot 3, NLP/P60 protein; spot 4, peptidoglycan-binding LysM; spot 5, unknown extracellular protein lr1267; spot 6, Apf1-like protein); (2) adhesion-involved proteins (spot 7, phosphopyruvate hydratase (a-enolase); spot 8, glycer-aldehyde 3-phosphate dehydrogenase; spot 9, elongation factor Tu; spot 10, elongation factor Ts; spot 11, molecular chaperone DnaK; spot 12, phosphoglycerate kinase; spot 13, phosphoglyceromutase; spot 14, trigger factor); (3) other pro-teins (spot 15, dextransucrase; spot 16, sucrose phosphory-lase; spot 17, mannitol dehydrogenase; spot 18, alcohol dehydrogenase; spot 19, phosphoketolase; spot 20, hypo-thetical protein Lreu_0552, spot 21, ribosome recycling factor). Since in a recent paper we proved28the ability of the strain to fix selenium into seven proteins in the form of selenocysteine (SeCys, U), the presence of selenopeptides in the extracellular secreted proteins from selenium-grown bacteria was manually checked in the raw data of the LC-MS/MS. This analysis revealed the presence of two peptides containing a SeCys and belonging, respectively, to glyceraldehyde 3-phosphate dehydrogenase (spot 8, NDGVDFVLEUTGFYTSAEK) and phos-phoketolase (spot 19, NQEUINLFVTSK) (Fig. 5).
Phosphoketolase has been observed to be induced in both in toto proteome27 and cell-envelope enriched proteome, and identified in this experiment analyzing extracellular proteome. It probably belongs to the so-called moonlighting proteins that display the ability to be present in different cellular districts.53 To this class belongs GAPDH as well. This glycolytic enzyme,
when extracellularly-exposed or released can act as an adhe-sine, since it is able to bind extracellular matrix proteins.54 It can be hypothesized that this enzyme could act as a sele-nium storage protein, as already suggested by Ogasawara and co-workers.55
Both glyceraldehyde 3-phosphate dehydrogenase and phos-phoketolase were already identified in the in toto proteome28as selenocysteine-containing proteins: this result corroborates once more the ability of the strain to fix selenium generating organic Se-forms and can explain the previously reported increase of Se concentration in the external medium after about 6 hours of growth.28 Furthermore, the fact that
SeCys-containing proteins are released in the extracellular environ-ment before cell lysis is an added value for a probiotic strain to be employed as a nutraceutical, since this mechanism increases selenium bioavailability in the human gut. In fact it has to be considered that only organic Se forms (selenocysteine and/or selenomethionine) can be metabolized and assimilated by humans without inducing toxic effects, actually representing the most bio-available form for human body.
Conclusions
Lactobacillus reuteri Lb2 BM DSM 16143 has been widely characterized for its ability to accumulate and metabolize selenium, with the final purpose to produce a nutraceutical supplement to be employed in the treatment of Se-deficiency. In this scenario, the use of comparative proteomic in different subcellular districts allowed us to have an overall picture of the effects of Se on bacterial physiology. In this study, in particular, the obtained results corroborate what described by our group in previous papers: (i) Se induces energy production through glycolysis and the pentose phosphate pathway at the expense
Fig. 5 Manual search of the selenocysteine-containing peptide NQEUINLFVTSK after interpretation of the MS/MS spectrum of the double charged ion of phosphoketolase at m/z 750.83.
of the ADI pathway; (ii) Se causes a slight stress response represented by the up-regulation of chaperones and stress proteins; (iii) it is interesting to underline the presence of SeCys residues in phosphoketolase and GAPDH in both the in toto and extracellular proteome, suggesting a Se-storage function.
The detection of SeCys in secreted proteins confers an added value since the host receives organic Se forms both during bacterial life in the human gut and once more after cell lysis.
Furthermore, Se seems to increase the probiotic potential of L. reuteri Lb2 BM DSM 16143; indeed, the up-regulation of choloylglycine hydrolase and the subsequent phenotypic analyses allowed us to demonstrate a positive effect of Se on the survival to bile salts. Even if the mechanisms of this phenomenon remain unknown, it is an appreciated ability since it guarantees the survival of the strain during the gastrointestinal transit.
Notes and references
1 H. Tjalsma, H. Antelmann, J. D. H. Jongbloed, P. G. Braun, E. Darmon, R. Dorenbos, J.-Y. F. Dubois, H. Westers, G. Zanen, W. J. Quax, O. P. Kuipers, S. Bron, M. Hecker and J. M. Van Dijl, Microbiol. Mol. Biol. Rev., 2004, 68, 207–233.
2 A. Pessione, C. Lamberti and E. Pessione, Mol. BioSyst., 2010, 6, 1419–1430.
3 A. S. Nouwens, S. A. Beatson, C. B. Whitchurch, B. J. Walsh, H. P. Schweizer, J. S. Mattick and S. J. Cordwell, Microbiol-ogy, 2003, 149, 1311–1322.
4 M. Gohar, N. Gilois, R. Graveline, C. Garreau, V. Sanchis and D. Lereclus, Proteomics, 2005, 5, 3696–3711.
5 F. Genovese, J. D. Coisson, A. Majumder, A. Pessione, B. Svensson, S. Jacobsen and E. Pessione, Food Res. Int., 2013, 54, 1072–1079.
6 E. Pessione, A. Pessione, C. Lamberti, J. D. Coisson, K. Riedel, R. Mazzoli, S. Bonetta, L. Eberl and C. Giunta, Proteomics, 2009, 9, 2695–2710.
7 N. Hansmeier, T.-C. Chao, J. Kalinowski, A. Puhler and A. Tauch, Proteomics, 2006, 6, 2465–2476.
8 H. Antelmann, E. Darmon, D. Noone, J.-W. Veening, H. Westers, S. Bron, O. P. Kuipers, K. M. Devine, M. Hecker and J. M. Van Dijl, Mol. Microbiol., 2003, 49, 143–156.
9 R. Mazzoli, E. Pessione, M. Dufour, V. Laroute, M. G. Giuffrida, C. Giunta, M. Cocaign-Bousquet and P. Loubie`re, Amino Acids, 2010, 39, 727–737.
10 M. J. J. B. Sibbald, A. K. Ziebandt, S. Engelmann, M. Hecker, A. De Jong, H. J. M. Harmsen, G. C. Raangs, I. Stokroos, J. P. Arends, J. Y. F. Dubois and J. M. Van Dijl, Microbiol. Mol. Biol. Rev., 2006, 70, 755–788.
11 A. Pessione, C. Lamberti, L. Cocolin, S. Campolongo, A. Grunau, S. Giubergia, L. Eberl, K. Riedel and E. Pessione, Proteomics, 2012, 12, 431–447.
12 H. B. Yu, R. Kaur, S. Lim, X. H. Wang and K. Y. Leung, Proteomics, 2007, 7, 436–449.
13 D. Bumann, S. Aksu, M. Wendland, K. Janek, U. Zimny-Arndt, N. Sabarth, T. F. Meyer and P. R. Jungblut, Infect. Immun., 2002, 70, 3396–3403.
14 M. Candela, S. Bergmann, M. Vici, B. Vitali, S. Turroni, B. J. Eikmanns, S. Hammerschmidt and P. Brigidi, J. Bacteriol., 2007, 189, 5929–5936.
15 J. Antikainen, V. Kuparinen, K. Lahteenmaki and T. Korhonen, J. Bacteriol., 2007, 189, 4539–4543.
16 H. H. Smits, A. Engering, D. van der Kleij, E. C. de Jong, K. Schipper, T. M. M. van Capel, B. A. J. Zaat, M. Yazdanbakhsh, E. A. Wierenga, Y. van Kooyk and M. L. Kapsenberg, J. Allergy Clin. Immunol., 2005, 115, 1260–1267.
17 D. Granato, G. E. Bergonzelli, R. D. Pridmore, L. Marvin, M. Rouvet and I. E. Corthesy-Theulaz, Infect. Immun., 2004, 72, 2160–2169.
18 G. E. Bergonzelli, D. Granato, R. D. Pridmore, L. F. Marvin-Guy, D. Donnicola and I. E. Corthesy-Theulaz, Infect. Immun., 2006, 74, 425–434.
19 B. Sanchez, P. Bressolier and M. C. Urdaci, FEMS Immunol. Med. Microbiol., 2008, 54, 1–17.
20 S. S. Awaisheh, M. S. Y. Haddadin and R. K. Robinson, Int. Dairy J., 2005, 15, 1184–1190.
21 A. N. Madhu, P. Giribhattanavar, M. S. Narayan and S. G. Prapulla, Biotechnol. Lett., 2010, 32, 503–506.
22 G. F. Combs, BioFactors, 2000, 12, 39–43.
23 M. B. Zimmerman and J. Kohrle, Thyroid, 2002, 12, 867–878. 24 G. V. Kryukov, S. Castellano, S. V. Novoselov, A. V. Lobanov, O. Zehtab, R. Guigo and V. N. Gladyshev, Science, 2003, 300, 1439–1443.
25 L. V. Papp, J. Lu, A. Holmgren and K. K. Khanna, Antioxid. Redox Signaling, 2007, 9, 775–806.
26 A. Belicova, L. Krizkova, J. Dobias, J. Krajkovic and L. Ebringer, Folia Microbiol., 2004, 49, 301–305.
27 C. Lamberti, E. Mangiapane, A. Pessione, R. Mazzoli, C. Giunta and E. Pessione, Proteomics, 2011, 11, 2212–2221. 28 E. Galano, E. Mangiapane, J. Bianga, A. Palmese, E. Pessione, J. Szpunar, R. Lobinski and A. Amoresano, Mol. Cell. Proteomics, 2013, 12, 2196–2204.
29 E. Pessione, R. Mazzoli, M. G. Giuffrida, C. Lamberti, E. Garcia-Moruno, C. Barello, A. Conti and C. Giunta, Proteomics, 2005, 5, 687–698.
30 K. Zuobi-Hasona, P. J. Crowley, A. Hasona, A. S. Bleiweis and L. J. Brady, Electrophoresis, 2005, 26, 1200–1205.
31 J. M. P. Deshusses, J. A. Burgess, A. Scherl, Y. Wenger, N. Walter, V. Converset, S. Paesano, G. L. Corthals, D. F. Hochstrasser and J.-C. Sanchez, Proteomics, 2003, 3, 1418–1424.
32 E. Mangiapane, C. Lamberti, A. Pessione, P. Ceruti, F. Novelli, E. Galano, R. Virkola, T. K. Korhonen and E. Pessione, J. Integr. OMICS, 2013, 3, 145–156.
33 V. Neuhoff, N. Arold, D. Taube and W. Ehrhardt, Electro-phoresis, 1988, 9, 255–262.
34 K. V. Rajagopalan, Biochem. Soc. Trans., 1997, 25, 757–761. 35 J. D. Schrag, W. Huang, J. Sivaraman, C. Smith, J. Plamonodon, R. Larocque, A. Matte and M. Cygler, J. Mol. Biol., 2001, 310, 419–431.
36 D. H. Haft and W. T. Self, Biol. Direct, 2008, 3, 4.
37 M. Srivastava, C. Mallard, T. Barke, L. E. Hancock and W. T. Self, J. Bacteriol., 2011, 193, 1643–1652.
38 M. Diez, M. Borsch, B. Zimmermann, P. Turina, S. D. Dunn and P. Graber, Biochemistry, 2004, 43, 1054–1064.
39 O. Gilad, B. Svensson, A. H. Viborg, B. Stuer-Lauridsen and S. Jacobsen, Proteomics, 2011, 11, 2503–2514.
40 S. Balasubramanian, T. R. Kannan and J. B. Baseman, Infect. Immun., 2008, 76, 3116–3123.
41 J. Membrillo-Hernandez, P. Echave, E. Cabiscol, J. Tamarit, J. Ros and E. C. C. Lin, J. Biol. Chem., 2000, 275, 33869–33875.
42 N. Asanuma, T. Yoshii and T. Hino, Arch. Microbiol., 2004, 181, 122–128.
43 R. V. Nair, G. N. Bennett and E. T. Papoutsakis, J. Bacteriol., 1994, 176, 871–885.
44 J. Fre´bortova, K. Matsushita, H. Arata and O. Adachi, Biochim. Biophys. Acta, 1998, 1363, 24–34.
45 D. Frees and H. Ingmer, Mol. Microbiol., 1999, 31, 79–87. 46 C. Beltramo, C. Grandvalet, F. Pierre and J. Guzzo, J. Bacteriol.,
2004, 186, 2200–2205.
47 P. Russo, M. de la Luz Mohedano, V. Capozzi, P. F. de Palencia, P. Lo´pez, G. Spano and D. Fiocco, Int. J. Mol. Sci., 2012, 13, 10680–10696.
48 B. V. Jones, M. Begley, C. Hill, C. G. M. Gahan and J. R. Marchesi, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 13580–13585.
49 M. Begley, C. Hill and C. G. M. Gahan, Appl. Environ. Microbiol., 2006, 72, 1729–1738.
50 S. I. Alam, S. Bansod, R. B. Kumar, N. Sengupta and L. Singh, BMC Microbiol., 2009, 9, 162.
51 M. P. St-Onge, E. R. Farnworth and P. J. H. Jones, Am. J. Clin. Nutr., 2000, 71, 674–681.
52 M. Hussain, G. Peters, G. S. Chhatwal and M. Herrmann, Infect. Immun., 1999, 67, 6688–6690.
53 J. C. Jeffery, Trends Genet., 2004, 19, 415–417.
54 R. A. Alvarez, M. W. Blaylock and J. B. Baseman, Mol. Microbiol., 2003, 48, 1417–1425.
55 Y. Ogasawara, G. M. Lacourciere, K. Ishii and T. C. Stadtman, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 1012–1016.
