Lactobacillus acidophilus-Rutin Interplay Investigated by Proteomics. - PDF Download Free (2024)

RESEARCH ARTICLE

Lactobacillus acidophilus—Rutin Interplay Investigated by Proteomics Maria Fiorella Mazzeo1*, Rosa Lippolis2, Alida Sorrentino1, Sarah Liberti1,3, Federica Fragnito1, Rosa Anna Siciliano1 1 Institute of Food Sciences, National Research Council, Avellino, Italy, 2 Institute of Biomembranes and Bioenergetics, National Research Council, Bari, Italy, 3 Department of Chemical Sciences, University of Naples Federico II, Naples, Italy * [emailprotected]

Abstract

OPEN ACCESS Citation: Mazzeo MF, Lippolis R, Sorrentino A, Liberti S, Fragnito F, Siciliano RA (2015) Lactobacillus acidophilus—Rutin Interplay Investigated by Proteomics. PLoS ONE 10(11): e0142376. doi:10.1371/journal.pone.0142376 Editor: Humberto Lanz-Mendoza, Instituto Nacional de Salud Pública, MEXICO Received: May 14, 2015 Accepted: October 21, 2015 Published: November 6, 2015 Copyright: © 2015 Mazzeo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was partially supported by funds from the Italian Ministry of Economy and Finance to CNR and ENEA for the project “Innovazione e sviluppo del Mezzogiorno - Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare”—Legge n.191/2009. All additional funding was from internal resources of CNR. Competing Interests: The authors have declared that no competing interests exist.

Dietary polyphenols are bioactive molecules that beneficially affect human health, due to their anti-oxidant, anti-inflammatory, cardio-protective and chemopreventive properties. They are absorbed in a very low percentage in the small intestine and reach intact the colon, where they are metabolized by the gut microbiota. Although it is well documented a key role of microbial metabolism in the absorption of polyphenols and modulation of their biological activity, molecular mechanisms at the basis of the bacteria-polyphenols interplay are still poorly understood. In this context, differential proteomics was applied to reveal adaptive response mechanisms that enabled a potential probiotic Lactobacillus acidophilus strain to survive in the presence of the dietary polyphenol rutin. The response to rutin mainly modulated the expression level of proteins involved in general stress response mechanisms and, in particular, induced the activation of protein quality control systems, and affected carbohydrate and amino acid metabolism, protein synthesis and cell wall integrity. Moreover, rutin triggered the expression of proteins involved in oxidation-reduction processes.This study provides a first general view of the impact of dietary polyphenols on metabolic and biological processes of L. acidophilus.

Introduction Dietary polyphenols are bioactive molecules that exert beneficial effects on human health, due to their anti-oxidant, anti-inflammatory, cardio-protective and chemopreventive properties [1,2]. These compounds are secondary plant metabolites found in different vegetables, fruits and beverages such as tea and wine [3]. They are usually classified in flavonoids (whose structures contain two benzene rings linked through a heterocyclic pyrone ring) and nonflavonoid phenolics, a heterogeneous group comprising benzoic acids, stilbenes, lignans, hydrolyzable tannins, gallotannins and ellagitannins. Moreover, polyphenols may be conjugated in nature with various carbohydrates and organic acids. Among flavonols, a class of flavonoids, quercetin is one of the most abundant found in several food matrices, and extensively studied for its relevant biological properties [4]. Rutin (quercetin-3-O-rutinoside) is one of the main

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

1 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

glycoconjugated forms of quercetin present in planta, and its antioxidant properties and pharmacological benefits for the treatment of chronic diseases such as cancer, diabetes, hypertension and hypercholesterolemia have been recently reviewed [5]. During digestion, polyphenols are absorbed in a very low percentage in the small intestine and reach intact the colon, where they are metabolized by gut microbiota. The transformation of polyphenols occurs via a complex set of chemical reactions (deglycosylation, demethylation, dehydroxylation, decarboxylation and reduction) and leads to the production of a relatively limited number of metabolites, mainly small phenolic acids, regardless of the complexity of natural polyphenols [6–8]. However, microbial metabolism is not only required for polyphenol absorption but also modulates their biological activity. Phenolic compounds produced by the microbial degradation could exhibit an enhanced bioavailability or biological activity compared to those of the native compounds and, in turn, these metabolites could modulate microbiota composition via their antimicrobial activity or pathogen inhibition. Notwithstanding the well documented impact of polyphenols on human health, molecular mechanisms at the basis of the bacteria-polyphenols interaction are still poorly understood and few intestinal bacterial species involved in their metabolism have been identified so far [8,9]. Bifidobacteria and lactobacilli, components of gut microbiota, seemed to be involved in polyphenol degradation as they produced different glycosyl-hydrolases able to efficiently hydrolyze glycoconjugated polyphenols [10]. In particular, the rhamnosidase from Lactobacillus acidophilus was able to efficiently degrade flavanone and flavonol glycosides such as hesperidin, naringin and rutin [11]. Therefore, as lactobacilli might contribute to bioavailability and absorption of these compounds, it is crucial to gain insight into the potential interaction between these microorganisms and polyphenols. A few proteomic studies were carried out to address the effects of polyphenols on lactobacilli metabolism and investigate the molecular mechanisms adopted by these bacteria to grow in the presence of polyphenols. Response mechanisms to tannic acid have been examined in L. hilgardii and L. plantarum [12–14]. Rivas-Sendra and coworkers highlighted cytoplasmic proteins presumably involved in the response of L. casei BL23 to p-coumaric acid [15]. As to L. acidophilus, proteomic studies were performed to assess how environmental stresses affected microbial metabolism [16,17], and to obtain a quantitative profile of bacteriocins present in probiotic preparations [18]. Regarding the probiotic properties of these bacteria, proteins involved in adhesion to intestinal epithelial cells [19,20] and in immunomodulation [21] or associated with the ability of L. acidophilus to reduce serum cholesterol [22] have been identified. Furthermore, changes in protein expression induced by the prebiotic lactitol have been reported [23]. On the other hand, no proteomic investigation has been carried out on L. acidophilus-polyphenols interplay. However, it has been documented that a specific intestinal strain of L. acidophilus showed exceptional resistance to different tea polyphenol extracts, that were able to slow down the growth of other bacteria [24]. Similarly, the polyphenolic extract of Sesbania grandiflora flower, containing rutin as a major flavonoid, exhibited a growth promoting property against L. acidophilus [25]. Furthermore, rutin had no inhibitory effect on representative gut bacteria species and induced a slight growth stimulation of lactobacilli while its aglycone quercetin exhibited a strong growth inhibitory effect [26]. In this context, we carried out the first differential proteomic study on a model system constituted by a potential intestinal probiotic L. acidophilus strain and rutin. These preliminary results could be considered an initial step to reveal the impact of rutin on L. acidophilus metabolism and functional processes.

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

2 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

Fig 1. Growth curves of L. acidophilus in control conditions and in the presence of rutin. (A) Growth curves of L. acidophilus obtained by plate counting. (B) Growth curves of L. acidophilus obtained measuring the optical density at 600 nm. Arrows indicate the times of bacterial cell collection for the further proteomic analyses. Values are the means of four independent experiments. doi:10.1371/journal.pone.0142376.g001

Materials and Methods Bacterial growth and proteome extraction L. acidophilus DSMZ 20079 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Germany) samples were prepared by using a two-step procedure. Bacteria from slants kept at 4°C were inoculated into DE MAN, ROGOSA, SHARPE broth (MRS, Thermo Scientific, Basingstoke, UK) and incubated for 24 h at 37°C. Then, the bacterial suspension (1%, 2.0×108 colony forming unit/mL (CFU/mL)) was used to inoculate fresh medium without (control sample) and with rutin (250 μg/mL final concentration). As reported by Duda-Chodak, this rutin concentration was unable to exert any antimicrobial activity on several bacteria species including lactobacilli [26]. For each condition, four bacterial growths were monitored by plate counting on MRS agar incubated at 37°C for 48 h and measuring the optical density at 600 nm (OD600) every two hours, in order to obtain the growth curves (S1 Table, Fig 1). Bacterial cells from two biological replicates for each condition were collected by centrifugation (7700 g for 15 min at 4°C) at early stationary phase defined as time 18 h for control sample (OD600 = 2.05, CFU = 4.37×108) and time 22 h for L. acidophilus grown in the presence of rutin (OD600 = 2.09, CFU = 4.28×108) and used to perform the proteomic experiments (S1 Table). Cell pellets from 50 mL of each bacterial culture were washed twice with PBS and submitted to enzymatic lysis with mutanolysin (134 units for 1.8×1010 cells, Sigma-Aldrich, St. Louise, MO, USA) in 20 mM Tris-HCl, 10 mM MgCl2, 0.5 M sucrose pH 7.4 containing a co*cktail of protease inhibitors (1/100 v/v, Sigma-Aldrich) for 2 h at 37°C. DNase I and Rnase A reactions (enzyme to substrate ratio of 1:100 v/v, Sigma-Aldrich) were carried out for 30 min at 37°C. Finally, protein precipitation was performed using 10 volumes of acidic acetone (1 mM HCl final concentration)/methanol (50/50 v/v) at −20°C for 18 h. Protein pellets were finally dissolved in buffer solution (8 M urea, 4% (w/v) CHAPS, 40 mM Tris–HCl, 1% DTT) and protein concentration was determined using the Bradford assay [27].

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

3 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

2-DE and image analysis Protein samples were separated by 2-DE [28,29]. Samples containing 250 μg proteins were diluted in IPG strip rehydration buffer (8 M urea, 2% (w/v) CHAPS, 0.5% IPG buffer, 2% DTT and 0.5% bromophenol blue) and loaded on 24-cm IPG strips pH 4–7 (GE Healthcare, Amersham Biosciences AB, Uppsala, Sweden). Isoelectric focusing (IEF) was carried out at 20°C on the Ettan IPGphor Isoelectric Focusing System (GE Healthcare) to 69.5 kVh totally. After focusing, the IPG strips were equilibrated for 15 min in the equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.5% bromophenol blue) containing 1% DTT, and for further 15 min in the same equilibration buffer containing 2.5% iodoacetamide. The second dimension gel electrophoresis (SDS-PAGE) was carried out using the vertical slab separation unit Ettan Dalt II System (GE Healthcare). hom*ogeneous SDS 12.5% polyacrylamide gel was used in a Laemmli system at a constant current of 15 mA/gel and at 10°C [30]. After separation, 2-DE gels were stained using Coomassie Blue Colloidal dye (Sigma-Aldrich). Stained gels were scanned with an Image Scanner (GE Healthcare) at 300 dpi resolution and analyzed with Image-Master 2D Platinum v.6 software (GE Healthcare) as already reported [31]. Briefly, spot detection was carried out using the optimized setting values for spot intensity, spot area and saliency, determined by applying real-time filters in order to minimize the detection of artifacts. After spot detection, manual spot editing was carried out to remove artifacts that escaped the filtering process. Three gels from the two biological replicates for each growth condition were used to create the two match sets. Relative spot volume (% vol), i.e. digitized staining intensity integrated over the area of the individual spot divided by the sum of volume of all spots in the gel and multiplied by 100, was used for spot quantification [32]. Spots present in all the gels of each group and exhibiting a significant intensity difference (> 1.5 fold change) between the two samples with a P value < 0.05, using the two tailored Student’s t-test for equal or unequal variance (depending on the calculated variance of spots), were considered to contain differentially expressed proteins.

Protein identification and functional classification Spots were excised from 2-DE gels and in-gel triptyc digestion was carried out following the procedure described by Shevchenko et al. [33]. MALDI-TOF-MS analyses were carried out on a Voyager DE PRO mass spectrometer (Applied Biosystems, Foster City, CA, USA) operating in positive-ion reflectron mode. Mass spectra were calibrated using as internal standards the monoisotopic peaks of angiotensin (m/z 931.5154) and adrenocorticotropic hormone (ACTH) fragment 18–39 (m/z 2465.1989) and data were processed using the DataExplorer 5.1 software (Applied Biosystems). Protein identification was achieved based on the mass spectral data using the Mascot Wizard tool for searches against the NCBInr database (http://www. matrixscience.com/). Parameters for all searches were as follows: all entries as taxonomic category, trypsin as enzyme, carbamidomethyl as fixed modification for cysteine residues, oxidation as variable modification for methionine residues, up to one missed cleavage and up to 50 ppm as mass tolerance. Identified proteins were classified on the basis of their biological functions using the bioinformatics resource KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome. jp/kegg/). Integrated function and protein interactions were explored using the database and web resource STRING v.9.1 (Search Tool for the Retrieval of Interacting Genes/Proteins, http:// string-db.org/). Active prediction methods used in our analysis were neighbourhood,

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

4 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

Fig 2. Representative 2-DE gels of L. acidophilus proteome. (A) 2-DE gel obtained from the proteome of L. acidophilus grown under control conditions. (B) 2-DE gel obtained from the proteome of L. acidophilus grown in the presence of rutin. Spots exhibiting significant differences in mean intensities are indicated. doi:10.1371/journal.pone.0142376.g002

coexpression, experiments, co-occurrence, databases and text mining, using custom confidence value of 0.600 [34].

Results Influence of rutin on bacterial growth The impact of rutin on L. acidophilus growth was clearly shown by the growth curves reported in Fig 1. The polyphenol had no inhibitory effect, but it slightly affected bacterial growth, with L. acidophilus cells reaching the early stationary phase with a delay of four hours in the presence of rutin (22 h) comparing to control cells (18 h). However, no substantial differences were observed in cell viability in the stationary phase (S1 Table). This observation suggested that a phase of adaptation to rutin was necessary to L. acidophilus, before it could initiate cell multiplication. Based on these results, the proteomic study was carried out on bacterial cells grown for 22 h in the presence of rutin and for 18 h in the control medium.

Proteomic analyses and functional classification of identified proteins 2-DE maps obtained from the analysis of L. acidophilus proteome contained about 430 spots (Fig 2). By comparing the 2-DE map of L. acidophilus grown in the presence of rutin to that of control sample, 38 spots displayed a mean intensity variation higher than a 1.5 factor and 35 proteins contained in these spots were identified (Table 1). The analysis of adjacent spots in the 2-DE maps led to the identification of the same protein in two cases, i.e. glyceraldehyde-3-phosphate dehydrogenase was identified in spots 264, 284 and 285, and ATP-dependent Clp protease subunit ClpE was identified in spots 50 and 54, thus indicating the presence of isoforms probably due to post-translational modifications. Two co-migrating proteins were identified in spot 382; this spot was not taken into account in the further analyses. Twenty-six proteins were present in higher abundance and eight proteins were present in lower abundance in the 2-DE map of cells grown in the presence of rutin. Spot 272, containing an outer surface protein, was detectable only in the 2-DE map of L. acidophilus grown in the presence of rutin.

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

5 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

Table 1. Identification of differentially expressed proteins. Spot

Accession Number (NCBInr)

Protein

Organism

MW

pI

Score

Matched Peptides

Coverage

5

gi|58337125

isoleucyl-tRNA synthetase

L. acidophilus NCFM

106576

5.12

207

20/30

29

30

gi|58336392

ribonucleoside triphosphate reductase

L. acidophilus NCFM

83982

5.62

491

47/76

63

33

gi|58336628

elongation factor G

L. acidophilus NCFM

76806

4.94

221

17/30

40

50

gi|58336963

ATP-dependent Clp protease, ATP-binding subunit ClpE

L. acidophilus NCFM

81748

5.00

221

23/32

39

54

gi|58336963

ATP-dependent Clp protease, ATP-binding subunit ClpE

L. acidophilus NCFM

81748

5.00

283

35/69

51

72

gi|58337255

pyruvate kinase

L. acidophilus NCFM

63136

5.23

227

27/59

58

90

gi|58337121

cell division protein FtsZ

L. acidophilus NCFM

48119

4.55

135

15/60

42

105

gi|58336743

molecular chaperone GroEL

L. acidophilus NCFM

57785

4.98

269

32/65

52

151

gi|58337766

glutamine synthetase

L. acidophilus NCFM

50672

5.17

227

27/71

48

157

gi|58337885

seryl-tRNA synthetase

L. acidophilus NCFM

49813

5.31

310

36/65

65

170

gi|58337265

30S ribosomal protein S1

L. acidophilus NCFM

44414

5.15

220

16/30

55

174

gi|58337282

ATP-dependent protease ATP-binding subunit HslU

L. acidophilus NCFM

52775

5.26

367

38/64

55

180

gi|58337501

pyridine mercuric reductase

L. acidophilus NCFM

49135

6.08

361

42/67

63

217

gi|58337130

tRNA-specific 2-thiouridylase MnmA

L. acidophilus NCFM

42426

5.50

231

20/67

59

242

gi|58337163

aspartate aminotransferase

L. acidophilus NCFM

43142

5.44

327

24/46

62

245

gi|58337903

multiple sugar ABC transporter ATPase

L. acidophilus NCFM

40533

5.87

264

22/44

56

264

gi|58337019

glyceraldehyde-3-phosphate dehydrogenase

L. acidophilus NCFM

36643

5.92

128

14/68

47

272

gi|489643333

outer surface protein

L. acidophilus NCFM

41656

6.08

202

17/37

45

274

gi|58336976

isomerase

L. acidophilus NCFM

38113

5.22

238

14/23

55

275

gi|58336405

D-lactate dehydrogenase

L. acidophilus NCFM

39177

4.96

215

28/63

76

282

gi|58337217

citrate lyase ligase

L. acidophilus NCFM

39455

5.57

246

28/74

62

284

gi|58337019

glyceraldehyde-3-phosphate dehydrogenase

L. acidophilus NCFM

36643

5.92

344

28/67

93

285

gi|58337019

glyceraldehyde-3-phosphate dehydrogenase

L. acidophilus NCFM

36643

5.92

341

30/52

76

292

gi|58336524

oxidoreductase

L. acidophilus NCFM

36225

5.57

241

22/29

64

293

gi|58337614

branched-chain amino acid aminotransferase

L. acidophilus NCFM

37926

5.31

222

22/50

53

372

gi|58337021

triosephosphate isomerase

L. acidophilus NCFM

27770

4.72

259

24/81

72 (Continued)

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

6 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

Table 1. (Continued) Spot

Accession Number (NCBInr)

382

gi|58337926

Protein elongation factor Pphosphoglycerate mutase

gi|58337936

Organism

MW

pI

Score

Matched Peptides

Coverage

L. acidophilus NCFM

21060

5.26

129

17/42

59

L. acidophilus NCFM

25036

5.17

168

14/42

74

384

gi|58337337

glutamine ABC transporter ATP-binding protein

L. acidophilus NCFM

28347

5.58

246

32/45

81

386

gi|58337608

elongation factor P

L. acidophilus NCFM

20849

5.07

72

6/14

29

387

gi|58338068

two-component response regulator

L. acidophilus NCFM

25504

5.12

99

7/21

30

388

gi|499573661

NAD-dependent dehydratase

L. acidophilus

23620

5.35

297

22/31

70

394

gi|489643392

ribosome recycling factor

L. acidophilus

20675

5.66

194

16/33

42

396

gi|58337527

heat shock protein GrpE

L. acidophilus NCFM

22044

5.79

163

18/33

63

399

gi|58337015

ATP-dependent Clp protease proteolytic subunit ClpP

L. acidophilus NCFM

21400

5.26

143

14/46

61

402

gi|58337281

ATP-dependent protease peptidase subunit HslV

L. acidophilus NCFM

18714

5.21

176

16/35

57

403

gi|58337284

hypothetical protein LBA0987

L. acidophilus NCFM

15561

6.07

190

16/50

70

407

gi|58337297

trp repressor binding protein

L. acidophilus NCFM

16992

4.81

145

13/52

72

433

gi|58336560

bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase

L. acidophilus NCFM

50162

5.57

140

20/70

41

doi:10.1371/journal.pone.0142376.t001

Functional classification of identified proteins was performed by KEGG and protein-protein interactions were analyzed using the web resource STRING (Table 2, Fig 3). The network is presented under evidence view, whereby different line colors represent the types of evidence for the association, whereas proteins are represented as nodes. Fig 3 shows the interaction between 22 out of 34 identified proteins, found to be linked either directly or indirectly through one or more interacting proteins, suggesting the existence of functional linkages. Notably, STRING analysis highlighted three main functional modules forming tightly connected clusters. The first functional module (circled in purple in Fig 3) included proteins involved in the energy metabolism (kpyK, tpiA, ldhD, LAB0698, Table 2), the second one (circled in green in Fig 3) included proteins responsible for protein folding and degradation (groEL, grpE, LBA0638, clpP, hslIV, hslIU, Table 2), the last one (circled in red in Fig 3) included ribosomal proteins and translation factors (fusA, efp2, LBA0968, LBA1267). These functional analyses highlighted that rutin induced in L. acidophilus metabolic changes mainly affecting carbohydrate and amino acid metabolism. Moreover, protein synthesis and molecular processes necessary to assure proper protein folding and avoid protein aggregation were activated by rutin.

Discussion Rutin is a polyphenol relevant for its antioxidant properties and its aglycone form (quercetin) has well documented positive effects on human health [4]. As most polyphenols, rutin is not absorbed in the small intestine and is metabolized in the gut by indigenous microbiota. Therefore, bioavailability and biological activity of rutin depend on the potential interaction with intestinal bacteria [7–9]. However, information on the molecular mechanisms underlying this

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

7 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

Table 2. Functional classification of the identified proteins and regulation of their abundance. Protein

Gene

Locus tag

Functional classification

Regulation a

CARBOHYDRATE METABOLISM glyceraldehyde-3-p dehydrogenase

LBA0698

Glycolysis / Gluconeogenesis

UPb(2.5)

triosephosphate isomerase

tpiA

LBA0700

Glycolysis / Gluconeogenesis / Fructose and mannose metabolism

DOWN(-1.8)

pyruvate kinase

kpyK

LBA0957

Glycolysis / Gluconeogenesis / Pyruvate metabolism

UP(1.8)

D-lactate dehydrogenase

ldhD

LBA0055

Pyruvate metabolism

DOWN(-1.7)

LBA0651

Pentose phosphate pathway

UP(1.6)

isomerase

AMINO ACIDS BIOSYNTESIS AND METABOLISM LBA0856

Lysine biosynthesis

UP(1.6)

branched-chain amino acid aminotransferase

aspartate aminotransferase ilvE

LBA1341

Valine, leucine and isoleucine biosynthesis or degradation

UP(1.6)

glutamine synthetase

glnA

LBA1501

Alanine, aspartate and glutamate metabolism / Arginine and proline metabolism

DOWN(-1.6)

ribonucleoside triphosphate reductase

rtpR

NUCLEOTIDE METABOLISM LBA0041

Purine metabolism

DOWN(-1.8)

AMINO SUGAR AND NUCLEOTIDE SUGAR METABOLISM bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase

glmU

LBA0219

DOWN(-1.8)

TRANSPORT SYSTEM glutamine ABC transporter ATP-binding protein

LBA1045

UP(7.8)

multiple sugar-binding ABC-transporter ATPase

LBA1645

DOWN(-2.0)

TRANSLATION isoleucyl-tRNA synthetase

ileS

LBA0817

DOWN(-1.8)

seryl-tRNA synthetase

serS

LBA1626

DOWN(-1.8)

ribosome recycling factor Rrf

LBA1267

UP(2.8)

30S ribosomal protein S1

rpsA

LBA0968

UP(2.9)

elongation factor G

fusA

LBA0289

UP(1.6)

elongation factor P

efp2

LBA1335

UP(1.6)

TRANSCRIPTION two-component response regulator

LBA1820

UP(2.0)

SIGNAL TRANSDUCTION citrate lyase ligase

citC

LBA0914

Two-component system

UP(1.6)

FOLDING, SORTING AND DEGRADATION ATP-dependent Clp protease, ATP-binding subunit ClpE

clpE

LBA0638

UPb(5.3)

ATP-dependent Clp protease proteolytic subunit

clpP

LBA0694

UP(1.6)

ATP-dependent protease ATP-binding subunit HslU

hslU

LBA0985

UP(1.7)

ATP-dependent protease subunit HslV

hslV

LBA0984

UP(1.6)

heat shock protein GrpE

grpE

LBA1248

UP(1.6)

molecular chaperone GroEL

groEL

LBA0406

tRNA-specific 2-thiouridylase MnmA

mnmA

LBA0822

UP(1.6) Sulfur relay system

UP(1.7)

OXIDATION-REDUCTION PROCESS oxidoreductase

LBA0182

UP(1.6)

trp repressor binding protein

LBA1001

UP(1.7)

pyridine mercuric reductase

LBA1220

UP(2.3) (Continued)

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

8 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

Table 2. (Continued) Protein

Gene

Locus tag

cell division protein FtsZ

ftsZ

LBA0812

Functional classification

Regulation a

MISCELLANEOUS NAD-dependent dehydratase

UP(1.6) UP(2.2)

hypothetical protein LBA0987

LBA0987

UP(2.1)

outer surface protein

LBA1239

spot detected only in 2-DE map of L. acidophilus grown with rutin

a

Changes in protein levels are reported as the ratio between the relative spot volume from L. acidophilus grown in the presence of rutin and L. acidophilus grown in control conditions (Vrutin/Vcontrol) for increased proteins and as the negative reciprocal values (-Vcontrol/Vrutin) for decreased proteins. Changes in protein levels >1.5 have been considered significant.

b

Average fold change for proteins contained in more than one spot has been calculated considering the relative spot volume of all the spots containing the same protein and a fold change >1.5 has been considered significant.

doi:10.1371/journal.pone.0142376.t002

interaction are still scarce. Lactobacilli could be leading actors in polyphenol degradation as they are able to efficiently hydrolyze the glycosidic moiety of flavonols and flavanones [3]. In this context, a differential proteomic study was performed to investigate the adaptive response of a potentially probiotic L. acidophilus strain to the presence of rutin in the growth medium. In our conditions, the presence of rutin induced a growth retardation, although cell viability in the stationary phase did not changed significantly (Fig 1). A similar effect was also displayed by other phenolic compounds on some Lactobacillus species, in particular tannic acid on L. plantarum [13], and p-coumaric acid on L. casei [15]. On the contrary, the

Fig 3. Interaction network (as displayed by EMBL STRING) of the identified proteins. Green lines indicate neighborhood, blue lines indicate co-occurrence, black lines indicate co-expression, purple lines indicate experiments, light blue lines indicate databases and yellow lines indicate text-mining. The network was obtained with a confidence score of 0.600. Main functional modules are circled. doi:10.1371/journal.pone.0142376.g003

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

9 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

polyphenol extract from Sesbania grandiflora flower, containing rutin as a major flavonoid, had a growth promoting property on L. acidophilus. However, this effect could be partially due to the synergic action of other polyphenols present in the extract [25]. Proteomic data were analyzed by STRING, which is a web resource aimed to predict proteinprotein interactions, considering both physical and functional associations and integrating information from numerous sources, including experimental repositories, computational prediction methods and public text collections [34]. STRING analysis led to define three main functional modules forming tightly connected clusters that included proteins involved in the energy metabolism, protein folding and degradation, and ribosomal proteins and translation factors (Fig 3). In particular, rutin induced stress conditions in bacterial cells, that elicited a response by activating the production of proteins belonging to the protein quality control systems such as the folding and degradation machineries to promote refolding and proper protein assembly, prevent aggregation phenomena and hydrolyze damaged proteins. These molecular mechanisms are regarded as general stress response systems in lactobacilli [35]. In fact, an increased abundance of heat shock protein GrpE, molecular chaperone GroEL, and ATP-binding subunits ClpE and ClpP of ATP-dependent Clp protease (included in the cluster circled in green in Fig 3) was observed in cells grown in the presence of rutin. Moreover, the overexpression of HslV protease and HslU ATPase that compose the two-component proteasome-related prokaryotic system (Table 2), the bacterial hom*ologue of the eukaryotic 20S proteasome core particle, well documented the need to degrade damaged or misfolded proteins in order to maintain cellular homeostasis [36]. In addition, rutin induced the activation of glycolysis as the key enzymes glyceraldehyde3-phosphate dehydrogenase and pyruvate kinase (included in the cluster circled in purple in Fig 3) were overexpressed in cells grown in the presence of this molecule. An increase of ATP production should be necessary to sustain the activity of the ATP-dependent folding and degradation systems. A similar trend has been also associated to the effect of tannic acid on L. plantarum [13]. The increased level of pyruvate kinase could also parallel with the higher expression level of branched-chain amino acid aminotransferase. This enzyme is involved in both biosynthesis and catabolism of branched-chain amino acids (BCAA) and catalyzes the final step of the conversion of pyruvate into BCAA in the biosynthetic pathway and the first step of BCAA degradation that leads to the production of α-keto acids in the catabolitic pathway. These are precursors of branched chain fatty acids, molecules that affect bacterial membrane fluidity. In Streptococcus mutans, BCAA aminotransferase played a key role in conferring acid tolerance and the biosynthesis of BCAA was proposed as a way to maintain proper intracellular pH conditions [37,38]. Overexpression of this protein was also reported in acid-stressed L. plantarum 423 cells [39]. In this light, it could be suggested that this enzyme might also play a role in the adaptive response to rutin in L. acidophilus. Our results also suggested that the presence of rutin could negatively affect cell wall integrity. In fact, a decreased amount of bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (GlmU) and D-lactate dehydrogenase in bacteria grown in the presence of rutin was found. GlmU is a bi-functional enzyme that catalyzes the last steps of the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), one of the main precursors of bacterial cell wall peptidoglycan, from fructose-6-phosphate [40,41] while D-lactate dehydrogenase catalyzes the conversion of pyruvate into D-lactic acid, the last residue incorporated in the muramoyl-pentadepsipeptide peptidoglycan precursor [42]. We observed an increased amount of proteins with oxidoreductase activity in cells grown with rutin that could trigger their expression to withstand oxidative stress, as already reported for other phenolic compounds [14]. However, the overexpression of oxidative stress related proteins could also take part in the general stress response mechanism.

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

10 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

Our data indicated that the presence of rutin strongly affected protein synthesis and cells needed to promote ribosomal activity. Worth noting, the abundance of 30S ribosomal protein S1, EF-P, EF-G and ribosome recycling factor Rrf, that are connected in the cluster circled in red in Fig 3, increased in L. acidophilus grown in the presence of rutin. EF-G catalyzes the movement of the tRNA-mRNA complex within the ribosome during translocation, while ribosome recycling factor Rrf, in conjunction with EF-G and Initiation Factor 3 (IF3), promotes the dissociation of the ribosome into its subunits in order to recycle ribosomes for a new round of translation. EF-P stimulates the first step of peptide bond formation through an interaction with the ribosome and initiator tRNA and rescues the ribosome stalling that occurs mainly during the synthesis of proteins containing consecutive prolines [43,44]. Recently, it has also been indicated a key role of EF-P in the translational regulation of a subset of proteins involved in stress tolerance [43,45]. Proteomic studies already showed that the growth in the presence of tannic acid induced an overexpression of ribosomal proteins and elongation factors in L. plantarum also associated to the prolongation of cell viability [13]. Moreover, the abundance of these proteins mostly increased in response to acid and bile exposure in other lactobacilli and bifidobacteria [46]. Interestingly we observed that cells grown in the presence of rutin expressed an outer surface protein that contained a glycoside hydrolase domain. This protein showed a high sequence identity with a PTS (PhosphoTransferase System) associated protein of other lactobacilli, a complex system involved in the uptake and metabolism of oligosaccharides [47]. As a biological function of this protein has not been established yet, it could not be ruled out a specific role in the degradation of the glycosidic moiety of conjugated polyphenols. In conclusion, this study provided a first overview of the lactobacilli-flavonoids interplay and showed that adaptive response to rutin modulated the expression level of proteins known to be involved in general stress response mechanisms, in particular to acid and oxidative stresses. Due to cross-protection phenomenon [35,48], drastic stress conditions faced by lactobacilli during the gastro-intestinal transit should prepare these bacteria to better tolerate diet polyphenols so that they might contribute to the metabolism of these compounds.

Supporting Information S1 Table. L. acidophilus growths in control conditions and in the presence of rutin. Four bacterial growth for each condition were monitored by optical density at 600 nm (OD sheet) and by plate counting (CFU sheet). (XLSX)

Acknowledgments We gratefully thank prof. Pietro Pucci for critically reading the manuscript. This article is especially dedicated, with affection, to the memory of our colleague and friend Giancarlo Cammarota, research scientist of the Institute of Food Sciences, who suddenly died in August 2015.

Author Contributions Conceived and designed the experiments: MFM RAS AS. Performed the experiments: MFM RL AS SL FF RAS. Analyzed the data: MFM RL AS RAS. Contributed reagents/materials/analysis tools: MFM RAS. Wrote the paper: MFM RL RAS.

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

11 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

References 1.

Crozier A, Jaganath IB, Clifford MN. Dietary phenolics: chemistry, bioavailability and effects on health. Nat Prod Rep. 2009; 26:1001–1043. doi: 10.1039/b802662a PMID: 19636448

2.

Vasanthi HR, ShriShriMal N, Das DK. Phytochemicals from plants to combat cardiovascular disease. Curr Med Chem. 2012; 19:2242–2251. PMID: 22414106

3.

Manach C, Scalbert A, Morand C, Rémésy C, Jiménez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr. 2004; 79:727–747. PMID: 15113710

4.

Russo M, Spagnuolo C, Tedesco I, Bilotto S, Russo GL. The flavonoid quercetin in disease prevention and therapy: facts and fancies. Biochem Pharmacol. 2012; 83:6–15. doi: 10.1016/j.bcp.2011.08.010 PMID: 21856292

5.

Sharma S, Ali A, Ali J, Sahni JK, Baboota S. Rutin: therapeutic potential and recent advances in drug delivery. Expert Opin Investig Drugs. 2013; 22:1063–1079. doi: 10.1517/13543784.2013.805744 PMID: 23795677

6.

Rechner AR, Smith MA, Kuhnle G, Gibson GR, Debnam ES, Srai SK, et al. Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products. Free Radic Biol Med. 2004; 36:212–225. PMID: 14744633

7.

van Duynhoven J, Vaughan EE, Jacobs DM, Kemperman RA, van Velzen EJ, Gross G, et al. Metabolic fate of polyphenols in the human superorganism. Proc Natl Acad Sci USA. 2011; 108:4531–4538. doi: 10.1073/pnas.1000098107 PMID: 20615997

8.

Selma MV, Espín JC, Tomás-Barberán FA. Interaction between phenolics and gut microbiota: role in human health. J Agric Food Chem. 2009; 57:6485–6501. doi: 10.1021/jf902107d PMID: 19580283

9.

Etxeberria U, Fernández-Quintela A, Milagro FI, Aguirre L, Martínez JA, Portillo MP. Impact of polyphenols and polyphenol-rich dietary sources on gut microbiota composition. J Agric Food Chem. 2013; 61:9517–9533. doi: 10.1021/jf402506c PMID: 24033291

10.

Rossi M, Amaretti A, Leonardi A, Raimondi S, Simone M, Quartieri A. Potential impact of probiotic consumption on the bioactivity of dietary phytochemicals. J Agric Food Chem. 2013; 61:9551–9558. doi: 10.1021/jf402722m PMID: 24007212

11.

Beekwilder J, Marcozzi D, Vecchi S, de Vos R, Janssen P, Francke C, et al. Characterization of rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus. Appl Environ Microbiol. 2009; 75:3447–3454. doi: 10.1128/AEM.02675-08 PMID: 19346347

12.

Bossi A, Rinalducci S, Zolla L, Antonioli P, Righetti PG, Zapparoli G. Effect of tannic acid on Lactobacillus hilgardii analysed by a proteomic approach. Appl Microbiol. 2007; 102:787–795.

13.

Cecconi D, Cristofoletti M, Milli A, Antonioli P, Rinalducci S, Zolla L, Zapparoli G. Effect of tannic acid on Lactobacillus plantarum wine strain during starvation: A proteomic study. Electrophoresis. 2009; 30:957–965. doi: 10.1002/elps.200800310 PMID: 19229842

14.

Curiel JA, Rodríguez H, de Las Rivas B, Anglade P, Baraige F, Zagorec M, et al. Response of a Lactobacillus plantarum human isolate to tannic acid challenge assessed by proteomic analyses. Mol Nutr Food Res. 2011; 55:1454–1465. doi: 10.1002/mnfr.201000621 PMID: 21638779

15.

Rivas-Sendra A, Landete JM, Alcántara C, Zúñiga M. Response of Lactobacillus casei BL23 to phenolic compounds. J Appl Microbiol. 2011; 111:1473–1481. doi: 10.1111/j.1365-2672.2011.05160.x PMID: 21951613

16.

Wang Y, Delettre J, Corrieu G, Béal C. Starvation induces physiological changes that act on the cryotolerance of Lactobacillus acidophilus RD758. Biotechnol Prog. 2011; 27:342–350. doi: 10.1002/btpr. 566 PMID: 21360838

17.

Wang Y, Delettre J, Guillot A, Corrieu G, Béal C. Influence of cooling temperature and duration on cold adaptation of Lactobacillus acidophilus RD758. Cryobiology. 2005; 50:294–307. PMID: 15925581

18.

Nandakumar R, Talapatra K. Quantitative profiling of bacteriocins present in dairy-free probiotic preparations of Lactobacillus acidophilus by nanoliquid chromatography-tandem mass spectrometry. J Dairy Sci. 2014; 97:1999–2008. doi: 10.3168/jds.2013-7470 PMID: 24565320

19.

Ashida N, Yanagihara S, Shinoda T, Yamamoto N. Characterization of adhesive molecule with affinity to Caco-2 cells in Lactobacillus acidophilus by proteome analysis. J Biosci Bioeng. 2011; 112:333– 337. doi: 10.1016/j.jbiosc.2011.06.001 PMID: 21763196

20.

Johnson B, Selle K, O’Flaherty S, Goh YJ, Klaenhammer T. Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM. Microbiology. 2013; 159:2269–2282. doi: 10. 1099/mic.0.070755-0 PMID: 24002751

21.

Kuwana R, Yamamoto N. Increases in GroES and GroEL from Lactobacillus acidophilus L-92 in response to a decrease in medium pH, and changes in cytokine release from splenocytes:

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

12 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

transcriptome and proteome analyses. J Biosci Bioeng. 2012; 114:9–16. doi: 10.1016/j.jbiosc.2012.02. 010 PMID: 22417456 22.

Lee J, Kim Y, Yun HS, Kim JG, Oh S, Kim SH. Genetic and proteomic analysis of factors affecting serum cholesterol reduction by Lactobacillus acidophilus A4. Appl Environ Microbiol. 2010; 76:4829– 4835. doi: 10.1128/AEM.02892-09 PMID: 20495044

23.

Majumder A, Sultan A, Jersie-Christensen RR, Ejby M, Schmidt BG, Lahtinen SJ, et al. Proteome reference map of Lactobacillus acidophilus NCFM and quantitative proteomics towards understanding the prebiotic action of lactitol. Proteomics. 2011; 11:3470–3481. doi: 10.1002/pmic.201100115 PMID: 21751373

24.

Almajano MP, Carbò R, Lopez Jimenez JA, Michael H, Gordon MH. Antioxidant and antimicrobial activities of tea infusions. Food Chem. 2008; 108:55–63.

25.

China R, Mukherjee S, Sen S, Bose S, Datta S, Koley H, et al. Antimicrobial activity of Sesbania grandiflora flower polyphenol extracts on some pathogenic bacteria and growth stimulatory effect on the probiotic organism Lactobacillus acidophilus. Microbiol Res. 2012; 167:500–506. doi: 10.1016/j.micres. 2012.04.003 PMID: 22583547

26.

Duda-Chodak A. The inhibitory effect of polyphenols on human gut microbiota. J Physiol Pharmacol. 2012; 63:497–503. PMID: 23211303

27.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248–254. PMID: 942051

28.

Gorg A, Postel W, Gunther S. The current state of two dimensional electrophoresis with immobilized pH gradients. Electrophoresis. 1988; 9:531–546. PMID: 3072185

29.

Hochstrasser DF, Harrington MG, Hochstrasser AC, Miller MJ, Merril CR. Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal Biochem. 1988; 173:424–435. PMID: 3189820

30.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227:680–685. PMID: 5432063

31.

Lippolis R, Gnoni A, Abbrescia A, Panelli D, Maiorano S, Paternoster MS, et al. Comparative proteomic analysis of four Bacillus clausii strains: proteomic expression signature distinguishes protein profile of the strains. J Proteomics. 2011; 74:2846–2855. doi: 10.1016/j.jprot.2011.06.032 PMID: 21810490

32.

Appel RD, Hochstrasser DF. Computer analysis of 2-D images. Methods Mol Biol. 1999; 112:363–381. PMID: 10027262

33.

Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996; 68:850–858. PMID: 8779443

34.

Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey C, Muller J, et al. STRING 8—a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res. 2009; 37:D412–416. doi: 10.1093/nar/gkn760 PMID: 18940858

35.

De Angelis M, Gobbetti M. Environmental stress responses in Lactobacillus: A review. Proteomics. 2004; 4:106–122. PMID: 14730676

36.

Shi L, Kay LE. Tracing an allosteric pathway regulating the activity of the HslV protease. Proc Natl Acad Sci USA. 2014; 111:2140–2145. doi: 10.1073/pnas.1318476111 PMID: 24469799

37.

Len AC, Harty DW, Jacques NA. Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance. Microbiology. 2004; 150:1353–1366. PMID: 15133097

38.

Santiago B, MacGilvray M, Faustoferri RC, Quivey RG Jr. The branched-chain amino acid aminotransferase encoded by ilvE is involved in acid tolerance in Streptococcus mutans. J Bacteriol. 2012; 194:2010–2019. doi: 10.1128/JB.06737-11 PMID: 22328677

39.

Heunis T, Deane S, Smit S, Dicks LM. Proteomic profiling of the acid stress response in Lactobacillus plantarum 423. J Proteome Res. 2014; 13:4028–4039. doi: 10.1021/pr500353x PMID: 25068841

40.

Barreteau H, Kovac A, Boniface A, Sova M, Gobec S, Blanot D. Cytoplasmic steps of peptidoglycan biosynthesis. FEMS Microbiol Rev. 2008; 32:168–207. doi: 10.1111/j.1574-6976.2008.00104.x PMID: 18266853

41.

Rodríguez-Díaz J, Rubio-del-Campo A, Yebra M. Metabolic engineering of Lactobacillus casei for production of UDP-N-acetylglucosamine. J Biotechnol Bioeng. 2012; 109:1704–1712.

42.

Ferain T, Hobbs JN Jr, Richardson J, Bernard N, Garmyn D, Hols P, et al. Knockout of the two ldh genes has a major impact on peptidoglycan precursor synthesis in Lactobacillus plantarum. J Bacteriol. 1996; 178:5431–5437. PMID: 8808932

43.

Ude S, Lassak J, Starosta AL, Kraxenberger T, Wilson DN, Jung K. Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches. Science. 2013; 339:82–85. doi: 10.1126/science. 1228985 PMID: 23239623

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

13 / 14

Proteomic Study of L. acidophilus - Rutin Interplay

44.

Peil L, Starosta AL, Lassak J, Atkinson GC, Virumäe K, Spitzer M, et al. Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P. Proc Natl Acad Sci USA. 2013; 110:15265–15270. doi: 10.1073/pnas.1310642110 PMID: 24003132

45.

Zou SB, Hersch SJ, Roy H, Wiggers JB, Leung AS, Buranyi S, et al. Loss of elongation factor P disrupts bacterial outer membrane integrity. J Bacteriol. 2012; 194:413–425. doi: 10.1128/JB.05864-11 PMID: 22081389

46.

Siciliano RA, Mazzeo MF. Molecular mechanisms of probiotic action: a proteomic perspective. Curr Opin Microbiol. 2012; 15:390–396. doi: 10.1016/j.mib.2012.03.006 PMID: 22538051

47.

Abou Hachem M, Andersen JM, Barrangou R, Møller MS, Fredslund F, Majumder A, et al. Recent insight into oligosaccharide uptake and metabolism in probiotic bacteria. Biocatal Biotransfor. 2013; 31:226–235.

48.

van de Guchte M, Serror P, Chervaux C, Smokvina T, Ehrlich SD, Maguin E. Stress responses in lactic acid bacteria. Antonie Van Leeuwenhoek. 2002; 82:187–216. PMID: 12369188

PLOS ONE | DOI:10.1371/journal.pone.0142376 November 6, 2015

14 / 14

Lactobacillus acidophilus-Rutin Interplay Investigated by Proteomics. - PDF Download Free (2024)
Top Articles
Latest Posts
Article information

Author: Msgr. Refugio Daniel

Last Updated:

Views: 6544

Rating: 4.3 / 5 (74 voted)

Reviews: 89% of readers found this page helpful

Author information

Name: Msgr. Refugio Daniel

Birthday: 1999-09-15

Address: 8416 Beatty Center, Derekfort, VA 72092-0500

Phone: +6838967160603

Job: Mining Executive

Hobby: Woodworking, Knitting, Fishing, Coffee roasting, Kayaking, Horseback riding, Kite flying

Introduction: My name is Msgr. Refugio Daniel, I am a fine, precious, encouraging, calm, glamorous, vivacious, friendly person who loves writing and wants to share my knowledge and understanding with you.