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
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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.
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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].
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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,
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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.
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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)
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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
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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)
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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
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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.
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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.
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