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DOI 10.1002/pmic.201400363

RESEARCH ARTICLE

Comparative proteomic analysis of biofilm and planktonic cells of Lactobacillus plantarum DB200 Maria De Angelis, Sonya Siragusa, Daniela Campanella, Raffaella Di Cagno and Marco Gobbetti Department of Soil, Plant and Food Science, University of Bari Aldo Moro, Bari, Italy

This study investigated the relative abundance of extracellular and cell wall associated proteins (exoproteome), cytoplasmic proteins (proteome), and related phenotypic traits of Lactobacillus plantarum grown under planktonic and biofilm conditions. Lactobacillus plantarum DB200 was preliminarily selected due to its ability to form biofilms and to adhere to Caco2 cells. As shown by fluorescence microscope analysis, biofilm cells became longer and autoaggregated at higher levels than planktonic cells. The molar ratio between glucose consumed and lactate synthesised was markedly decreased under biofilm compared to planktonic conditions. DIGE analysis showed a differential exoproteome (115 protein spots) and proteome (44) between planktonic and biofilm L. plantarum DB200 cells. Proteins up- or downregulated by at least twofold (p < 0.05) were found to belong mainly to the following functional categories: cell wall and catabolic process, cell cycle and adhesion, transport, glycolysis and carbohydrate metabolism, exopolysaccharide metabolism, amino acid and protein metabolisms, fatty acid and lipid biosynthesis, purine and nucleotide metabolism, stress response, oxidation/reduction process, and energy metabolism. Many of the above proteins showed moonlighting behavior. In accordance with the high expression levels of stress proteins (e.g., DnaK, GroEL, ClpP, GroES, and catalase), biofilm cells demonstrated enhanced survival under conditions of environmental stress.

Received: July 30, 2014 Revised: December 30, 2014 Accepted: February 24, 2015

Keywords: Biofilm / Comparative proteomic analyses / Environmental stress / Lactobacillus plantarum / Microbiology

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Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction

Bacteria grow and proliferate either as single cells, termed the planktonic phenotype, or organized into biofilms: functional, complex microbial communities attached to abiotic or biotic surfaces [1]. Biofilms contain water channels that are Correspondence: Professor Marco Gobbetti, Department of Soil, Plant and Food Science, University of Bari Aldo Moro, Via G. Amendola 165/a, 70126 Bari, Italy E-mail: [email protected] Fax: +39 080 5442911 Abbreviations: CFS, cell-free supernatant; CFU, colony-forming unit; EMEM, Eagle’s minimum essential medium; FAA, free amino acids; KEGG, Kyoto Encyclopedia of Genes and Genomes; MRS, de Man, Rogosa, and Sharpe; TF, trigger factor

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thought to operate as a distribution system for nutrients and O2 [2]. Gradients of substrates and metabolites are present within biofilms and drive pronounced microenvironmental and physiological heterogeneity [2]. Recently, three of the main mechanisms playing a pivotal role in survival within biofilms were reviewed: biofilmspecific protection against oxidative stress, the expression of efflux pumps, and the protection provided by matrix polysaccharides [3]. Most studies have focused on pathogenic bacteria [1, 4–6], and compared to planktonic cells, higher levels of surface proteins are found in the cells of biofilms composed of many bacterial pathogens [1]. Moreover, a marked difference in the relative abundance of proteins associated with

Colour Online: See the article online to view Figs. 1 and 2 in colour.

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metabolic functions has been demonstrated between planktonic and biofilm cells [1]. Lactobacillus is dominant among the biofilm inhabitants of the human and animal intestine [7]. The formation of biofilms by Lactobacillus species has been previously described [8–12], and the genes responsible for quorum sensing, adhesion, and biofilm formation have been identified [9, 11–13]. Compared to planktonic lactobacilli, the stress responses of lactobacilli populating biofilms were found to be increased [8, 14]. Lactobacillus spp. synthesizes the cell surface and extracellular proteins that are putatively responsible for adhesion, including molecular chaperones, enzymes, lipoproteins, and surface layer proteins [15,16]. Interestingly, the relative abundance of these proteins increases during cultivation of Lactobacillus plantarum with the peptide pheromone plantaricin A (PlnA), especially in association with Lactobacillus sanfranciscensis [17, 18]. Nevertheless, the molecular mechanisms underlying the formation of biofilms are still not completely understood. Indeed, a comparative proteomic analysis between planktonic and biofilm cells of L. plantarum (a spoilage, starter, and/or probiotic bacterium) that elucidates the mechanisms behind the stress response could be useful for the development of appropriate tools for the control of lactobacilli biofilms in efforts to prevent food spoilage or to enhance food fermentation and probiotic performance. Therefore, in this study, we compared the proteome and phenotypic performances of L. plantarum DB200 grown as biofilms and as planktonic cells.

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crystal violet in an isopropanol–methanol–PBS solution (1:1:18 v/v/v). Excess stain was rinsed off by placing the pegs into a 96-well plate filled with 200 ␮L sterile water per well. After the pegs were air dried (approximately 2 h), the dye bound to the adherent cells was resolubilized with 200 ␮L ethanol– acetone (8:20). The absorbance of each well (a volume of 135 ␮L) was measured at 570 nm.

2.3 Culturing of Caco-2 cells and adhesion of L. plantarum strains to Caco-2 cells Caco-2 cells were cultured in Eagle’s minimum essential medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 U/mL). Caco-2 cells were inoculated at a density of 5 log per well into a 12-well microtiter plate and incubated at 37⬚C in a 5% CO2 atmosphere [20]. Lactobacillus plantarum DB200, CC3M8, POM1, 4.1, and LP40 grown at 30⬚C for 16 h were used for the adhesion assay. The L. plantarum cells were added (approximately 7 log per well) to the Caco-2 cells (approximately 5 log per well) [20]. After 2 h of incubation at 37⬚C, the bacterial cells were withdrawn from the wells, and the Caco-2 cells were washed twice with 1 mL of sterile PBS. The bacterial cells that adhered to the Caco-2 cells were removed by treatment with 1 mL 0.5% v/v Triton X-100 for 3 min on ice. Serial dilutions of the cell suspensions were plated onto modified MRS agar at 30⬚C for 48 h.

Materials and methods 2.4 Production of planktonic and biofilm cells

2.1 Bacterial strains and culture conditions Lactobacillus plantarum DB200, CC3M8, POM1, 4.1, and LP40 were propagated in modified de Man, Rogosa, and Sharpe agar (MRS, Oxoid, Basingstoke, Hampshire, England). Modifications included the omission of meat extract and the addition of fresh yeast extract (5%, w/v) and 28 mM maltose at a final pH of 5.6.

2.2 In vitro biofilm assays The method to assess in vitro biofilm formation by L. plantarum DB200, CC3M8, POM1, 4.1, and LP40 was adapted from Lebeer et al. [19]. The assay was performed on polystyrene pegs suspended into the wells of a microtiter plate. Lactobacillus plantarum strains were grown at 30⬚C for 16 h until the early stationary phase of growth was achieved. The pegs were placed into wells containing 200 ␮L of modified MRS, and the bacterial cells (approximately 7 log colonyforming units, CFU/mL) were incubated for 72 h at 37⬚C. The control wells contained sterile-modified MRS alone. After incubation, the pegs were washed once in PBS. Attached bacteria were stained for 30 min with 200 ␮L 0.1% w/v  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Lactobacillus plantarum DB200 showed the highest production of biofilms and the highest adhesion to Caco-2 cells and was thus selected for a comparative proteomic analysis. Planktonic and biofilm cells were produced as described by Kubota et al. [14]. Briefly, planktonic cells of L. plantarum DB200 were statically produced in glass tubes containing 10 mL of modified MRS broth under aerobic conditions for 24 h at 30⬚C. To prepare biofilms, the cells were grown for 24 h at 30⬚C under aerobic conditions on glass cover slips (12 mm ø, Thermo Fisher Scientific, Waltham, MA, USA) in the wells of static 6-well polystyrene microtiter dishes (Iwaki 1820–042N, Asahi Techno Glass Co., Ltd., Chiba, Japan) containing 4 mL of modified MRS broth. At the end of the incubation period, the spent medium was removed and analyzed for the contents of carbohydrates, organic acids, and free amino acids (FAA). The glass cover slips were carefully rinsed with 4 mL of 0.85% saline solution to eliminate nonadherent cells, and the remaining materials were defined as biofilms. To recover cells from the biofilm, 4 mL of 0.85% saline solution was added to each well containing the glass cover slips, and the cells were detached by pipetting up and down at least ten times. These cells were used for phenotypic and proteomic analyses. www.proteomics-journal.com

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2.5 Cell morphology and viability Lactobacillus plantarum DB200 was analyzed using LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Cambridge Bioscience, Cambridge, UK) according to the manufacturer’s instructions. Stained bacterial suspensions were observed using a LEICA LDMC (Leica Microsystems SpA, Milan, Italy) with a 60× objective. ImR Plus image analysis ages were analyzed using Image-Pro software (Media Cybernetics Inc., Silver Spring, MD, USA).

2.6 Growth, acidification, consumption of carbohydrates, synthesis of organic acids, and concentration of FAA After 24 h of incubation at 30⬚C, the cell density of L. plantarum DB200 grown under planktonic or biofilm conditions was determined by plating onto modified MRS agar at 30⬚C for 48 h. The concentration of carbohydrates and organic ¨ acids was determined by HPLC using an AKTA PurifierTM system (GE Healthcare, Uppsala, Sweden). The FAA content was analyzed using a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, United Kingdom) [21]. The concentration of carbohydrates, organic acids, and FAAs was expressed as milliliter of medium for both planktonic and biofilm cells.

2.7 Protein extraction and CyDye labelling Proteomic analyses were performed for cells of L. plantarum DB200 grown for 24 h at 30⬚C under planktonic and biofilm conditions. Cytoplasmic and cell wall associated proteins and proteins from the cell-free supernatant (CFS) were analyzed. The CFS was used to map extracellular proteins. Freeze-dried CFS was rehydrated in 25 mL of H2 O and transferred into Amicon-Ultra-15 centrifugal filter units (Millipore Co., Carrigtwahill, Cork, Ireland). The filtered and concentrated extracellular proteins were dissolved in Milli-Q water and used for DIGE analysis. Cell wall associated proteins were obtained as described by Calasso et al. [18]. Cell pellets were resuspended in 3 mL of extraction solution (50 mM Tris-HCl, pH 7.5, 20% w/v of sucrose, and 1 mg/mL of lysozyme) and incubated at 37⬚C for 2 h at 150 rpm. After centrifugation (8452 rpm, 15 min, 20⬚C), supernatants were recovered, dialyzed for 24 h at 4⬚C against 2 mM PBS (pH 7.0) and concentrated approximately 20-fold by freeze-drying. The protein concentration was determined by the Bradford method [22]. General aminopeptidase activity toward leucine paranitroanilide (Leu-pNA) was used as a marker of the integrity of spheroplasts [23]. No aminopeptidase activity was detected in the extracellular and cell wall associated protein preparations. Spheroplasts were broken with glass beads, and the cytoplasmic proteins were extracted as previously described [23]. The CFS, cell wall associated and cytoplasmic protein  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

fractions were labelled using Cy2, Cy3, or Cy5 dye (CyDye DIGE Fluor minimal dyes, GE Healthcare) according to the EttanTM DIGE protocol.

2.8 DIGE and DeCyder analyses Equal amounts of total protein (150 ␮g) were used for each electrophoretic run. IEF was performed using 18-cm Immobiline strips to provide a nonlinear pH 3–10 gradient with a IPGphore system (GE Healthcare). For the second dimension, 12.5% homogeneous SDS-PAGE gels were used. A voltage of 15 mA was applied to the gel using an Ettan DALTsix electrophoresis unit (GE Healthcare). The gels were scanned using the Typhoon FLA 9500 laser scanner (GE Healthcare) at wavelengths of 473 (for Cy2), 532 (Cy3), and 635 nm (Cy5) and the corresponding voltages of 420, 410, and 400 V. After scanning, the gels were silverstained. Images and statistical analyses for cropped DIGE gels were performed using the DeCyder 2D 7.0 software (GE Healthcare). Spot boundaries were detected, and spot volumes were calculated. Furthermore, spot volumes of the Cy3 and Cy5 samples were compared with the spot volume of the Cy2 sample (internal standard) to generate standard spot volumes to correct for intergel variations. In the biological variation analysis module, the Cy2 images of four replicate gels were matched, and the standard spot volume ratios between all four gels were compared. Protein spots that exhibited at least twofold differences in the average spot volume ratios between the planktonic and biofilm cells were selected for identification [24].

2.9 Protein identification Proteins were digested automatically with the Proteineer DP robot (Bruker Daltonik, Bremen, Germany) and the Control 1.2 software according to the modified protocol of Shevchenko et al. [25]. Modified porcine trypsin (Promega, Madison, WI, USA) at a final concentration of 8 ng/␮L in 50 mM ammonium bicarbonate was added to dried gel pieces, and the digestion proceeded at 37°C for 8 h. For peptide extraction, 0.5% TFA was added, and the resulting digestion solutions were transferred by centrifugation into V-bottom 96-well polypropylene microplates. MALDI samples were prepared by mixing equal volumes of digestion solution and matrix solution consisting of ␣-cyano-4-hydroxycinnamic acid in 50% aqueous acetonitrile and 0.25% TFA. The mixture was deposited onto a 600-␮m AnchorChip prestructured MALDI probe (Bruker Daltonik) [26] and allowed to dry at room temperature. Positive-ion MALDI mass spectra were obtained using a Bruker Ultraflex III in reflectron mode equipped with a Nd:YAG smart beam laser [27]. Monoisotopic masses were obtained using a SNAP average algorithm and an S/N threshold of 2. Bruker flexAnalysis software (version 3.3) was used to perform spectral processing and peak list generation. www.proteomics-journal.com

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Tandem mass spectral data were submitted for database searching using a locally run copy of the Mascot program (Matrix Science Ltd., version 2.4) through the Bruker ProteinScape interface (version 2.1). To study changes in metabolic enzymes in response to biofilms, all identified proteins were mapped to Kyoto Encyclopedia of Genes and Genomes pathways using both the enter gene ID and/or EC number functions (www.genome.jp/kegg/pathway.html) [18].

2.10 Heat, acid, and ethanol resistance and catalase activity Lactobacillus plantarum DB200 cells were grown in modified MRS broth under planktonic or biofilm conditions and harvested by centrifugation at 10 000 rpm for 10 min at 4⬚C. The bacteria were washed twice with 50 mM sterile PBS (pH 7.0) and resuspended into fresh modified MRS at a final cell density of 9.4 log CFU/g. Samples (0.5 mL) were transferred into capillary glass tubes and heated in a water bath at 55 or 65⬚C for 10 min or 72⬚C for 15 sec. After heat treatment, the samples were chilled on ice for 5 min and plated onto modified MRS agar at 37⬚C for 48 to 96 h. To determine acid and ethanol resistance, the growth of L. plantarum DB200 cells for 24 h at 30⬚C was assessed under planktonic or biofilm conditions using modified MRS broth supplemented with acetic acid (1.5%, v/v) [8] or ethanol (10–12.5%, v/v). The initial OD at 620 nm was approximately 0.150 (corresponding to approximately 7.2 log CFU/mL). Furthermore, survival under extreme acidic or ethanol stress conditions was determined. Biofilms formed on glass cover slips or planktonic bacterial cells (approximately 9.0 log CFU/mL) were inoculated into 1.1 mL of acid (8, 10, and 11%, v/v) or alcohol (25 and 30%, v/v) solutions for 30 min at room temperature [8]. Saline solution (0.85% NaCl, v/v) was used as the control. After treatment, the samples were plated onto modified MRS agar at 37⬚C for 48 to 96 h. The catalase activity absorbance assay was performed as previously described [28]. Briefly, cells were washed in PBS and diluted (1:10, v/v) in PBS supplemented with H2 O2 (final concentration, 40 mM). The decrease in absorbance at 240 nm was measured over time at 30⬚C. One unit of catalase activity was defined as the decrease in absorbance at 240 nm of 1 unit/min.

2.11 Statistical analysis Data from the phenotypic and proteomic analyses were subjected to a one-way ANOVA (SAS, 1985), and pair-comparison of treatment mean values was achieved by Tukey’s procedure at p < 0.05 using the statistical software Statistica for Windows (Statistica 6.0 per Windows 1998).  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Results

3.1 Biofilm formation and adhesion to Caco-2 cells All L. plantarum cells tested adhered to the polystyrene pegs (Supporting Information Fig. 1A) and Caco-2 cells (Supporting Information Fig. 1B), but their densities varied depending on the strain. The highest biofilm formation on the polystyrene pegs was found for strains DB200 and POM1. The level of adhesion of L. plantarum strains ranged from approximately 4.20 to 6.1 log CFU/5 log Caco-2 cells per well. Because L. plantarum DB200 demonstrated the highest ability to form biofilms and adhere to Caco-2 cells, this strain was selected for further analysis.

3.2 Kinetics of growth and morphology, acidification, consumption of carbohydrates, synthesis of organic acids and concentration of FAAs The cell density of L. plantarum DB200 cultivated in a biofilm was not significantly different from that of planktonic cells (p = 0.874; 9.26 ± 0.071 vs. 9.38 ± 0.112 log CFU/mL, respectively). However, the biofilm cells were longer than the planktonic cells (Supporting Information Fig. 2) and showed the highest level of autoaggregation. The consumption of glucose during growth in modified MRS broth was similar (p = 0.503) between the planktonic and biofilm cells (14.40 ± 0.57 mg/mL). Compared to planktonic cells (16.21 ± 0.50 mg/mL), a lower (p = 0.031) synthesis of lactic acid (10.81 ± 0.41 mg/mL) was observed in the biofilm state; in contrast, the synthesis of acetic acid was not detected. The concentration of total FAAs was similar (p = 0.472) between the planktonic (ca. 1.83 mg/mL) and biofilm (ca. 1.84 mg/mL) cultures of L. plantarum DB200.

3.3 DIGE analysis and protein identification by MALDI-TOF-MS/MS A total of 145 protein spots that showed at least twofold decreases or increases (p < 0.05) during biofilm growth were analyzed by MALDI-TOF-MS/MS (Fig. 1, Table 1, and Supporting Information Fig. 1). An additional 14 protein spots were not identifiable due to their low abundance. Changes in exoproteome and proteome composition, metabolic functions, and the moonlighting activity of some proteins were observed (Table 1 and Fig. 2). Compared to planktonic cells, L. plantarum DB200 grown in biofilms showed a decrease in 18 spots identified as hydrolases. Sortase A (SrtA) was found at the highest level in the cells grown in biofilms, and proteins involved in the cell cycle were found at the lowest levels during biofilm growth. Six protein spots that are mainly cell wall associated and involved in adhesion were markedly increased during growth under biofilm conditions. Compared to planktonic www.proteomics-journal.com

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Figure 1. Representative overlay image of DIGE gel containing extracellular (A), cell wall associated (B), and cytoplasmic (C) proteins, which were synthesized by L. plantarum DB200 during growth as planktonic or biofilm cells onto modified MRS broth at 30⬚C for 24 h. Proteins from planktonic and biofilm cells were labelled in green and red, respectively. Spots appearing in yellow indicated proteins found in both planktonic and biofilm cells. Spot designation corresponds to that of the proteins in Table 1.

cells, DB200 cells grown in biofilms exhibited increases in the relative abundance of some proteins involved in the transport of peptides, DNA, amino acids, and metal ion transport. In contrast, the levels of oligopeptide ABC transporter and ABC transporter ATP-binding protein were decreased. Compared to planktonic cells, the relative abundance of some proteins involved in glycolysis and carbohydrate metabolism, amino acid metabolism, protein biosynthesis, and protein secretion was affected to differing degrees under biofilm conditions. Additionally, some proteins (e.g., Eno 1, GAPDH, PGK, TPI,  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

and PGAM2) (Table 1) demonstrated moonlighting behavior. Proteins responsible for the synthesis of exopolysaccharide were found at the highest levels during biofilm growth. Six proteins related to fatty acid and lipid biosynthesis were overexpressed during growth under biofilm conditions. Protein-protein interaction networks revealed that all of the proteins found at the highest levels in biofilm culture strongly correlated with one another (Supporting Information Fig. 3). www.proteomics-journal.com

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Table 1. Properties, homologous protein, putative function, relative volume ratio (B/P, biofilm/planktonic), and accession numbers of extracellular (E), cell wall associated (CW) and cytoplasmic (C) proteins showing an increased or decreased (ࣙ or ࣘ of 2-fold, p < 0.05) level of synthesis in L. plantarum DB200 grown as planktonic or biofilm cells on modified MRS broth at 30⬚C for 24 h a)

Function

Homologous protein

Spot

Estimated Mr (kDa) and pI

Cell wall and catabolic process

Extracellular transglycosylase (TG) Extracellular transglycosylase (TG) LysM domain Extracellular transglycosylase (TG) Extracellular transglycosylase, with LysM peptidoglycan binding domain (TG)

2E 8E

58.0/9.25 41.5/9.05

Putative cell surface hydrolase, membrane-bound (CSH) Cell surface hydrolase, membrane-bound (CSH) Extracellular lipase/acylhydrolase with GDSL-like motif (GLIP) Cell surface hydrolase (CSH) Cell surface hydrolase (CSH)

Cell cycle and adhesion

Transport

Cell surface hydrolase, membrane-bound (CSH) Sortase A (SrtA) Extracellular zinc metalloproteinase, M10 family (Zmp) Cell surface protein (CSP) Cell surface protein, CscB family (CSP) Cell surface protein (CSP) S9 family serine peptidase(S9-SP) Possible hydrolase (Hyd) Cell surface SD repeat protein, membrane-anchored (CSP) Cell surface protein precursor (CSP) Cell surface protein, LPXTG-motif cell wall anchor (CSP) Cell surface adherence protein, collagen-binding domain, LPXTG-motif cell wall anchor (CSP) Mannose-specific adhesin, LPXTG-motif cell wall anchor (MSA) Mannose-specific adhesin, LPXTG-motif cell wall anchor (MSA) Cell division protein FtsZ (CpFtsZ) Cell division initiation protein DivIVA (DivIVA) Cell division initiation protein DivIVA (DivIVA) Cell division initiation protein DivIVA (DivIVA) ABC-transporter (Bacteriocin ABC transporter) (PlnG) Extracellular protein, peptide binding protein OppA-like protein (OppA) Oligopeptide ABC transporter, substrate binding protein (OppD)

 C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

b)

B/P

c)

p Value A.N.

0.01 0.048 0.02 0.028

gi|28379317 gi|28377225

77CW 25.3/8.50 82CW 23.7/5.36

0.4 0.5

0.019 0.050

R9WZC8 M4KL58

87CW 23.2/5.26 40CW 36.0/6.45

0.04 0.029 0.05 0.044

U2XMN2

47CW 35.0/8.35

0.5

0.038

R9X6S9

48CW 35.0/8.50 49CW 34.5/9.88

0.5 0.042 0.05 0.009

R9X1Y1

51CW 53CW 54CW 71CW

0.2 0.05 0.05 0.2

32.5/9.60 31.7/8.50 31.7/8.30 26.5/9.92

75CW 25.5/9.15 76CW 25.3/8.90 80CW 86CW 88CW 83CW 92CW 1CW

23.7/9.34 23.4/6.70 23.0/4.30 23.7/8.30 21.5/8.10 122.8/3.67

0.027 0.050 0.028 0.031

R9X0U8 R9X7J0

20 0.026 0.05 0.037

F9UKZ1 R9X1F5

M7CR61

0.05 0.01 0.05 0.4 0.1 2

0.020 0.017 0.029 0.034 0.042 0.036

U2IG48 F9UNJ1 V7YXB3 gi|227895627 E1TNN4 M4KGR1

5CW 66/3.90 19CW 46.6/4.59

2.5 4.5

0.028 0.034

YP_003063952 H3NZJ3

42CW 35.5/4.48

3

0.042

H3P5T2

3CW

103/4.90

2.2

0.039

F9UN23

46CW 35.0/4.50

2.1

0.031

S2VGS2

23CW 44.0/4.44 25C 31.5/4.50

0.3 0.5

0.025 0.014

YP_003063419 YP_003063415

33CW 37.0/4.07 52CW 32/5.07

0.3 0.5

0.050 0.038

F9UQB9

70CW 26.5/4.67

0.5

0.014

R9X6Q9

2CW

80/5.10

2.1

0.008

F9UU09

4CW

67.0/5.38

3

0.029

E1TQQ5

11CW 60.3/4.00

0.5

0.015

YP_003062651

12CW 13CW 14CW 15CW

0.3 0.1 0.1 0.2

0.017 0.050 0.019 0.028

60.3/5.20 60.3/5.30 60.3/5.00 60.3/5.10

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Proteomics 2015, 00, 1–14 Table 1. Continued

Function

Homologous protein

Glycolysis and carbohydrate metabolism

Oligopeptide ABC transporter, substrate binding protein (OppD) Putative competence-damage inducible protein (cinA) Manganese ABC superfamily ATP binding cassette transporter, substrate binding protein (mtsA) Branched-chain amino acid ABC transporter (livA) Competence protein (ComEA) ComE operon protein 1, DNA receptor (ComE) ABC transporter, ATP-binding protein (ABC-ATP) Bacterial type II secretion/trafficking system extracellular protein, competence protein (ComGD) Enolase (Eno1) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

Exopolysaccharide metabolism

Amino acid and protein metabolisms

a)

Spot

b)

B/P

c)

p Value A.N.

16CW 60.3/5.35 27C 31.5/7.30

0.1 0.031 0.05 0.039

YP_003062655

24CW 44.8/4.60

2.1

0.010

Q88UZ3

44CW 35.2/8.90

2

0.034

R4Q243

55CW 31.3/9.60

2.4

0.018

C6VKH6

68CW 26.6/8.85 74CW 25.8/9.05

2 2

0.011 0.026

U2I3B7 R9X4P3

79CW 25.3/5.45

0.05 0.032

F9URT1

95CW 18.0/8.30

2

F9UQG8

4E 11E

48.03/4.61 36.6/4.00

0.05 0.033 0.05 0.028

Q88YH3 YP_003062238

38.6/5.30 38.6/5.50 36.6/5.20 36.6/5.50 36.6/5.57 22.0/8.80

0.05 0.05 0.1 0.3 0.3 0.07

0.028 0.015 0.112 0.341 0.020 0.024

YP_003924665

21.5/9.90

0.05 0.016

YP_003062238

44.0/4.51 41.2/5.04 26.6/5.20 25.9/3.99 25.3/4.64 26.0/4.94

2 2.4 0.1 0.2 0.4 0.5

0.025 0.036 0.036 0.014 0.024 0.026

YP_003062241 R9X1Q1 YP_003923932 NP_784536 Q88YH4 Q88T35

84/5.30 62.9/5.35 50/6.08 33/4.67 33/4.20 32/7.50

2 2 0.4 2.6 2.2 0.05

0.012 0.028 0.012 0.015 0.030 0.021

C6VM05 YP_003063179 YP_003063474 CAA50277

32/8.00 24/5.10 30.9/5.25 31.1/9.70 33/6.15

0.4 2 2 2 3.3

0.050 0.026 0.024 0.033 0.014

Q88V41 YP_003064118 R9X8J8 D7VC13 YP_003062211

73.8/5.10 47.1/4.49 44.3/5.94 44.3/6.00 41.3/6.50

2.1 2.5 5 5.5 5

0.023 0.037 0.011 0.006 0.024

Q88WU9 YP_003063166 YP_003063360

38.6/5.65

0.05 0.012

YP_003063651

38.4/6.10

2.2

29CW 30CW 34CW 36CW 37CW Chitin-binding protein/carbohydrate-binding 16E protein (CBP) Chitin-binding protein/carbohydrate-binding 91CW protein (CBP) Phosphopyruvate hydratase (EnoA1) 22CW Phosphoglycerate kinase (PGK) 27CW Triosephosphate isomerase (TPI) 69CW Triosephosphate isomerase (TPI) 73CW Triosephosphate isomerase (TPI) 78CW 2,3-bisphosphoglycerate-dependent 72CW phosphoglycerate mutase 2 (PGAM2) Formate C-acetyltransferase (PflB2) 1C Pyruvate kinase (PKI) 3C Catabolite control protein A (CcpA) 10C L-lactate dehydrogenase (LDH) 35CW 20C NAD-independent L-lactate dehydrogenase 22C (NAD-I-LDH) Acetate kinase (AckA) 23C Phosphoglyceromutase (PGM) 32C dTDP-4-dehydrorhamnose reductase (RfbD) 61CW Capsular exopolysaccharide family (Etk) 65CW UTP-glucose-1-phosphate 19C uridylyltransferase (GalU) Threonine-tRNA ligase (ThrS) 7CW 30S ribosomal protein S1 (S1) 18CW Elongation factor Tu (Tuf) 20CW 21CW Phenylalanine-tRNA ligase alpha subunit 25CW (PheS) Aspartate-semialdehyde dehydrogenase 32CW (ASADH) 14C

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Estimated Mr (kDa) and pI

0.017

YP_007412872

D7V8W9

0.010

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Table 1. Continued

Function

Homologous protein

Ornithine carbamoyltransferase (OTC) Release factor glutamine methyltransferase (Prmc) Foldase protein (PrsA 1) Elongation factor Ts (Ts) Transcription elongation factor GreA (GreA) Aspartyl/glutamyl-tRNA amidotransferase subunit A (Asp/Glu-ADT subunit A) Acetolactate synthase (ALS) Glycine hydroxymethyltransferase (STM) Ribosome recycling factor (RRF)

Fatty acid and lipid biosynthesis

Purine and nucleotide metabolism

50S ribosomal protein L5 (L5) 50S ribosomal protein L10 (L10) Acyl carrier protein (FadD) 3-oxoacyl-ACP synthase (FabH) Putative linoleate isomerase (LI) Glycerol-3-phosphate acyltransferase PlsX (GPAT) Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta 1 (AccC2) Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta 1 (EC 6.4.1.2) (AccD2) Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta 1 (EC 6.4.1.2) (AccD1) Acetyl-CoA carboxylase, biotin carboxyl carrier protein (AccB3) Guanosine 5’-monophosphate oxidoreductase (GMPR) Bifunctional pyrimidine regulatory protein PyrR uracil phosphoribosyltransferase (PyrR1)

Stress response

Oxidation/reduction processes

Adenylate kinase (AK) Molecular chaperone DnaK, partial (DnaK) Trigger factor (TF) Molecular chaperone GroEL (GroEL) Heat-inducible transcription repressor (HrcA) Peptidylprolyl isomerase (PPIase) ATP-dependent Clp protease proteolytic subunitl (ClpP) Alkaline shock protein (ASP) Small HSP 10 kDa chaperonin (GroES) Glutathione reductase (GshR) Oxidoreductase (OR) Oxidoreductase (OR) Catalase (KAT) NADH peroxidase (NPXase) Thioredoxin reductase (TXNRD) Phosphoglycerate dehydrogenase (PGDH)

 C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

a)

Spot

Estimated Mr (kDa) and pI

15C 38.4/6.20 41CW 36.0/5.34 60CW 31/4.90

b)

B/P

c)

p Value A.N.

2.5 2 0.1

0.027 0.036 0.011

ZP_0707884 R9X3V2

66CW 67CW 97CW 5C

31.0/9.85 31.0/5.00 17.9/4.61 55/5.40

11.2 0.1 0.2 2.6

0.043 0.047 0.008 0.040

Q88×05 YP_003063300 YP_003062893 YP_003062527

7C 9C 35C 37C 36C 38C 89CW 2C 4C 16C

61.1/6.20 50/6.00 20/5.70 19/6.15 20/6.90 17.9/5.40 21.9/5.19 63/5.00 55/6.00 37.3/7.00

2.4 0.4 2.2 20 0.5 2 2 3.3 0.5 0.05

0.046 0.029 0.048 0.050 0.039 0.017 0.045 0.021 0.042 0.006

YP_008120650 YP_003063569 YP_003063298

11C

50/6.35

2.8

0.031

F9UP42

28C

30/7.10

2.0

0.015

C6VQ34

30C

29.5/7.20

2

0.042

C6VLJ0

41C

17.5/4.40

20

0.003

YP_003062993

YP_003062446 YP_003062144 YP_003062989 YP_003062992 AAY27085 YP_003062952

31CW 38.6/5.57

0.2

0.017

YP_003064139

17C 37/5.65 90CW 21.5/4.40

0.5 0.036 0.05 0.047

YP_003063753

94CW 39C 31C 6CW 8CW 9CW 10CW 26CW

0.05 0.5 0.5 2.6 2.5 1.2 2.2 0.05

21.3/8.40 17.5/5.40 24.6/5.41 66/4.66 73.8/4.40 73.8/4.30 65.3/4.70 39/5.60

0.016 0.028 0.050 0.038 0.027 0.040 0.012 0.050

YP_004888979 AFM80219 YP_003063359 YP_003062187 Q88VL8

93CW 21.5/4.53 29C 29.5/4.70

0.2 20

0.017 0.041

YP_003063454 YP_003062235

42C 43C 44C 5E 62CW 24C 64CW 6C 8C 18C 26C

2 2.2 2 0.01 2.25 2.2 2.5 2.4 0.5 20 0.3

0.028 0.005 0.034 0.033 0.049 0.035 0.007 0.022 0.050 0.014 0.028

YP_003062359 YP_003064271 Q88YM6 YP_003062644 YP_003064295

17/5.10 16.5/5.40 10.3/4.91 47.0/5.10 31.6/5.50 31.6/5.55 31.2/5.40 55.2/5.90 52/5.65 33.5/4.70 31.5/7.00

NP_786627 YP_003064442 YP_003063632 YP_003062215 YP_003062234

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Proteomics 2015, 00, 1–14 Table 1. Continued a)

Function

Homologous protein

Spot

Energy metabolism

F0F1 ATP synthase subunit beta (F0F1 ATPase) Putative manganese-dependent inorganic pyrophosphatase (PPase) Manganese-dependent inorganic pyrophosphatase (PPase) Extracellular protein Extracellular protein Extracellular protein Uncharacterized protein Peptidoglycan-binding protein (PGB) Extracellular protein

17CW 51.5/4.57

Unknown function

Extracellular protein, lysozyme-like domain

Hypothetical protein

Not identified

Extracellular protein, membrane-anchored Extracellular protein Extracellular protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein N574_0118545, partial Not identified Not identified Not identified Not identified Not identified Not identified Not identified Not identified Not identified Not identified Not identified Not identified Not identified Not identified

Estimated Mr (kDa) and pI

b)

c)

B/P

p Value A.N.

20

0.006

Q88UU3

45CW 35.1/4.48

0.2

0.028

R4PQV1

13C

48/4.70

2.6

0.031

YP_003063129

1E 3E 6E 9E 17E 10E 43CW 15E 18E 39CW 84CW 96CW 14E 38CW 21C 33C 34C 7E 12E 13E 28CW 50CW 56CW 57CW 58CW 59CW 63CW 81CW 85CW 12C 40C

59.9/8.50 51.99/9.17 44.42/6.09 41.37/8.95 22.0/5.00 35.64 /9.29 35.4/9.29 23.0/4.00 22.2/3.90 36.5/6.00 23.6/4.35 17.8/9.20 26/9.00 36.5/5.29 32/5.15 21/4.45 20/5.10 44.42/6.35 35/4.50 30/4.20 40/3.90 33.0/4.35 31/4.07 31/7.50 31/7.70 31/6.50 31.4/4.08 23.7/4.00 23.5/8.60 50/8.00 17.5/5.60

0.05 0.02 0.04 0.01 20 0.05 0.3 0.05 0.05 0.2 4 0.05 0.01 0.05 2.4 2.6 2.2 0.05 0.03 0.05 2 0.2 0.05 0.05 0.05 2 0.2 0.5 0.4 0.4 0.2

0.014 0.023 0.022 0.009 0.048 0.036 0.037 0.024 0.050 0.033 0.028 0.037 0.014 0.042 0.040 0.015 0.030 0.042 0.008 0.050 0.048 0.027 0.035 0.007 0.031 0.032 0.028 0.037 0.026 0.044 0.031

U2W6G1 R4Q5J2 R4Q1P4 D7V9V9 ZP_03959644 U2WJB8 C6VKK0 R9X1J1 C6VPI3 R9X2C6 YP_003061855 YP_003063477 YP_003063471 ETC78453

a) Spot designations correspond to those of the gels shown in Figs. 1 and 2. b) Biofilm/planktonic relative volume of protein spots. c) Accession number, AN.

Compared to planktonic cells, biofilm-grown L. plantarum DB200 showed higher levels of DnaK, trigger factor (TF), GroEL, ClpP, alkaline shock protein, small HSP, and GroES (Table 1). In contrast, ClpL was found to be decreased. Oxidoreductases and catalase were oversynthesized under biofilm conditions, whereas glutathione reductase was at the highest level in planktonic cells. Some stress proteins (e.g., DnaK, TF, and GroES) showed moonlighting behavior. With the exception of ClpP, very consistent interaction networks were identified for several proteins associated with biofilm cells, including those related to amino acid and protein metabolism and L-LDH (Supporting Information Fig. 4). Based on the levels of DnaK and GroEL, the lowest amount of the heat-inducible transcription repressor HrcA was found in the biofilm cells. The HrcA protein was the only protein  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

that negatively correlated with the DnaK and GroEL proteins.

3.5 Heat, acid, ethanol resistance, and catalase activity Compared to planktonic cells, L. plantarum DB200 grown in biofilm demonstrated increased heat tolerance by an order of magnitude of approximately 1 log cycle (Fig. 3) and also showed a higher level of tolerance to acidity (Fig. 4A and B) and ethanol stress (Fig. 4C and D). Furthermore, DB200 grown under biofilm conditions showed higher levels of catalase activity (0.8 vs. 0.05 U/min, p = 0.03) compared to planktonic cells.

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Figure 2. Schematic representation of the changes of extracellular, cell wall associated, and cytoplasmic proteins (grouped based on Kyoto Encyclopedia of Genes and Genomes functions) of L. plantarum DB200 cells grown as planktonic or biofilm cells onto modified MRS broth at 30⬚C for 24 h. Compared to planktonic, proteins showing an increased (↑) or decreased (↓) (ࣙ or ࣘ of 2.0-fold, p < 0.05) relative abundance by biofilm cells are represented. Protein names and spot numbers correspond to those of Table 1.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 3. Survival of L. plantarum DB200 grown as planktonic (dark rectangular) or biofilm (light grey rectangular) cells onto modified MRS broth at 30⬚C for 24 h.

4

Discussion

In accordance with previous studies [8], most biofilm cells were found to be lengthened and subject to increased autoaggregation compared to planktonic cells. Although the cell density did not vary between the planktonic and biofilm cells, the lactate yield (molar ratio between glucose consumed and lactate synthesised) was markedly decreased under biofilm conditions, which could be due to the higher capacity of biofilm

cells to metabolise lactic acid [29]. In this study, we performed a comparative proteomic analysis between planktonic and biofilm cells and showed that the relative abundance of 159 protein spots was affected. The metabolic activity of bacteria is typically decreased during growth under biofilm conditions [5]. Indeed, the relative abundance of extracellular and cellwall hydrolases in the biofilm cells of L. plantarum DB200 was markedly decreased. The only exception was found for SrtA. The role of SrtA in surface protein sorting has been

Figure 4. Growth (A and C) and survival (B and D) of L. plantarum DB200 grown as planktonic (dark rectangular) or biofilm (light grey rectangular) cells onto modified MRS broth, added of acetic acid (A and B) or ethanol (C and D), at 30⬚C for 24 h.

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M. De Angelis et al.

described for Staphylococcus aureus [30], with SrtA recognizing proteins bearing the conserved C-terminal cell-wall sorting signal LPXTG (Leu-Pro-any-Thr-Gly). Recently, it was demonstrated that SrtA is required for the appropriate subcellular localization of specific sortase-dependent proteins and for their covalent coupling to the cell envelope of L. plantarum WCFS1 [31]. Studies focused on pathogenic bacteria and lactobacilli have revealed that sortases assemble virulence factors on bacterial cell surfaces, a process that is crucial for adhesion and pathogenicity processes, biofilm formation, and iron uptake [32]. The relative abundance of proteins with the LPXTGmotif, which is responsible for cell adhesion, increases under biofilm conditions compared to planktonic cultures. In addition, sortase-dependent mucus-binding proteins play a role in the autoaggregation of Lactobacillus reuteri [33] and vaginal L. plantarum CMPG5300 [34]. The mannose-specific adhesin gene msa is a potential probiotic gene involved in bacterial persistence and the competitive exclusion of pathogens at the intestinal level [35], and the present study is the first to show that SrtA and LPXTG-motif proteins, including msa, are induced during biofilm growth. In accordance with the cellular shape of L. plantarum DB200, the lowest levels of the cell division protein FtsZ and the cell division initiation protein DivIVA were found in the biofilm cells. FtsZ supports septum formation for cell separation in a variety of bacterial species. However, little is known about septum formation under biofilm conditions; an exception is Neisseria meningitidis, in which FtsZ was found to be downregulated during biofilm formation [4]. The relative abundance of glycolytic enzymes has been shown to be differentially affected under biofilm conditions, mainly depending on the species and strain of pathogenic bacteria [1, 4, 5, 36]. In agreement with these results, the relative abundance of certain glycolytic enzymes (e.g., Eno1, GAPDH, TPI, PGAM2, and NAD-I-LDH) was decreased under biofilm conditions. In contrast, other glycolytic enzymes (e.g., Eno A1, PGK, LDH, and PKI) were increased in the cellwall fraction. Moonlighting activities may be hypothesized for many of the above proteins, as their abundance is predominantly affected at the extra-cytoplasmic level. Some of these proteins have also been identified in the exoproteome of other lactobacilli [37, 38]. In accordance with the cytoplasmic level of some glycolytic enzymes (e.g., PKI and PGM), the lowest level of catabolite control protein A (CcpA) was found in the biofilm culture; however, a positive role for the master regulator catabolite control protein A in biofilm development was previously reported [39]. PrsA, the counterparts of which in other gram-positive bacteria encode functions involved in protein postexport, was found to be increased in the biofilm culture of DB200; this protein also appears to be particularly abundant in Pneumococcus biofilms [5]. GreA, a transcription factor that is responsible for the protection of cellular proteins against aggregation and environmental stress due to its chaperon activity, was found to be decreased during the cultivation of L. plantarum DB200 under biofilm conditions. Because the highest expression of FadD, FabH, AccC2, AccD2, AccD, and  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2015, 00, 1–14

AccB3 were found under biofilm conditions, a different fatty acid and lipid biosynthesis pathway might be hypothesized. Overall, a marked increase in heat stress proteins (e.g., DnaK, TF, GroEL, ClpP, HSP, and GroES) was found for biofilms compared to planktonic cells. Interestingly, all of the above heat stress proteins increased when L. plantarum was cultivated with pheromone plantaricin A and/or cocultivated with L. sanfranciscensis [18]. DnaK and GroEL also showed adhesive and immune-modulatory properties [38]. In agreement with the high levels of heat stress proteins, the biofilm cells showed the highest survival under heat stress conditions. In general, heat stress proteins show cross-protection toward other environmental shocks [40]. Thus, the high expression levels of these proteins may contribute to the increased acid and ethanol stress resistance of biofilm cells. The upregulation of proteins such as catalase that are involved in the detoxification of ROS in biofilm cells of L. plantarum DB200 is in agreement with proteomic and microarray studies on the biofilms of S. aureus and N. meningitidis [4]. It is likely that massive genetic and proteomic efforts will be required in the future to elucidate the biofilm and coordinated behavior of lactobacilli. Bacterial performance is mostly the consequence of very complex community interactions. Within such a context, this study shows the proteomic adaptation of L. plantarum DB200 during biofilm formation, although the proteome changes were found to be strictly related to phenotypic traits. The authors have declared no conflict of interest.

5

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