Uncovering Surface-Exposed Antigens of ... - ACS Publications

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Uncovering Surface-Exposed Antigens of Lactobacillus rhamnosus by Cell Shaving Proteomics and Two-Dimensional Immunoblotting Eva Espino,† Kerttu Koskenniemi,‡,∥ Lourdes Mato-Rodriguez,†,⊥ Tuula A. Nyman,§ Justus Reunanen,‡ Johanna Koponen,§,# Tiina Ö hman,§ Pia Siljamak̈ i,§,† Tapani Alatossava,† Pekka Varmanen,*,† and Kirsi Savijoki† †

Department of Food and Environmental Sciences, ‡Department of Veterinary Biosciences, and §Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland S Supporting Information *

ABSTRACT: The present study reports the identification and comparison of all expressed cell-surface exposed proteins from the well-known probiotic L. rhamnosus GG and a related dairy strain, Lc705. To obtain this information, the cell-surface bound proteins were released from intact cells by trypsin shaving under hypertonic conditions with and without DTT. Liquid chromatography tandem mass spectrometry (LC−MS/MS) analyses of the purified peptides identified a total of 102 and 198 individual proteins from GG and Lc705, respectively. Comparison of both data sets suggested that the Msp-type antigens (Msp1, Msp2) and the serine protease HtrA were uniquely exposed at the cell surface of GG, whereas the Lc705-specific proteins included lactocepin and a wider range of different moonlighting proteins. ImmunoEM analyses with the GG and Lc705 antibodies suggested that the whole-cell immunization yielded antibodies toward surface-bound proteins and proteins that were secreted or released from the cell-surface. One of the detected antigens was a pilus-like structure on the surface of GG cells, which was not detected with Lc705 antibodies. Further 2-DE immunoblotting analysis of GG proteins with both L. rhamnosus antisera revealed that majority of the detected antigens were moonlighting proteins with potential roles in adhesion, pathogen exclusion or immune stimulation. The present study provides the first catalog of surface-exposed proteins from lactobacilli and highlights the importance of the specifically exposed moonlighting proteins for adaptation and probiotic functions of L. rhamnosus. KEYWORDS: Lactobacillus rhamnosus, surfaceome proteins, surface shaving, immunoEM, 2-DE immunoblotting, LC−MS/MS

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tives of the two pheno−genotypic groups.2 GG belongs to the host-adapted group that is suggested to include effective health promoter strains,2,3 which are of human origin and equipped with an ability to colonize the ileum and colon and to survive the conditions of the human GIT.4 These strains are regarded as factors contributing to pathogen exclusion, immune modulation, and enhanced contact with the mucosa.5−8 Although Lc705 is considered as a dairy adapted strain, it is also able to exert certain bioprotective properties as well as some health benefits when administered in combination with other probiotic strains.9−12 Proteins expressed at the cell surface (the surfaceome) are involved in signaling events, transport, and adherent growth and therefore are the likely factors contributing to inter- and intracellular interactions and to adaptation in changing environments. Decoding several L. rhamnosus genomes, including those of GG and Lc705, has revealed the presence of genes coding for cell surface exposed proteins, from which

INTRODUCTION Lactobacilli have been extensively used in the food and pharmaceutical industries for many years. This genus of lactic acid bacteria (LAB) has also attracted special interest because certain health benefits are ascribed to the consumption of specific strains of Lactobacillus species, including Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei.1 Of these species, L. rhamnosus has recently been the subject of an extensive pangenome analysis, which divided this species into two groups. The first group is characterized by antimicrobial activities, stress resistance, and flexibility to adapt to stable nutrient-rich environments, including milk. The second group has the ability to adapt to the gastrointestinal tract (GIT), which contains varying nutrient resources, bacterial population densities, and host effects.2 The widely marketed probiotic organism, L. rhamnosus GG (hereafter GG), which has many of the desired features of a successful probiotic strain, and the L. rhamnosus strain Lc705 that is routinely used as an adjunct starter culture in dairy products, including cheese and yogurt, are known representa© XXXX American Chemical Society

Received: October 7, 2014

A

dx.doi.org/10.1021/pr501041a | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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the species-specific pilus proteins SpaCBA from GG and the cell surface protein complex SpcA-SpcB from Lc705, GG, and L. rhamnosus HN001, with likely protein−protein interaction mediating functions, are the best characterized to date.13−19 The presence of the unique SpaCBA pilus in GG, contributing to direct interaction with the host, biofilm formation, and balancing proinflammatory cytokine expression (IL-8), is thought to explain the more adhesive nature and the prolonged persistence of GG in the human GIT compared to Lc705.13,14 In addition, membrane teichoic acids, cell-wall peptidoglycans as well as several cytoplasmic proteins are reported to have strong immunomodulatory properties and elicit specific immune responses necessary for establishment in the gut.20−25 L. rhamnosus has been the subject of many proteomic studies aiming to relate the genome to the phenotype.26−31 Our previous study using Ge-liquid chromatography tandem mass spectrometry (Ge-LC−MS/MS) analysis to compare all the expressed GG and Lc705 proteins suggested strain-specific differences among the cell surface bound proteins.30 The present study was undertaken to complement these findings and to explore surfaceome differences between GG and Lc705 in more detail. To that end, we used cell surface shaving proteomics to identify all surface-bound proteins from GG and Lc705 cells. These analyses were complemented with immunoproteomics to identify antigenic surface proteins from GG. In addition, the cross-recognition of GG proteins using antibodies raised against cells of Lc705 and the more distant Lactobacillus delbrueckii was employed to identify antigens common to different Lactobacillus species.

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Figure 1. Cell density of GG and Lc705 propagated in MRS broth as a function of time. The error bars indicate standard deviations among four biological replicates. At the time points indicated by the arrows, equal amounts of bacteria were withdrawn for surfaceome shaving and 2DE immunoblotting analyses.

of the trypsin-digested cells from each condition was determined by plating serial dilutions (10−12, 10−14, and 10−16) on MRS agar. Colony forming units (CFUs)·mL−1 were determined after incubation for 48 h at 37 °C under anaerobic conditions. This was repeated twice with three biological replicas. To improve the identification of disulfide bond-containing proteins, trypsin-shaving was also tested under reducing (5 mM DTT) and nonreducing conditions in 50 mM TEAB and in 50 mM TEAB containing 17% sucrose using the optimal digestion time (15 min at 37 °C). Cells were first removed by centrifugation (4000g, 2 min, RT), and the digestions were filtrered through 0.2 μm pore size acetate membranes (Spin-X; Costar; Corning Incorporated) by centrifugation (16 000g, 2 min, RT) for further purification. Digestions (flow-through) incubated for 16 h at 37 °C were terminated by adding 0.6% TFA.

MATERIALS AND METHODS

Bacterial Strains and Growth Conditions

L. rhamnosus GG, L. rhamnosus Lc705, and L. delbrueckii subsp. lactis, ATCC15808, were propagated on MRS agar (Becton, Dickinson and Co.) and cultivated anaerobically (using Anaerocult A, Merck KGaA, Darmstadt, Germany) at 37 °C. Separate colonies were inoculated in duplicate (for cell surface shaving) or in quadruplicate (for 2-DE immunoblotting) in fresh MRS broth (BD) to grow the strains first at 37 °C under microaerophilic conditions. Overnight cultures were diluted 100-fold in fresh MRS medium and samples for surface shaving (GG and Lc705 cells) and 2-DE immunobloting (GG cells) were withdrawn at OD600 ≈ 2.5 (midlogarithmic growth phase) and at OD600 ≈ 4.7 (late-logarithmic growth phase), respectively (Figure 1).

LC−MS/MS Analyses

The peptides resulting from trypsin-shaving were purified using C18 ZipTips (Millipore) and analyzed using an Ultimate 3000 Nano-LC system (Dionex, Sunnyvale, CA) coupled to a QSTAR Elite hybrid quadrupole TOF mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA) with nanoESI ionization.33,34 Briefly, the samples were first concentrated and desalted on a C18 trap column (10 mm × 150 μm, 3 μm, 120 Å; PROTECOL, SGE Analytical Science, Griesheim, Germany) followed by peptide separation on a PepMap100 C18 analytical column (15 cm × 75 μm, 5 μm, 100 Å; LC Packings, Sunnyvale, CA) using linear gradient of acetonitrile (in 0.1% formic acid) from 0% to 40% in 120 min with flow rate of 200 nL·min−1. MS data were acquired automatically using Analyst QS 2.0 software. Information-dependent acquisition method consisted of a 0.5 s TOF-MS survey scan of m/z 400−1400. From every survey scan, two most abundant ions with charge states +2 to +4 were selected for product ion scans, and each selected target ion was dynamically excluded for 60 s. Smart IDA was activated with automatic collision energy and automatic MS/MS accumulation. All LC−MS/MS analyses were performed with three biological replicates.

Surface Shaving of Live GG and Lc705 Cells

Cell surface proteins from GG and Lc705 were released from living cells using trypsin in the absence and presence of 17% sucrose and/or DTT.32 Briefly, the intact cells (1.8 mL) harvested by centrifugation (4000g, 10 min, 4 °C), were washed once with 50 mM TEAB (triethylammonium bicarbonate). The washed cells harvested by centrifugation at 3000g for 5 min (4 °C) were suspended gently in 100 μL of TEAB with or without 17% sucrose (pH 8.5). Enzymatic digestion was performed with modified porcine trypsin (47 ng·μL−1) (Promega) at 37 °C for different time periods (15, 30, 45, and 60 min) to screen for the optimal digestion time that would result in the maximal protein release without cell lysis. Digestions were terminated by the addition of trifluoracetic acid (TFA) to a final concentration of 0.6% (Applied Biosystems), and the released protein/peptide concentrations were measured with a NanoDrop spectrophotometer (ND 1000, Fisher Scientific) at 280 nm. The viability B



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(Sigma-Aldrich), 4% CHAPS (Sigma-Aldrich), and 30 mM Trizma base (Sigma-Aldrich Co.). The protein concentration was determined using the 2-D Quant Kit (GE Healthcare) according to the manufacturer’s protocol. Protein samples (40 μg in total) were subjected to isoelectric focusing (IEF) with IPG strips (11 cm, pH 4−7, Bio-Rad) followed by second dimension separation with 12.5% Criterion gels (Bio-Rad) using a previously described method.28 For immunoblotting, the gels were equilibrated in Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) for 5 min. After electrophoresis, the proteins were electroblotted onto nitrocellulose membranes (Trans-Blot transfer medium, Bio-Rad) using the Trans-Blot semidry transfer cell (Bio-Rad) at 15 V for 40 min. After electroblotting, the gels were stained with silver to assess the efficiency of protein transfer.40 Next, the membranes were stained with SYPRO Ruby stain (Lonza, Rockland, ME) to detect all the GG proteins and then immunoblotted with specified antibodies using the protocol described by Harlow and Lane.39 Briefly, the fluorescent 2-DE images were acquired with a FLA-5100 laser scanner with laser excitation at 473 nm and a resolution of 50 μm. After this, the membranes were rinsed several times with PBS, treated with 3% BSA (Sigma-Aldrich) overnight at 5 °C and immunoblotted with GG, Lc705, or ATCC15808 antibodies (1:4000 each) and AP-conjugated goat antirabbit IgG (ZYMED Laboratories, San Francisco, CA). The AP-bound immune complexes were stained using a BCIP/NBT Kit (Zymed Lab. Inc.) according to the instructions provided. Each immunoblot was repeated at least three times. The images of the 2-DE immunoblots were acquired with a FLA-5100 laser scanner using an LPG filter at 532 nm with a resolution of 50 μm. The detected fluorescent and antigen proteomes were analyzed and compared using the SameSpots software (TotalLab Ltd., version 4.5). The immunoblot images were first aligned with their corresponding fluorescent 2-DE image, and then the fluorescent 2-DE images were aligned with each other. Slight distortions in the protein migration patterns were corrected with the aid of manually drawn and automated vectors comparing each image to the 2-DE reference image. After automatic spot detection, background subtraction, and spot intensity normalization, all protein spots detected as antigens were cut out from the silver-stained 2-DE gels, and the proteins were identified using the peptide-mass fingerprinting (PMF) and the LC−MS/MS-based identification methods as described previously.26,28,40 The PMF data were searched against the GG protein database using the Biotools 3.0 (Bruker Daltonik) interface with the following search criteria: trypsin digestion with one missed cleavage allowed, carbamidomethyl modification of cysteine as a fixed modification, and oxidation of methionine as a variable modification. For the PMF spectra, the maximum peptide mass tolerance was ±80 ppm. A successful identification was reported when a significant match (p < 0.05) was obtained. The presence of known contaminants containing matrix, trypsin, and keratin peptide masses were excluded from the database searches. In the case of the LC−MS/MS, identifications were considered reliable when a significant match (p < 0.05) with a minimum of two peptides (ion score > 40) was obtained. The LC−MS/MS data were also assessed for the presence of trypsin and keratin contamination as described above.

For protein identification, the LC−MS/MS data obtained from GG and Lc705 peptides were searched against their respective databases; the GG protein database with 2944 entries (FM179322) and the Lc705 database with 2992 entries (FM179323 and pLC; FM179324)13 using the Mascot (version 2.4.0) search engine through the ProteinPilot (version 4.0) interface. The search criteria for Mascot searches were trypsin digestion with one allowed missed cleavage, carbamidomethyl modification of cysteine as a fixed modification, oxidation of methionine as a variable modification, a peptide mass tolerance of 50 ppm, an MS/MS fragment tolerance of 0.2 Da, and peptide charges of 1+, 2+, and 3+. A successful identification was reported when a significant match (p < 0.05) was obtained with a Mascot ion score ≥40 was obtained. In the case of single peptide hits, ion scores >45 were required for positive identification. The LC−MS/MS data were also searched against all protein entries in the Swiss-Prot database (p < 0.05) to assess the presence of contaminating peptides originating from trypsin and keratin autolysis. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository35 with the data set identifier PXD001201. To estimate the false discovery rates (FDRs), all Mascot searches were repeated using identical search parameters and validation criteria against GG and Lc705 decoy databases containing all protein sequences in both forward and reverse orientations. Sequences were reversed using the Compid tool,36 and the FDR percentages were calculated using the formula 2nreverse/(nreverse + nforward) given by Elias and co-workers.37 The calculated FDRs were 0% in each data set. Proteome Bioinformatics

The theoretical isoelectric point (pI) and molecular weight (Mw) values for the identified surfaceome proteins were defined with the ProMoST program.38 The presence of protein orthologs and paralogs in GG and Lc705, the cell membrane, and wall anchoring domains and motifs, and the secretory signals that direct proteins to the extracellular milieu were obtained from the previous comparative proteome cataloging study.30 Raising Antibodies against GG, Lc705, and ATCC15808

Whole bacterial cells of three different Lactobacillus strains, GG, Lc705, and ATCC15808, were used as immunogens, for two New Zealand white rabbits each. Whole bacterial preparations in phosphate buffered saline (PBS) were mixed with Freunds’s complete adjuvant and used for the first three immunizations and with Freunds’s incomplete adjuvant for the rest of the immunizations.39 The amount used per immunization was 200 μL/rabbit. The immunization protocol was as follows: three immunizations in two week periods followed by monthly periods for a total immunization period of three months. After each immunization and before the first immunization (for negative control sera), animals were bled for test samples. At the end of the immunization protocol, rabbits were bled by cardiac puncture according to the procedure of the University Animal Center (Oulu, Finland). 2-DE Immunoblotting and Protein Identification

Proteins were extracted and purified from GG cells (5 mL cell samples) as described previously.26 Proteins were purified using the 2-D Clean-Up Kit (GE Healthcare) and solubilized in a buffer composed of 7 M urea (Sigma-Aldrich), 2 M thiourea C



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Immunogold Labeling and TEM Analyses

Prior to immunogold labeling, half of the bacterial culture was washed in 0.1 M Na-acetate, pH 4.0, and fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in Na-acetate, pH 4.0. Transmission electron microscopy (TEM) experiments with intact and fixed GG and Lc705 cells were conducted essentially as described previously.18 Briefly, bacteria were washed once in PBS and suspended in PBS before being applied to the grids. The grids were then washed with 0.02 M glycine in PBS followed by blocking with 1% BSA in PBS (PBSb). After being blocked, the grids were incubated with 1:100 diluted antisera raised against intact bacteria, after which they were washed with 0.1% BSA in PBS. Next, the grids were incubated on droplets of protein A-gold (10 nm gold particles) diluted in PBSb, and washed with PBS. After fixation with 1% glutaraldehyde, the grids were washed with water, followed by negative staining with 1.8% methylcellulose and 0.4% uranyl acetate (pH = 4.0). The grids were examined and micrographs were taken using a JEOL 1200 EX II transmission electron microscope (TEM).

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RESULTS AND DISCUSSION

Optimizing the Surface Shaving Method for GG and Lc705

Previous total proteome cataloging of GG and Lc705 predicted that proportionally more surfaceome proteins with likely roles in adaptation would be expressed by Lc705.30 To complement these findings and to gain a deeper insight into the composition of the GG and Lc705 surfaceomes we applied surface shaving proteomics involving trypsin digestion to release surface attached proteins and LC−MS/MS analysis to identify the released peptides. The main challenges in identifying surfaceexposed proteins are to maximize the number of proteins identified and to avoid cell lysis, which may lead to the release of cytoplasmic proteins.41,42 These challenges were addressed first by harvesting cell samples from logarithmic cultures (Figure 1) and then by searching for the optimal digestion time for cell surface shaving. The tested trypsin digestion times were 15−60 min, and the largest amount of peptides was released after 30 min. However, the number of viable GG cells was markedly decreased compared to control samples without trypsin, suggesting that the increased peptide concentration most likely was due to cell lysis (data not shown). In the case of Lc705, a decrease in the number of viable cells was only observed with the longest trypsin digestion times (45−60 min) (data not shown), suggesting that Lc705 cells are more robust than GG in the conditions used. Because the surface shaving with the shortest reaction time released a reasonable amount of peptides without affecting the viability of the GG cells (data not shown), the 15 min incubation time was used to release surface-exposed proteins from both the GG and Lc705 cells. Digestions were next tested under conditions (17% sucrose and/or 5 mM DTT) that make some surface-attached proteins more accessible to trypsin, while protecting the cells from autolysis.42 Trypsin digestions performed in the absence and presence of sucrose (17%) and/or DTT (5 mM) resulted in increased numbers of viable GG and Lc705 cells compared to the control digestions (Figure 2A). This result could be explained by the proteolytic cleavage of certain surfaceome proteins, reducing cellular adhesion and aggregation leading to the increased number of colony-forming individual cells. In the case of Lc705, the amount of proteins and peptides released was highest in the presence of DTT and sucrose, whereas for

Figure 2. Effect of different trypsin digestion conditions on cell viability and the extent of peptide release. (A) Cell viability was measured by plating trypsin-shaved cells on MRS agar plates and by calculating colony forming units (CFUs) after 2 days of incubation at 37 °C under anaerobic conditions. (B) Using a NanoDrop, the concentrations of released peptides (ng·μL−1) were measured after 15 min digestion with trypsin. All experiments were repeated twice with three biological replicates.

GG cells the maximal protein and peptide release was obtained with sucrose (Figure 2B). Thus, to maximize the number of identifiable proteins without “contaminating” the surfaceome protein fractions with cytoplasmic proteins, all shaving experiments were conducted under hypertonic conditions exerted by sucrose and with both reducing and nonreducing conditions. An equal amount of tryptic peptides released from three biological replicas (in the absence and presence of DTT) of GG and Lc705 were submitted to LC−MS/MS analyses. The LC− MS/MS analyses and database searches identified 102 and 198 proteins from GG and Lc705, respectively (Supplemental Tables 1 and 2; the PRIDE database with data set identifier PXD001201). Additional searches against the entire Swiss-Prot protein database identified only few keratin and/or trypsin peptides, demonstrating very low levels of keratin contamination. The number of single peptide-based identifications was high in both cases (30 proteins in GG and 80 in Lc705 samples), which was likely to have resulted from the small size of the identified proteins and the short trypsin digestion time (15 min) that was used to release peptides from intact cells. In the case of both strains, the overlap in protein identifications among biological replica samples was extensive, with more than 50% of the identified proteins shared between any two replicate samples (Figure 3A). The number of specifically identified proteins obtained in the presence DTT was higher from Lc705 than GG (Figure 3B). These included D



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The identified GG and Lc705 proteins were next classified into different functional groups based on their COG (orthologous groups of proteins) codes,43 which shows that most of the identified proteins were associated with translation and that Lc705 has more proteins than GG in most of the COG groups (Figure 4B). Further comparison revealed that the number of uniquely identified proteins was 34 in GG and 130 in Lc705, whereas more than 60 proteins were commonly displayed at the cell surface of both strains (Figure 4C). With the exception of two GG proteins, all the uniquely identified proteins had evolutionary counterparts in both strains (Supplemental Table 2). All surfaceome identifications obtained in the present study were also compared with those reported in our previous total proteome cataloging study30 revealing that the same set of proteins was identified by GeLC−MS/MS. The identified proteins assumed to be anchored to the cell wall/cell membrane or secreted out of the cell fell into seven groups according to the predicted anchoring mechanism (Figure 4D). In the case of both strains, the highest number of identifications was obtained with integral membrane proteins (IMPs) with one or more TMDs and with proteins secreted to extracellular milieu. In Lc705, one of the specifically identified IMPs was the pili retraction protein T (PilT). PilT is a hexameric ATPase required for type IV pilus retraction, which is known to mediate intimate attachment to and signaling in host cells, type II secretion, biofilm formation, natural transformation, and phage sensitivity in Gram-negative bacteria.44−47 The serine-type lactocepin PrtR, an LPxTG domain containing protein, was also specifically identified from Lc705. The GG strain harbors the PrtR encoding gene; however, the corresponding protein was not identified, suggesting that the protein is not produced or its amount remained below detection limit. Since this cell-envelope associated protein is known for its casein-degradation activity,48 it could be that higher PrtR levels facilitate the adaptation of Lc705 in milk environment containing casein. In addition, in other probiotics such as Lactobacillus casei and Lactobacillus paracasei, lactocepin is reported to possess additional functions,

Figure 3. Protein identification results after surfaceome shaving of GG and Lc705 cells. (A) The number of protein identifications (Mascot ≥ 40, p < 0.05) that are unique and common among different biological replicate samples prepared in the absence or presence of DTT. (B) Comparison of the surface shaving identifications obtained under nonreducing (−DTT) or reducing (+DTT) conditions.

proteins, such as ClpB ATPases, exoribonuclease R, βlactamase, PTS system transporters, certain regulators, peroxidases, and reductases, which could form potential redox-active intra/interprotein disulfide bridges. The presence of DTT in the shaving reactions, making these proteins more accessible to trypsin, is the likely reason for their identification only under reducing conditions. Thus, the conducted surfaceome analyses of GG and Lc705 with or without DTT implies that Lc705 is more efficient in exporting proteins to the cell surface Specifically Expressed Surface Anchored Proteins Have Likely Roles in Adaptation and Probiotic Functions

To investigate specific differences between the expressed GG and Lc705 surfaceomes, all the data sets were compiled, and the identified proteins were divided according to their predicted subcellular location and function. The first categorization revealed that proportionally more cytoplasmic proteins (77− 88%) were identified from both strains, whereas the rest of the identifications (23% in GG and 12% in Lc705) were predicted to contain motifs signaling the protein for secretion or anchoring to the cell membrane or the cell wall (Figure 4A).

Figure 4. Classification of the identified GG and Lc705 surfaceome proteins. (A) The division of all identified GG and Lc705 proteins on the basis of their predicted subcellular location. (B) A comparison of all the identified GG and Lc705 proteins as a function of their COG categories. COGs are as follows: C, energy production and conversion; D, cell cycle control, cell division and chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination, and repair; M, cell wall/ membrane/envelope biogenesis; N, cell motility; O, post-translational modification and protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites in biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown. T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms. N/A indicates query proteins that do not belong to any of the currently defined COGs. (C) The number of unique and common identifications between the GG and Lc705 datasets. (D) A diagram showing the number of the identified GG and Lc705 proteins predicted to be exported out of the cell or anchored to the cell-membrane or cell-wall via different domains or motifs. IMP, integral membrane proteins with one or more TMDs; Nterm, Cterm, proteins suggested to be anchored through an N-terminal or C-terminal anchor containing a predicted N-terminal signal peptide but lacking an apparent signal peptidase cleavage site; LPxTG, proteins with a C-terminal LPxTG cell wall anchoring signal for covalent attachment to peptidoglycan by sortase; Secreted, proteins secreted into the extracellular environment; Lipobox, lipoproteins with an N-terminal lipobox that mediates the covalent binding of a conserved Cys residue to a lipid; LysM, proteins with a C-terminal lysine mediating non-covalent attachment to peptidoglycan; Sec, proteins harboring a secretory signal peptide that directs proteins into the extracellular milieu. E



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Table 1. Identified GG and Lc705 Surfaceome Proteins with Potential Adhesive/Probiotic Properties; Proteins Predicted to Have Moonlighting Functions Are Marked with an Asterisk gene name

gene_ID

accession number

protein function

null

LGG_01945

ref|YP_003171691.1|

oppA

LGG_01652

ref|YP_003171398.1|

abc

LGG_00689

ref|YP_003170435.1|

atpB atpB atpF atpF f tsH f tsH pbp2B2

LC_01196 LGG_01177 LC_01198 LGG_01179 LC_02518 LGG_02514 LGG_01706

ref|YP_003173890.1| ref|YP_003170927.1| ref|YP_003173893.1| ref|YP_003170930.1| ref|YP_003175208.1| ref|YP_003172260.1| ref|YP_003171452.1|

prtR

LC_02738

ref|YP_003175428.1|

wzr

LC_02035

ref|YP_003174725.1|

null p75

LC_02014 LGG_00324

ref|YP_003174704.1| ref|YP_003170070.1|

p40 p60 dnaK* dnaK* groEL* groEL* null

LGG_00031 LGG_02016 LC_01585 LGG_01604 LC_01812 LGG_01830 LGG_01865


null null null null null cydA

LC_00239 LC_00784 LGG_00790 LGG_02880 LGG_00106 LGG_00016

ref|YP_003172929.1| ref|YP_003173474.1| ref|YP_003170536.1| ref|YP_003172626.1| ref|YP_003169852.1| ref|YP_003171954.1|

dacA

LC_00245

ref|YP_003172935.1|

dacA

LGG_00254

ref|YP_003170000.1|

tuf * tuf * pepO* eno3* eno3* pgi* gapB*

LC_01356 LGG_01342 LC_01494 LC_00989 LGG_00936 LC_01162 LC_00986


cadA

LC_02794

ref|YP_003175484.1|

guaB*

LC_00240

ref|YP_003172930.1|

guaB*

LGG_00249

ref|YP_003169995.1|

cad

LGG_02307

ref|YP_003172053.1|

L-LDH L-LDH Flot1

LC_02527 LGG_02523 LGG_02157

ref|YP_003175217.1| ref|YP_003172269.1| ref|YP_003171903.1|

null

LGG_00789

ref|YP_003170535.1|

ABC transporter, oligopeptide transporter periplasmic component ABC transporter, oligopeptidebinding protein ABC transporter, substratebinding protein ATP synthase A chain ATP synthase A chain ATP synthase B chain ATP synthase B chain cell division protein FtsH cell division protein FtsH cell division protein/penicillinbinding protein 2 cell envelope-associated proteinase, lactocepin PrtR cell envelope-related transcriptional attenuator truncated antigen (NLP/P60) cell wall-associated glycoside hydrolase (NLP/P60 protein) surface antigen surface antigen (NLP/P60) chaperone protein dnaK chaperone protein dnaK chaperonin GroEL chaperonin GroEL conserved extracellular matrix binding protein conserved membrane protein conserved protein conserved protein conserved protein conserved protein cytochrome d ubiquinol oxidase subunit I D-alanyl-D-alanine carboxypeptidase D-alanyl-D-alanine carboxypeptidase elongation factor Tu (EF-TU) elongation factor Tu (EF-TU) endopeptidase O enolase enolase glucose-6-phosphate isomerase glyceraldehyde-3-phosphate dehydrogenase heavy metal translocating P-type ATPase inosine-5′-monophosphate dehydrogenase inosine-5′-monophosphate dehydrogenase lipoprotein (pheromone precursor) L-lactate dehydrogenase L-lactate dehydrogenase membrane protease subunit, stomatin/prohibitin family protein methyl-accepting chemotaxis-like protein F

[ms] score

no. matched peptides

pI

Mw (Da)

536

12

10.15

61163

45

1a

9.88

108

2

74 54 198 67 73 98 56

domain

no. TMDs

E

lipobox

0

66490

E

lipobox

0

10.5

34518

R

lipobox

1

4 3 4 3 2 2 1

9.53 9.53 7.68 7.68 5.13 5.13 9.56

26509 26461 17676 17690 78224 78238 77432

C C C C O O M

IMP IMP IMP IMP Cterm Cterm Nterm

5 5 1 1 2 2 1

49

1

4.93

155717

O

LPxTG

2

45

1

9.47

32740

K

Nterm

1

41 148

1 4

8.39 6.96

16142 49739

M M

Sec Sec

0 1

149 379 357 109 244 399 209

8 34 11 5 5 11 8

7.7 9.06 4.57 4.57 7.32 7.32 5.12

42600 40915 67221 67221 21085 21071 252730

M M O O J J null

Sec Sec

LPxTG

0 1 0 0 0 0 2

83 198 42 44 88 60

1 4 1 1 2 1

9.43 4.13 4.13 10.74 10.83 9.47

29083 17876 17886 20688 25490 54616

null null null null null C

IMP Sec Sec Sec Sec IMP

4 1 1 1 0 9

104

3

10.17

46901

M

Sec

1

185

4

10.18

46988

M

Sec

1

463 3423 164 165 61 80 193

12 58 4 4 5 1 4

4.56 4.56 5.33 4.39 4.39 4.73 5.63

43560 43560 71709 47127 47127 49365 36724

J J O G G G G

48

1

4.8

64041

P

46

1

5.99

52619

E

0

67

1

5.99

52619

F

0

138

6

10.03

32688

T

lipobox

0

226 43 62

8 1 2

5.09 5.09 4.92

35531 35504 55348

G C S

IMP IMP IMP

1 1 1

112

2

9.11

14892

NT

Sec

1

COG

0 0 0 0 0 0 0 IMP

6



Article

Table 1. continued gene name

gene_ID

accession number

protein function

null pgk* gpm2* yacL

LGG_01113 LC_00987 LGG_01983 LC_02332

ref|YP_003170859.1| ref|YP_003173677.1| ref|YP_003171884.1| ref|YP_003175022.1|

oxaA1

LC_01707

ref|YP_003174397.1|

EF-G*

LC_02494

ref|YP_003175184.1|

EF-G*

LGG_02493

ref|YP_003172239.1|

secA* f ruA

LC_00948 LC_01372

ref|YP_003173638.1| ref|YP_003174062.1|

f ruA

LGG_01359

ref|YP_003171105.1|

null

LGG_01093

ref|YP_003170839.1|

pdhC*

LC_01336

ref|YP_003174026.1|

pyk* pyk* nrdE*

LC_01390 LGG_01375 LC_01491

ref|YP_003174080.1| ref|YP_003171121.1| ref|YP_003174181.1|

htrA lytR

LGG_02806 LC_00275

ref|YP_003172552.1| ref|YP_003172965.1|

null tpiA* tpiA* null

LC_02883 LC_00988 LGG_00935 LGG_00886

ref|YP_003175573.1| ref|YP_003173678.1| ref|YP_003170681.1| ref|YP_003171964.1|

null

LC_00935

ref|YP_003173625.1|

phage-related protein phosphoglycerate kinase phosphoglycerate mutase PIN/TRAM domain protein, Pili retraction protein pilT preprotein translocase subunit YidC protein translation elongation factor G (EF-G) protein translation elongation factor G (EF-G) protein translocase subunit secA PTS system, fructose-specific IIABC component PTS system, fructose-specific IIABC component putative protein without homology pyruvate dehydrogenase complex E2 component pyruvate kinase pyruvate kinase ribonucleoside-diphosphate reductase, alpha subunit serine protease transcriptional regulator, LytR family transporter, RND superfamily triosephosphate isomerase triosephosphate isomerase XRE family transcriptional regulator [Lactobacillus rhamnosus GG] Xre-like DNA-binding protein

[ms] score

no. matched peptides

pI

Mw (Da)

COG

44 128 272 78

2 6 10 1

7.77 5.73 6.97 9.7

16702 42214 25041 41600

NT G G R

IMP

1 0 0 4

42

1

10.08

36542

U

IMP

6

290

8

4.5

76936

J

0

252

3

4.5

76936

J

0

181 110

4 3

5.2 6.7

89469 70028

U G

IMP

0 9

107

3

6.7

70072

G

IMP

9

279

5

10.26

24944

null

Nterm

1

84

2

4.48

57416

C

0

286 302 40

6 10 1

5.07 5.07 6.43

62849 62849 82192

G G F

0 0 0

60 114

3 2

6.06 10.06

45202 40272

O K

IMP Sec

1 1

111 121 408 95

2 4 8 1

9.62 4.63 4.63 9.41

38772 26865 26865 33333

M G G S

IMP

IMP

1 0 0 1

77

1

9.15

33290

S

IMP

1

domain Sec

no. TMDs

a

The MS/MS spectra, including fragment ion assignments, for the proteins with single peptide matches are provided deposited at http:// proteomecentral.proteomexchange.org with the data set identifier PXD001201.

found at the cell surface as well as secreted to the growth medium in other probiotics, have been reported to contribute to antiapoptotic and cell protective effects on human intestinal epithelial cells.51−54 In addition, Msp1 was recently shown to appear in two isomeric forms in GG; a cytoplasmic protein that has undergone o-glycosylation and a nonglycosylated form of the protein after it is exported out of the cell.55

including selective degradation of pro-inflammatory chemokines, which reduce immune cell infiltration and inflammation in experimental IBD models.49 Lc705 also uniquely displays a conserved membrane protein (LC_00239) with reasonably high identification scores (Mascot score > 80, p < 0.05), for which the biological role remains to be shown. From GG, the specifically identified surface-associated proteins included the membrane anchored HtrA, surface, and secreted antigens Msp1 and Msp2, a conserved matrix binding protein (LGG_01865) consisting of an LPxTG domain, a superinfection immunity protein with N-terminal membrane anchoring domain (LGG_01093), and stomatin/prohibitin family IMP protease subunit (LGG_02157). For these GG proteins, with the exception of the superinfection immunity protein, the evolutionary counterparts are found in Lc705. It could be that the expression level of these proteins in Lc705 was below detection limit or that they were not expressed under the tested conditions, which explains why these proteins could not be identified in the present study. The specific identification of the serine-type HtrA suggests that this housekeeping protease known to be associated with the bile stress response in GG and other probiotics27,50 helps the cells to survive the conditions of GIT. The Msp1 and Msp2 antigens specifically identified from GG are also likely to promote the probiotic functions of this strain since these antigens, both

Higher Number of Moonlighting Proteins Is Displayed by the Lc705 Strain

Comparing the subcellular location of the identified proteins suggests that Lc705 exposes more cytoplasmic proteins at the cell surface than GG (Supplemental Table 3). Many of these proteins in several other bacteria are described as nonclassically exported moonlighting proteins, which in the cytoplasm participate in central metabolic functions and stress responses, but after reaching the extracellular environment they take part in the virulence, adhesion, and/or exclusion of other bacteria.56,57 In the present study, 14 and seven moonlighting proteins with suggested adhesive and immunostimulatory functions were identified from Lc705 and GG, respectively (Table 1). The specifically identified moonlighting proteins from Lc705 included the conserved glyceraldehyde-6-phosphate dehydrogenase (GapDH), glucose-6-phosphate isomerase (Pgi), phosphoglycerate kinase (Pgk), pyruvate dehydrogenase (Pdh), protein translocase subunit A (SecA), chaperG



Article

mechanism occurring during vegetative growth, which releasing proteins such as those related to translation (ribosomal proteins) results in decoration of the cell walls of intact bacteria due to the high intrinsic affinity of these proteins to the bacterial cell wall structures.67 Specific mechanisms are thought to include post-translational modification(s) influencing the subcellular localization of the protein or reversible and pHdependent association of certain moonlighting proteins.57,69−71 For example, the moonlighting proteins such as enolase, GAPDH, and Ef-TU proteins, attach to the cell surface under acidic conditions but are released into the medium under neutral or alkaline pH; a process that is likely to affect the adhesion of probiotics to the gastrointestinal surface. The expression of GapDH and Eno at the cell surface has also been shown to be coordinated via HtrA-mediated functions in some bacteria like Streptococcus mutans.72 Our findings suggest that Lc705 is more efficient in nonclassical protein export. Alternatively, the pH of the TEAB buffer (pH 8.0) used for washing the cells has released some of the moonlighting proteins prior to trypsin shaving. Lc705 acidifies the culture medium more rapidly than GG;30 the Lc705 cell samples had lower pH compared to those withdrawn from GG for shaving analyses, which may have helped the Lc705 to retain more moonlighting proteins attached to the cell surface compared to GG. In summary, the surfaceome shaving analyses indicated that the nonclassically exported cytoplasmic proteins account for the majority of the identifications and that both strains benefit from the specific appearance of these proteins at the cell surface.

onin GroES, and endopeptidase O (PepO). These proteins in probiotics including Lactobacilus crispatus and L. plantarum and some pathogenic bacterial species are reported to bind one or several components of the host, including plasminogen, fibrinogen, fibronectin, actin, myosin, EGF-receptor, C5acomplement, salivary mucin, blood antigens, and other bacterial cells.57 In addition, stress-response related cytoplasmic proteins such as the Clp family chaperones ClpX, ClpY, ClpB1/2, ClpC, and ClpE were also identified at the cell surface of Lc705 (Supplemental Table 3). These proteins are involved in central housekeeping duties and some of them (ClpX, ClpC, and ClpE), after association with the ClpP subunit, contribute to the degradation of proteins.58 Other proteolysis-related enzymes such as the aminopeptidases C and N, known cytoplasmic components of the casein degradation machinery in LAB,48 were also specifically identified at the cell surface of Lc705 (Supplemental Table 3). It could be that the export of these house-keeping chaperones and the components of the proteolytic machinery to the cell surface of Lc705 contribute to the adaption to the milk environment containing casein and casein-derived oligopeptides. The unique identifications obtained with GG samples included only the phosphoglycerate mutase (Gpm), which has been identified as human plasminogen receptor in different microorganisms including probiotic bifidobacteria.59,60 It has been suggested that the appearance of this moonlighting protein interferes the plasmin(ogen) system of the human host, which facilitates the colonization of the human GIT and improves the possibility of closer interactions with the host host cells.60 It remains to be shown if specific expression of Gpm at the cell surface of GG contributes to the adhesion of GG. The commonly identified surfaceome proteins from GG and Lc705 included the chaperones DnaK and GroEL and the translation elongation factors Ef-G and EF-Tu, enolase (Eno), inosine-5′-monophosphate dehydrogenase (GuaB), pyruvate kinase (Pyk), and triosephosphate isomerase (TpiA), all having an ability to bind several host components or other bacteria, or to modulate the host immune response.57 Of these, Tpi, Ef-Tu, Eno, and GroEL were identified with higher Mascot scores from GG than from Lc705, suggesting their higher abundances at the cell surface in GG. GroEL is reported to bind intestinal cells and mucus, stimulate IL-8 secretion, and aggregate Helicobacter pylori cells,61 whereas both Ef-Tu and Tpi, besides binding several host factors, are also able to exclude and displace pathogens, including Clostridium sporogenes and Enterococcus faecalis.62 A recent study demonstrated that GroEL is one of the major proteins secreted by Lactobacillus casei (ATCC334) biofilms, and it contributes to antiinflammatory effects in vivo.63 In the case of Lc705, higher identification scores were obtained with DnaK that, in addition to plasminogen binding activity, stimulates antigen-presenting cells by binding to CCR5 and competing with HIV for CCR5 binding.64,65 Among the commonly identified moonlighting proteins, the ribosomal proteins formed the biggest group (∼44% in GG; ∼20% in Lc705) (Supplemental Table 3). Ribosomal proteins have also been identified at the cell surface of other lactobacilli31,66 and Gram-positive bacteria,67 and a novel moonlighting function (contributory role in abiotic stress resistance) for a ribosomal protein in Aspergillus glaucus was recently demonstrated.68 At present, both specific and nonspecific mechanisms are thought to underlie the export of these proteins to the exterior of the bacterial cells. Cell lysis is considered the nonspecific

Examining the Cross-Reactivity of GG Proteins with GG, Lc705, and L. delbrueckii Antibodies

For exploring antigens specifically displayed at the cell surface of GG and antigens that are common to L. rhamnosus, antibodies against whole GG and Lc705 cells were raised in rabbits. ImmunoEM was first tested with intact GG and Lc705 cells, and it revealed that the GG antisera recognized proteins both at the cell surface and in the extracellular milieu of the GG cells, whereas slightly fewer immunoreactive Lc705 proteins were detected with Lc705 antibodies (Figure 5). The immunoreactive proteins found at the cell surface were considered to represent only the bona f ide surface-anchored proteins because the majority of the moonlighting proteins were most likely released under the experimental conditions used for immunolabeling. According to this protocol, prior to TEM and before fixing the labeled cells with glutaraldehyde, the native cells were incubated at a pH above 7.0 for 1−2 h, which is a condition known to release most of the moonlighting proteins.57,71 The immunoreactive proteins found in the extracellular space were suggested to include both the bona f ide secreted proteins and the moonlighting proteins that were released upon exposure to an alkaline pH. This was tested further by washing the cells with a low pH buffer (an acetate buffer, pH 4.0) and fixing the cells prior to immunogoldlabeling. This procedure was expected to block the protein secretion and result in the detection of only bona f ide cell surface proteins and the moonlighting proteins that remained attached. Consistent with this expectation, Figure 5 shows that most of the cross-reactive proteins were found at the surface of the cells, whereas practically no proteins were detected in the space surrounding the cells. These analyses suggest that the GG and Lc705 antisera recognized the bona f ide cell surface and secreted proteins and/or the moonlighting proteins at the cell H



Article

Figure 5. TEM images of intact and fixed GG and Lc705 cells labeled with antibodies raised against GG (A) and Lc705 (B) and 10 nm protein A gold particles (pAg). The Spa pilus specific to GG is shown by an arrow. Scale bars, 200 and 500 nm.

surface and in the extracellular space. Interestingly, the GGspecific antibodies most likely also cross-reacted with the Spafamily proteins forming the pili13 at the cell surface of GG cells because this structure was not detected on GG cells treated with Lc705 antibodies (Figure 5A). Next, proteins purified from whole cells of GG were subjected to 2-DE followed by immunoblotting with GG and Lc705 antisera. GG proteins for immunoblotting were harvested from the late logarithmic growth stage because a previous study suggested that the expression of pili and other potential adhesins of GG are maximal during this growth stage.73 For this purpose, the purified GG proteins were separated by 2-DE and then electroblotted onto nitrocellulose membranes, which were then stained with SYPRO Ruby to visualize the transferred 2-DE proteome (Figure 6). The fluorescent 2-DE images were scanned for SameSpots analysis, which enabled the detection of approximately 350 to 400 spot features in the GG proteome over the pI range of 4−7 (Figure 6A). These membranes were then subjected to immunoblotting with GG,

Figure 6. Representative 2-DE proteome of GG proteins on a nitrocellulose membrane followed by immunoblotting the same membrane with GG antibodies. (A) GG proteins (40 μg in total) were electroblotted to a nitrocellulose membrane and stained with SYPRO Ruby for visualization. (B) The 2-DE immunoproteome obtained after treating the same nitrocellulose membrane with GGspecific antibodies. (C) The overlaid 2-DE images composed of both the fluorescent 2-DE proteome and the 2-DE immunoproteome indicating proteins that cross-reacted with GG-antibodies. White and green spots, immunoreactive proteins; red spots, nonimmunoreactive proteins. The possible SpaC ladder is circled.

Lc705, and ATCC15808 antisera to assess the antibody repertoire generated in rabbits against the three strains. The fluorescent 2-DE images and the 2-DE immunoproteomes detected with each of the three antisera were analyzed by I



Article

whereas six GG proteins were recognized exclusively by one or both of the Lc705 and ATCC15808 antibodies. Among all the identified antigens, three, two, and one were specifically detected with the GG, Lc705, and ATCC15808 antibodies, respectively.

SameSpots, which revealed highly similar antigen profiles for the L. rhamnosus antisera, with 39 and 40 proteins crossreacting with GG and Lc705 antibodies, respectively (Figure 7A,B). Antibodies specific to a more distant Lactobacillus

Majority of the Antigens Were Identified as Known Moonlighting Proteins

For identification, corresponding proteins were cut out from the silver-stained 2-DE gel of GG proteins and subjected to ingel tryptic digestion and mass spectrometric identification (Supplemental Figure 1 and Table 2). A total of 45 proteins were identified, from which eight were identified in several protein spots, suggesting that these proteins have undergone charge modifications (Table 2). All the identified proteins were predicted to have cytoplasmic locations with likely roles in carbohydrate and nucleotide metabolism, protein synthesis, stress responses, or cell division (Table 2). Nine of the identified antigens were associated with the adhesive and immunostimulatory moonlighting proteins GapDH, GroEL, Pgk, Pyk, and Ef-TU. These proteins were all recognized by all three antisera, but DnaK and Tpi could be detected only with the GG and Lc705 antibodies. Proteins detected only with the Lc705 antibodies were Eno and inosine-5′-monophosphate dehydrogenase (IMP), whereas FruB, which in some bacteria is reported to bind human plasminogen,59 was detected only with the ATCC15808 antibodies (Table 2). It has been reported that serum IgG antibodies of healthy and immunocompromised patients recognize the Eno, Pgk, Pyk, Ef-Ts, Ef-Tu, and GroEL proteins expressed by a probiotic L. acidophilus and that differences in their recognition efficiency may indicate significant differences in immune system and commensal bacteria cross-talk in these groups.24 Proteins that were specifically detected only with GG antibodies included an oxidoreductase, the cochaperonin protein complex GroES, and the universal stress protein UspA (Table 2), suggesting that these moonlighting proteins were exposed at the cell surface of only the GG cells in vivo. It is known that GroES interacts with GroEL, which makes GroEL function properly as a chaperonin in the intracellular compartment of a bacterial cell. The secreted GroES in some bacterial pathogens like Mycobacterium tuberculosis has been found to bind plasminogen and promote the recruitment of osteoclasts, which was thought to account for the clinical features of spinal tuberculosis.74 It is also indicated that the moonlighting proteins EF-Tu, GroEL, DnaK, GapDH, and GroES are overexpressed in the cell wall proteome of the highly adhesive strain L. plantarum WHE 92, suggesting the involvement of these proteins in the adhesion process.75 In another study, the increased production of two universal stress proteins UspA at the cell surface of Lactobacillus Hon2N was reported when cells were stressed with lipopolysaccharides.76 The translationassociated proteins, RpsA, RplL, and MetG, were detected by each three Lactobacillus antisera, indicating that these antigens are common to lactobacilli. It is noteworthy that none of the pilus proteins or other surfaceome proteins harboring cell wall or cell membrane anchoring domains or motifs was detected among the identified antigens. The major reason for this is the limited solubility of these proteins, which hinders their analysis by 2-DE. However, 2-DE immunoblots with GG antiserum, but not with the other antisera, revealed a horizontal string of multiple isospot pairs varying in isoelectric point and molecular mass in the upper left

Figure 7. 2-DE immunoblotting of GG proteins with antisera against GG (A), Lc705 (B), and ATCC15808 (C) whole cells. The immunogenic proteins were localized in GG cells after being identified and are listed in Table 2. The possible SpaC ladder is circled.

species and L. delbrueckii subsp. lactis ATCC15808, were also tested, which revealed the presence of fewer cross-reacting antigens (24 detected antigens) among the GG proteins (Figure 6C). Although slight differences between the antigen profiles were detected with different biological replicate antisera (Table 2), the GG proteins treated with control antibodies (diluted 1:100−1:1000) taken from naive rabbits before each immunization injection resulted in no cross-reacting protein spots in 2-DE immunoblots (data not shown). SameSpots analysis indicated a total of 45 distinct antigenic protein spots and 19 protein spots that were found to crossreact with each of the three antisera and are considered to represent the Lactobacillus-specific antigens (Figure 7). Most of the antigenic proteins were recognized by the GG antibodies, J



Article

Table 2. Identified L. rhamnosus GG Proteins Found to Cross-React with GG, Lc705, and ATCC15808 Antibodies; * Refers to Moonlighting Proteins with Predicted Adhesive/Immunostimulatory Functions antiserac,d spot number

protein name and function

1 2 3

Adk AtpD DivIVA

4

DkgA

5 6 7 8

DnaK* Eno* FruB* EF-G*

9

GapDH*

10

GapDH*

11

GapDH*

12

GapDH*

13 14 15 16 17 18

Gpm GroEL* GroEL* GroEL* GroES IMP*

19

LacD

20 21 22 23

Ldh Ldh MalY MetG

24

MreB

25

NagA

26 27 28 29

Pgk* Pgk* Pgk* PpaC

30 31

PtsH PtsI

32 33 34 35

PurA Pyk* Pyk* RplL

36 37 38

RpsA RpsA Ssb

39 40 41 42

Tig TpiA* TpiA* Tsf

43 44

Tuf* Tuf*

adenylate kinase ATP synthase B chain cell-division initiation protein, DivIVA oxidoreductase, aldo/keto reductase family chaperone protein dnaK enolase 1-phosphofructokinase protein translation elongation factor G (EF-G) Glyceraldehyde 3-phosphate dehydrogenase glyceraldehyde 3-phosphate dehydrogenase glyceraldehyde 3-phosphate dehydrogenase glyceraldehyde 3-phosphate dehydrogenase phosphoglycerate mutase 60 kDa chaperonin GROEL 60 kDa chaperonin GROEL 60 kDa chaperonin GROEL 10 kDa chaperonin GROES inosine-5′-monophosphate dehydrogenase tagatose 1,6-diphosphate aldolase L-lactate dehydrogenase L-lactate dehydrogenase aminotransferase methionyl-tRNA synthetase/ protein secretion chaperonin, CsaA rod shape-determining protein MreB N-acetylglucosamine-6phosphate deacetylase phosphoglycerate kinase phosphoglycerate kinase phosphoglycerate kinase manganese-dependent inorganic pyrophosphatase phosphocarrier protein HPr phosphoenolpyruvate-protein phosphotransferase adenylosuccinate synthetase pyruvate kinase pyruvate kinase LSU/50S ribosomal protein L7/ L12P SSU/30S ribosomal protein S1P SSU/30S ribosomal protein S1P single-stranded DNA-binding protein trigger factor triosephosphate isomerase triosephosphate isomerase protein translation elongation factor Ts (EF-Ts) elongation factor Tu (EF-TU) elongation factor Tu (EF-TU)

score

identification methoda

seg. cov. %

no. matched peptidesb

GG

Lc705

ATCC15808

23.7/6.5 53.1/4.3 28.8/4.3

94 377 91

MS MS/MS MS

40 21 27

9/11 6 6/11

+ − +

+ + +

− ± ±

LGG_00259

31.6/6.4

131

MS

28

8/2

+

−

−

LGG_01604 LGG_00936 LGG_01360 LGG_02493

67.2/4.6 47.1/4.4 32.3/6.0 76.9/4.5

584 610 50 320

MS/MS MS/MS MS MS

28 34 13 47

10 8 4/11 32/14

+ − − −

± + − ±

− − ± +

LGG_00933

36.7/5.6

165

MS/MS

19

2

+

+

+

LGG_00933

36.7/5.6

548

MS/MS

20

8

+

+

+

LGG_00933

36.7/5.6

320

MS/MS

22

5

+

+

+

LGG_00933

36.7/5.6

130

MS/MS

13

2

+

+

+

LGG_02138 LGG_02239 LGG_02239 LGG_02239 LGG_02240 LGG_00249

26.0/5.2 57.4/4.7 57.4/4.7 57.4/4.7 10.0/4.7 52.6/6.0

185 252 214 710 94 125

MS MS/MS MS MS/MS MS/MS MS

40 20 43 33 33 16

12/7 3 23/25 12 2 10/9

+ + + + + −

+ + + + − +

+ + + + − −

LGG_02575

36.3/4.9

208

MS/MS

31

3

+

+

−

LGG_02523 LGG_02523 LGG_00857 LGG_02584

35.5/5.1 35.5/5.1 43.7/5.8 74.9/5.8

682 260 350 295

MS/MS MS/MS MS/MS MS/MS

27 16 22 12

11 4 6 6

+ + ± ±

+ + − +

+ − ± −

LGG_01265

34.9/4.5

303

MS/MS

24

4

+

+

−

LGG_01862

42.3/4.9

179

MS/MS

20

4

±

+

−

LGG_00934 LGG_00934 LGG_00934 LGG_01424

42.2/5.7 42.2/5.7 42.2/5.7 33.5/4.5

449 358 198 150

MS/MS MS/MS MS/MS MS/MS

36 30 26 18

9 8 5 2

+ + + ±

+ + + +

+ ± − −

LGG_01821 LGG_01820

9.3/4.6 63.3/4.8

52 176

MS MS

40 27

3/19 14/5

+ +

+ +

− −

LGG_00133 LGG_01375 LGG_01375 LGG_02276

47.1/6.0 62.8/5.1 62.8/5.1 12.6/4.2

385 48 100 136

MS/MS MS MS MS

16 14 16 54

4 6/20 8/5 7/10

± + + +

+ + + ±

− + + −

LGG_01389 LGG_01389 LGG_00012

47.2/4.9 47.2/4.9 20.9/4.9

178 454 54

MS MS/MS MS

27 29 12

14/13 9 4/17

+ + −

+ + ±

+ + −

LGG_01351 LGG_00935 LGG_00935 LGG_01627

49.8/4.3 26.9/4.6 26.9/4.6 31.6/4.7

397 102 78 86

MS/MS MS MS MS

26 36 31 32

7 7/8 6/13 6/19

+ ± ± +

+ + + +

± − − +

LGG_01342 LGG_01342

43.6/4.6 43.6/4.6

887 166

MS/MS MS/MS

48 29

14 3

+ +

+ +

+ +

locus ID

Mw (kDa)/pI

LGG_02466 LGG_01184 LGG_01290

K



Article

Table 2. continued antiserac,d spot number 45

protein name and function Usp

universal stress protein, UspA family

locus ID

Mw (kDa)/pI

score

LGG_02151

16.8/4.8

104

identification methoda MS

seg. cov. %

no. matched peptidesb

GG

Lc705

ATCC15808

24

6/10

+

−

−

a

Identification type: MS, MALDI−MS; MS/MS, and LC−MS/MS. bMS identifications: matched/unmatched peptides. c+, identification by the antisera of the both two biological replicate antisera; ±, identification by the other of the two biologiacal replicate antisera; −, no identification by the antisera. dGG, L. rhamnosus GG; Lc705, L. rhamnosus Lc705: ATCC15808, L. delbrueckii subsp. lactis ATCC 15808.

roles in the bacterial response to bile, the degradation of milk casein or stimulation of the host immune functions. The most distinct difference between the two data sets was associated with moonlighting proteins with predicted functions in adhesion, immune stimulation, or pathogen exclusion. Complementary 2-DE immunoblotting with antibodies generated against whole GG and Lc705 cells and the more distantly related L. delbrueckii indicated that GG and Lc705 were antigenically more similar, whereas less cross-reacting proteins were detected with L. delbrueckii antibodies. More than 50 distinct antigens were identified, among which Eno and IMP with adhesive and immune modulatory functions were specifically detected by Lc705 antibodies, whereas those specifically detected with GG antibodies included GroES, Usp, and an oxidoreductase, each with predicted stress-related activities. These findings indicated that antibodies were specifically formed against these GG and Lc705 proteins in vivo, and that the strain-specific moonlighting antigens with adhesive and immunostimulatory functions could provide both these strains with an ability to adapt to their specific ecological niches.

corner of the blot corresponding to the proteome region containing high-molecular weight and acidic proteins (Figure 7). These spots, in ladder and string-like formations in the immunoblot, were not detected in silver stained gels and thus could not be identified. The locations of these spots in the immunoblot correlate reasonably well with the SpaC protein (MW 90 kDa, pI 5.1) that is typically detected in 1D-gels as a ladder representing the various extended lengths of pili.13 The GG antibodies failed to recognize Eno and IMP, which, however, were identified after cell surface shaving (Table 1). Proteins such as GapDH and GroES were detected with GG antibodies and GapDH also with Lc705 antisera, but these proteins were not among those identified by cell surface shaving from GG. However, cell surface shaving enabled identification of both proteins from Lc705, suggesting that they are more abundant in this strain during logarithmic phase of growth. It could be that the export of some of the moonlighting proteins is dependent on the growth stage or pH of the surrounding environment. For example, Lc705 has been shown to display increased ability to acidify the growth medium compared to GG.30 This was shown to result in cell samples with lower pH compared to GG samples withdrawn at the same growth stage, which may influence the interactions of the moonlighting proteins with the cell surface structures and could explain the discrepancy between the obtained surface shaving and 2-DE immunoblotting results. However, the 2-DE immunoblotting was conducted with samples withdrawn at later stages of growth, which may have a great impact on the moonlighting protein composition. Nonclassical protein secretion has been shown to be increased during stationary phase of growth in some bacteria, which was not resulting from cell lysis.77 Cell-wall proteome studies of probiotic Lactobacillus salivarius have demonstrated that transition from logarithmic to stationary phase of growth correlates with increased abundance of DnaK, Ef-Ts, and Pyk associated with in vitro adherence.78 Another explanation could be that antibodies were not formed against some of these proteins, such as Eno and IMP, during immunization due to interfering compounds hampering their recognition by the host cells.

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ASSOCIATED CONTENT

S Supporting Information *

Supplemental Figure 1. A silver-stained 2-DE gel representing the proteome of L. rhamnosus GG. Proteins (1−45) marked with a red circle were identified as proteins that cross-react with GG-, Lc705-, or ATCC15808-specific antibodies (Table 2). Supplemental Table 1. The surfaceome proteins identified (Mascot ions score ≥40 and p < 0.05) from GG and Lc705 cells after trypsin shaving in the presence and absence of DTT. Supplemental Table 2. All the identified GG surfaceome proteins (Mascot ions score ≥40 and p < 0.05) with the physicochemical features and information related to function, subcellular localization, presence of membrane, or cell wall anchoring domains and motifs. Supplemental Table 3. Comparison of the identified GG (in white) and Lc705 (in gray) surfaceome proteins. The commonly identified proteins are shown in bold letters. This material is available free of charge via the Internet at http://pubs.acs.org.

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CONCLUSIONS The present study provides the first in-depth analysis of the surface-associated proteome from the well-known probiotic Lactobacillus rhamnosus strain GG and the closely related dairy strain Lc705, for which previously defined genomes and protein catalogs are available. The cell surface bound proteins released by trypsin shaving were analyzed by LC−MS/MS, which identified a total of 102 and 198 individual proteins from GG and Lc705, respectively. Comparison of the surfaceome data sets indicated strain-specific differences among bona fide cell surface proteins such as HtrA, PrtR, and Msp with suggested

AUTHOR INFORMATION

Corresponding Author

*Tel: +358 2 941 570 57. Fax: +358 2 941 584 60. E-mail: pekka.varmanen@helsinki.fi. Present Addresses ∥

Ductor Corp., 00790 Helsinki, Finland. EDLONG Dairy Technologies, Elk Grove Village, Illinois 60007, United States. # Glykos Finland, Ltd., 00370 Helsinki, Finland. ⊥

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Academy of Finland (grants 139296 to P V., 252803 to J.R., and 135628 and 140950 to T.N.) and the EMFOL (Erasmus Mundus Food of Life) scholarship to E.E. We thank the Electron Microscopy Unit of the Institute of the Biotechnology (University of Helsinki), for providing laboratory facilities.

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