iTRAQ-based proteomic analysis of responses of Lactobacillus plantarum FS5-5 to salt tolerance

Abstract

Lactobacillus plantarum FS5-5 (L. plantarum FS5-5) is a salt-tolerant probiotic strain, which had been isolated from northeast Chinese traditionally fermented Dajiang. We analyzed the underlying molecular mechanisms of L. plantarum FS5-5 after salt stress by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics and bioinformatics analysis. L. plantarum FS5-5 was treated with low (1.5, 3.0, 4.0, and 5.0% (w/v) NaCl) and high (6.0, 7.0, and 8.0% (w/v) NaCl) salt stress. Differentially expression proteins (DEPs) of all groups were measured by quantitative proteomic approach of iTRAQ with LC–MS/MS. Furthermore, DEPs were identified by Mascot and GO, and bioinformatics analysis was conducted by KEGG. Thirty DEPs (P < 0.05) between low salt stress and control condition (0% (w/v) NaCl) were mapped and classified into nine functional groups; 122 DEPs (P < 0.05) between high salt stress and control condition were mapped and classified into 15 functional groups. In all groups, most proteins were involved in amino acid metabolism, carbohydrate metabolism, nucleotide metabolism, and ATP-binding cassette (ABC) transporter. We found that six proteins (metS, GshAB, GshR3, PepN, GshR4, and serA) involved in amino acid metabolism, three proteins (I526_2330, Gpd, and Gnd) involved in carbohydrate metabolism, and one protein (N876_0118940) involved in peptidoglycan hydrolysis were upregulated after salt stress. Conclusively, optimal L. plantarum FS5-5 growth was dependent on the collective action of different regulatory systems, with each system playing an important role in adapting to salt stress. There may be some relationship between the upregulated proteins of L. plantarum FS5-5 and salt stress.

Introduction

Lactobacillus is a large group of lactic acid bacteria (LAB), which includes more than 150 different species. Lactobacillus plantarum (L. plantarum) is one of the most widespread Lactobacillus species and is commonly used in fermentation industry as the starter (Behera et al. 2018). During the fermentation process, L. plantarum forms organic acids, amino acids, small peptides, and other flavor compounds and generate peroxide and bacteriocins and other natural antibacterial substances, thus, imparting a special flavor, quality, and nutritional value to the fermented products. In addition, probiotic functions of L. plantarum, such as anti-oxidation, cholesterol-lowering, and immunity-enhancing, have been confirmed by a large number of researchers (Gill et al. 2000; Jones et al. 2012; Li et al. 2013). In the food industry, L. plantarum is commonly used as the starter for fermenting vegetables and dairy, meat, and soy products. However, L. plantarum is exposed to various stress conditions, such as temperature, acid, salt, starvation, osmotic pressure, and oxidative stress during the industrial fermentation and food processing (Li et al. 2012), and increasing attention has been paid to understand the adaption mechanisms of L. plantarum to these stressful conditions in recent years (Belfiore et al. 2013; Bengoa et al. 2018; Engelhardt et al. 2018).

Salt stress is an important challenge for L. plantarum in a variety of fermented foods. High salt concentration can damage the morphology and physiology of the cells. Therefore, adaptation to salt stress is important for L. plantarum for thriving and proliferating in their natural ecosystems and in industrial applications (Zhao et al. 2014). In recent years, increasing studies have been performed to determine the changes in L. plantarum under salt stress (Zhao et al. 2014). L. plantarum manages thriving in a high osmotic pressure environment by activating various adaptation strategies. Some membrane proteins directly or indirectly regulate cell membrane permeability of salt ions, thereby regulating the osmotic pressure (Kleerebezem et al. 2003; Wang et al. 2011). The Na⁺/H⁺ antiporter on the plasma membrane regulates microbial efflux and influx of Na⁺ and H⁺ (Padan et al. 2005). The Na⁺/H⁺ antiporter is powered by a transmembrane proton electrochemical gradient that drives extracellular Na⁺ to maintain intracellular Na⁺ balance. Secondly, L. plantarum can also absorb or synthesize amino acids, small peptides, polyols, and disaccharides to maintain the balance of intracellular and extracellular osmotic pressure to resist various environmental stresses (Roberts 2005). Under high osmotic pressure, as enzymes that catabolize the compatible solute are inhibited, the compatible solute can accumulate in the cell at a high concentration, thus, leading to osmotic protection function. In addition, L. plantarum can maintain the balance of osmotic pressure by regulating the expression of some stress proteins and altering the composition of the cell membrane/cell wall (Romeo et al. 2001). Although a high number of studies have been conducted on the salt stress response of L. plantarum, the description of L. plantarum at the gene level under various salt concentration stresses is not much comprehensive.

Isobaric tags for relative and absolute quantitation (iTRAQ) is a new quantitative proteomic approach and has been widely used in the identification, characterization, and expression analysis of the proteins (Gao et al. 2017; Lin et al. 2017). Multiple peptides representing the same protein may be identified with iTRAQ, which affords higher confidence for both identification and quantification of the protein (Li et al. 2017).

L. plantarum FS5-5 (CGMCC no. 10331) is a salt-tolerant strain isolated from Northeast Chinese traditionally fermented Dajiang. The objectives of this study were to analyze the response of L. plantarum FS5-5 to salt stress (1.5, 3.0, 4.0, 5.0, 6, 7, and 8% (w/v) NaCl) at the protein and gene transcription levels by iTRAQ multidimensional coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) proteomic approach. We propose that the results will provide an important molecular basis and reference information for future study of L. plantarum salt tolerance.

Materials and methods

Strains, growth conditions, and salt stress

L. plantarum FS5-5, isolated from Northeast Chinese traditionally fermented Dajiang in the Liaoning province of China, showed higher capacity to high salt tolerance based on our previous report (Song et al. 2016). For salt stress response analysis, the strain, which was freeze-dried and stored at − 80 °C, was reconstituted at 37 °C (optimum growth temperature for L.plantarum FS5-5) in de Man, Rogosa, and Sharpe medium (MRS) for 24 h, and this procedure was repeated three times. Bacterial suspension (1%; 10⁶ colony forming units (CFU)/mL) was inoculated in MRS with 0, 1.5, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0% (w/v) NaCl and incubated at 37 °C to reach the exponential phase (at 6, 6, 6, 7, 8, 10, 13, and 18 h, respectively). Cell pellets were prepared by centrifugation at 4000 ×g at 4 °C for 10 min and washed three times with phosphate buffer. The final bacterial solution concentration reached 10⁹ CFU/mL and was stored at − 80 °C until further use.

Transmission electron microscope (TEM) analysis

L. plantarum FS5-5 cells with 0, 1.5, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0% (w/v) NaCl were cultured at 37 °C to reach the exponential phase (6, 6, 6, 7, 8, 10, 13, and 18 h, respectively) and fixed in buffered 2.5% glutaraldehyde. Cells were collected after centrifugation at 4000 ×g at 4 °C for 10 min and washed three times with physiological saline to remove excess fixative. The cells were fixed in unbuffered 1% osmium tetroxide and washed with physiological saline. Then, the samples were dehydrated in ethanol at concentrations of 50, 70, 80, 90, and 95% and dehydrated in a graded series of acetone solutions and gradually impregnated in EPON resin with heat polymerization. Semi-thin survey sections were sliced with glass knives, stained with 1% phosphotungstic acid, and used to orient sections (Feliciano and Rivera 2016). Thin sections were viewed under a TEM (H-7650, Hitachi Ltd. Japan).

Protein extraction and quantification

Proteins were extracted according to a previously reported method (Xia et al. 2016) with slight modifications. Cells (500 μg) at the logarithmic stage of each NaCl concentration were mixed with 1 mL of lysis solution, comprised of 8-M urea solution (1 mL), 30-mM HEPES (1 mL), 1-mM phenylmethane sulfonyl fluoride (PMSF; 1 mL), 2-mM EDTA (1 mL), and 10-mM dithiothreitol (DTT; 1 mL), and the total protein was further lysed by ultrasonication (pulse on, 2 s; pulse off, 3 s; power 180 W), followed by centrifugation at 20,000 ×g for 30 min at 4 °C. The supernatant was collected, and DTT was added to the supernatant at a final concentration of 10 mM and incubated at 56 °C in water bath for 1 h. After removing the mixture from the water bath, iodoacetamide (IAM) was added rapidly to the mixture at a final concentration of 55 mM and incubated in darkness for 1 h. Next, acetone was added at four times the volume of the mixture, incubated at − 20 °C for 3 h, centrifuged at 20,000 ×g for 30 min at 4 °C, precipitate was collected, dissolution buffer (final triethyl ammonium bicarbonate (TEAB) concentration, 50%; and final sodium dodecyl sulfate (SDS) concentration, 0.1%) was added to the precipitate, ultrasonic treatment was administered as mentioned above, and the mixture was centrifuged at 20,000 ×g for 30 min at 4 °C. The supernatant was collected, and protein concentration was quantified using the Bradford method (Braford 1976).

Protein digestion and iTRAQ labeling

The proteins extracted (100 μg) from each samples were digested with trypsin, and the peptide samples were lyophilized and labeled using the iTRAQ Reagent-8Plex Multiplex Kit (AB SCIEX, Foster City, CA, USA) according to the manufacturer’s instructions. Each sample was tagged as follows: the sample without NaCl was labeled with tags 113, sample with 1.50% (w/v) NaCl was labeled with tags 114, sample with 3.0% (w/v) NaCl was labeled with tags 115, sample with 4.0% (w/v) NaCl was labeled with tags 116, sample with (w/v) 5.0% NaCl was labeled with tags 117, sample with 6.0% (w/v) NaCl was labeled with tags 118, sample with 7.0% (w/v) NaCl was labeled with tags 119, and sample with (w/v) 8.0% NaCl was labeled with tags 120.

Strong cation exchange (SCX) chromatography separation

The labeled peptide samples were preliminary separated by SCX chromatography (Luna SCX 100A, Phenomenex, USA) according to a previously reported method (Yang et al. 2017) with slight modifications. iTRAQ-labeled peptides were dissolved in ten times the volume of buffer A (25% (v/v) acetonitrile (ACN), 10-mM KH₂PO₄; pH 3.0) and centrifuged at 15,000 ×g for 10 min. The supernatant was collected and purified on SCX column. The peptide samples were eluted at a flow rate of 1 mL/min with a gradient of 0–5% buffer B (25% (v/v) ACN, 2-M KCl, and 10-mM KH₂PO₄; pH 3.0) for 1 min, 5–30% buffer B for 10 min, 30–50% buffer B for 5 min, 50% buffer B for 10 min, 50–100% buffer B for 5 min, and 100% buffer B for 10 min. The eluent was collected after 214 nm, mixed according to the peaks, and then desalted on strata-X C18 column (Phenomenex, Torrance, CA, USA) according to a previously reported method (Xia et al. 2016).

Nano LC–MS/MS analysis

The peptide samples were separated using Nano-LC (DIONEX, USA) with a C18 chromatography column (100 mm × 75 mm, 300 A, 5 μg, C18; Phenomenex, USA) equilibrated with buffer A (0.1% formic acid in Milli-Q water) according to a reported method (Yang et al. 2017) with slight modifications. The peptide samples were loaded onto the C18 chromatography column and eluted at a flow rate of 400 nL/min, with a gradient of 5% buffer B (0.1% formic acid in ACN) for 10 min, 5–30% buffer B for 30 min, 30–60% buffer B for 5 min, 60–80% buffer B for 3 min, 80% buffer B for 7 min, and finally 5% buffer B for 10 min. The Q Exactive mass spectrometer (Thermo Fisher Scientific, USA) was used for data acquisition and performed as previously reported (Yu et al. 2017).

Protein identification and quantification

Mascot version 2.3.0 (Matrix Science, Boston, MA, USA) and Proteome Discoverer version 1.4 (Thermo Scientific, USA) software packages were used for iTRAQ protein identification and quantification analysis. The obtained raw data files were searched against the 1578_UNI_Lactobac database (downloaded on August 7, 2015; number of sequences, 461,115). The search parameters of Mascot for protein identification were as follows: Fixed modification, carbamidomethyl (C); variable modification, oxidation (M), Gln → Pyro→Glu (N-term Q), iTRAQ 8 plex (K), iTRAQ 8 plex (Y), iTRAQ 8 plex (N-term); peptide tol, ± 15 ppm; MS/MS tol, ± 20 mmμ; max missed cleavages, 1; enzyme, trypsin. Quantification analysis for each SCX elution was further performed using Proteome Discoverer; the parameters of which were as follows: protein ratio type, median; minimum peptides, 1; normalization method, median; P value, < 0.05. An identified protein was considered significantly upregulated or downregulated in abundance if the fold change (FC) met the threshold criterion of an iTRAQ ratio of 1.2 (P < 0.05). P value was calculated according to the equations of Cox and Mann.

Bioinformatics analysis

The differentially expressed proteins were mapped to Gene Ontology (GO) terms (http://www.geneontology.org) for functional classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (http://www.kegg.jp/) for predicting the main metabolic pathways.

qRT-PCR analysis

To yield more accurate and reliable quantitative results of genes, the most stable reference gene was selected from the five housekeeping genes (16S rRNA, gapdh, gapB, dnaG, and gyrA). The primers for housekeeping genes and target genes are listed in Table 1, and qRT-PCR was performed as previously reported (Wu et al. 2016). The 2^−ΔΔCT method was used to calculate the relative changes in gene expression (Livak and Schmittgen 2001) by comparing the CT values for 1.5, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0% (w/v) NaCl cultures with the 0% (w/v) NaCl culture. All samples were measured in triplicates.

Table 1 Primer sequences for quantitative PCR

Statistical analysis

Data analysis was performed using the SPSS version 19.0 statistical software (SPSS Inc., Chicago, IL, USA). To examine intrasample variation, mean and standard deviation (SD) were determined. Gene expression data were also analyzed using analysis of variance (ANOVA). A protein was considered differentially expressed when it exhibited a FC of > 1.2 or < 0.83 and P value of < 0.05.

Results

Morphological changes in salt tolerance

The morphological changes of L. plantarum FS5-5 in 0, 1.5, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0% (w/v) NaCl were clearly observed by TEM (Fig. 1). The cell wall of cells subjected to low salt stress under 1.5, 3.0, 4.0, and 5.0% (w/v) was separated, and some cells exhibited a clear cavity compared with cells under 0% (w/v) NaCl. The appearance of cells subjected to salt stress under 6.0, 7.0, and 8.0% (w/v) NaCl was significantly damaged compared with that of cells under 0% (w/v) NaCl. The results revealed that high salt concentration could alter the osmotic pressure of cells and lead to cell shrinkage and breakage.

Fig. 1

TEM images of L. plantarum FS5-5 exposed in MRS medium at different NaCl concentrations. a 0.0%, b 1.5%, c 3.0%, d 4.0%, e 5.0%, f 6.0%, g 7.0%, h 8.0%

Protein quantification and identification

Protein samples were quantified using the Bradford method, and the concentration of protein was 1.55 μg/μL with 0% NaCl, 1.07 μg/μL with 1.5% NaCl, 1.21 μg/μL with 3.0% NaCl, 1.47 μg/μL with 4.0% NaCl, 1.26 μg/μL with 5.0% NaCl, 1.07 μg/μL with 6.0% NaCl, 1.25 μg/μL with 7.0% NaCl, and 1.32 μg/μL with 8.0% NaCl. The total ion current diagram (Fig. 2) was searched against the 1578_UNI_Lactobac database by Mascot version 2.3.0 and Proteome Discoverer version 1.4 software. A total of 2056 proteins were specifically identified from 85,516 MS/MS spectra, and 11,215 peptides were identified using the false discovery rate (FDR) of < 1% as the cutoff. Significant differences in protein expression were determined using two criteria of “P ≤ 0.05 and FC > 1.2 or FC < 0.83” for comparative analysis among the strains under 1.5, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0% (w/v) NaCl and under 0% (w/v) NaCl (Fig. 3).

Fig. 2

The total ion current diagram. The x-axis represents the elution time. The y-axis represents the signal strength

Fig. 3

Distribution of differently changed proteins. Proteins with corrected P values less than 0.05 and FCs larger than 1.20 or smaller than 0.83 were considered to be significantly differential; 114/113 represents the comparison between the samples with 1.5% (w/v) NaCl and the control samples; 115/113 represents the comparison between the samples with 3% (w/v) NaCl and the control samples; 116/113 represents the comparison between the samples with 4% (w/v) NaCl and the control samples; 117/113 represents the comparison between the samples with 5% (w/v) NaCl and the control samples; 118/113 represents the comparison between the samples with 6% (w/v) NaCl and the control samples; 119/113 represents the comparison between the samples with 7% (w/v) NaCl and the control samples; 120/113 represents the comparison between the samples with 8% (w/v) NaCl and the control samples

From the results, we found that the amount of DEPs increased with the increase in NaCl concentration, implying that a high number of metabolic pathways are altered in L. plantarum FS5-5 to resist high salt stress.

Bioinformatics analysis of differential protein species identified by iTRAQ

The function and metabolic pathways of differential protein species were analyzed by mapping to GO terms and KEGG pathways. A total of 30 DEPs (P < 0.05) between low salt stress and control conditions were mapped and classified into nine functional groups (Fig. 4a), 122 DEPs (P < 0.05) between high salt stress and control condition were mapped and classified into 15 functional groups (Fig. 4b), and 22 common DEPs (P < 0.05) in all high salt groups were mapped and classified into eight functional groups (Fig. 4c). The detailed information is provided in Tables 2–4. Most DEPs under low salt stress were involved in carbohydrate metabolism (26.67%), nucleotide metabolism (23.33%), ATP-binding cassette (ABC) transporter (20%), amino acid metabolism (10%), lipid metabolism (6.67%), vitamin metabolism (3.33%), phosphotransferase system (PTS; 3.33%), ribosomal protein (3.33%), and peptidoglycan hydrolysis (3.33%); most DEPs under high salt stress were involved in amino acid metabolism (17.21%), carbohydrate metabolism (17.21%), nucleotide metabolism (16.39%), ABC transporter (13.11%), ribosomal protein (9.02%), lipid metabolism (4.92%), replication and repair (6.56%), and PTS (3.28%). Moreover, most common DEPs were involved in nucleotide metabolism (31.82%), ABC transporter (27.27%), amino acid metabolism (9.09%), carbohydrate metabolism (13.64%), vitamin metabolism (4.55%), ribosomal protein (4.55%), and PTS (4.55%).

Fig. 4

Functional groups classification and proportion of differential protein species. Different colors represent different functional groups of differential protein species. a Differential protein species between low salt stress (1.5%, 3.0%, 4.0%, 5.0% w/v) NaCl) and control conditions. b Differential protein species between high salt stress (6.0%, 7.0%, 8.0% (w/v) NaCl) and control conditions. c Common differential protein species between all salt stress and control conditions

Table 2 The detailed information of differential protein species under high salt stress. Proteins with corrected P values less than 0.05 and FCs larger than 1.20 or smaller than 0.83 were considered to be significantly differential; 118/113 represents the comparison between the samples with 6% (w/v) NaCl and the control samples; 119/113 represents the comparison between the samples with 7% (w/v) NaCl and the control samples; 120/113 represents the comparison between the samples with 8% (w/v) NaCl and the control samples

Table 3 The information list of changed expression proteins under the low NaCl concentrations. Proteins with corrected p values less than 0.05 and fold changes larger than 1.20 or smaller than 0.83 were considered to be significantly differential; 114/113 represents the comparison between the samples with 1.5% (w/v) NaCl and the control samples; 115/113 represents the comparison between the samples with 3% (w/v) NaCl and the control samples; 116/113 represents the comparison between the samples with 4% (w/v) NaCl and the control samples; 117/113 represents the comparison between the samples with 5% (w/v) NaCl and the control samples

Table 4 The information list of changed expression proteins under all NaCl concentrations. Proteins with corrected P values less than 0.05 and fold changes larger than 1.20 or smaller than 0.83 were considered to be significantly differential; 114/113 represents the comparison between the samples with 1.5% (w/v) NaCl and the control samples; 115/113 represents the comparison between the samples with 3% (w/v) NaCl and the control samples; 116/113 represents the comparison between the samples with 4% (w/v) NaCl and the control samples; 117/113 represents the comparison between the samples with 5% (w/v) NaCl and the control samples; 118/113 represents the comparison between the samples with 6% (w/v) NaCl and the control samples; 119/113 represents the comparison between the samples with 7% (w/v) NaCl and the control samples; 120/113 represents the comparison between the samples with 8% (w/v) NaCl and the control samples

From the results, we found that most DEPs were involved in amino acid, carbohydrate, and nucleotide metabolism, indicating that L. plantarum FS5-5 resistance to salt stress was closely related to these metabolic pathways.

Transcriptional expression analysis by qRT-PCR

The stability of the five housekeeping genes was evaluated by the 2^−ΔΔCT method. The results are shown in Table 5. There was a certain correlation between the expression of gapdh, gapB, dnaG, and gyrA and salt stress (P < 0.05), but 16S rRNA showed higher stability than other housekeeping genes (P > 0.05). Therefore, 16S rRNA was selected as the internal reference gene for this experiment.

Table 5 Fold change in the gene expression of housekeeping genes under different salt concentrations

To determine whether the significant changes observed in specific proteins under high salt stress also occurred at the level of gene expression, qRT-PCR was performed to evaluate the mRNA levels of the proteins whose expression changed significantly. The results are shown in Fig. 5. The gene expression of N876_0118940, involved in peptidoglycan hydrolysis, and metS, involved in amino acid metabolism, was upregulated (P < 0.05) in cells at all NaCl levels, except for metS at 7.0% (w/v) NaCl. However, the mRNA levels of araT, involved in amino acid metabolism, in cells at all NaCl levels were repressed (P < 0.05), except at 5.0% (w/v) NaCl. The mRNA levels of carA (carbamoyl-phosphate synthase small subunit), purB (adenylosuccinate lyase), purA (adenylosuccinate synthase), guaC (GMP reductase), purH (phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase), and pyrF (orotidine-5′-phosphate decarboxylase), involved in nucleotide metabolism, were repressed at all NaCl levels (P < 0.05), except for purH at 1.5% (w/v) NaCl. The mRNA levels of mapB and pflF, involved in carbohydrate metabolism, were repressed at all NaCl levels (P < 0.05). The results revealed that the mRNA expression of selected genes was almost consistent with the expression of the corresponding proteins.

Fig. 5

Trend graph of corresponding gene transcriptional expression by qRT-PCR analysis in Lactobacillus plantarum FS5-5 cells after being exposed to 0% NaCl, 1.5% (w/v) NaCl, 2.0% (w/v) NaCl, 3.0% (w/v) NaCl, 4.0% (w/v) NaCl, 5.0% (w/v) NaCl, 6.0% (w/v) NaCl, 7.0% (w/v) NaCl, or 8.0% (w/v) NaCl. The x-axis represents the gene transcription of Lactobacillus plantarum FS5-5 cultivated in MRS at 0% (w/v) NaCl (control) or 1.5% (w/v) NaCl, 3.0% (w/v) NaCl, 4.0% (w/v) NaCl, 5.0% (w/v) NaCl (low salt stress), 6.0% (w/v) NaCl, 7.0% (w/v) NaCl, 8.0% (w/v) NaCl (high salt stress), and the y-axis represents the normalized fold expression of genes. Error bars represent the SD of three independent experiments, and the asterisks indicate a significant difference (*P < 0.05, **P < 0.01)

Discussion

Proteins involved in amino acid metabolism

GshAB, which plays an important role in the biosynthesis and metabolism of glutathione, not only shows the catalysis as GshA but also shows catalytic effect as GshB and finally generates GSH. GSH plays important roles in bacterial cells, such as protecting proteins and DNA from oxidative damage and promoting transmembrane transport of amino acids. Moreover, GSH also can be converted to reduced glutathione by reductase (such as GshR3 and GshR4) and aminopeptidase (such as PepN). The reduced glutathione not only can protect cells from oxidative damage but also can alleviate cell toxicity and stress damage (Vila Sanjurjo et al. 2004). In this study, GshAB, GshR3, PepN, and GshR4 were overexpressed (P < 0.05) in L. plantarum FS5-5 in response to high salt stress but remained unaltered in response to low salt stress. In particular, GshR4 was overexpressed by two-fold in bacteria at 6.0, 7.0, and 8.0% (w/v) NaCl than that in cells at 0% (w/v) NaCl. A previous study has shown that GSH protects Lactococcus lactis from osmotic stress (Zhang et al. 2010). Similarly, overexpression of glutathione reductase and aminopeptidase (GshAB, GshR3, and GshR4 and PepN, respectively) may regulate the content of glutathione and reduced glutathione to protect L. plantarum FS5-5 from salt stress damage. Glutathione reductase can oxidize glutathione (GSSG) to glutathione catalyzed (GSH), which plays important roles in cellular antioxidant mechanisms. Previous studies have demonstrated that GSH protects cells from various environmental stresses, such as osmotic pressure, oxidative, and acid stress (Zhang et al. 2010; Zou et al. 2014; Wang 2015). It can be speculated that high salt concentration can induce the expression of GshAB, GshR3, PepN, and GshR4, thereby protecting L. plantarum FS5-5 from salt or other adverse environmental factors.

Proteins involved in carbohydrate metabolism

Carbohydrate metabolism is an important process for microbes as it provides the necessary energy for metabolism and significantly supports complete growth (Li et al. 2017a, 2017b). Glycolysis, as the main form of carbohydrate metabolism, provides energy to LAB under anaerobic conditions (Veith et al. 2017). pgmB (beta-phosphoglucomutase), which encodes a phosphoglucomutase, can catalyze the interconversion of D-glucose 1-phosphate (G1P) and D-glucose 6-phosphate (G6P) to yield beta-D-glucose 1,6-(bis) phosphate (beta-G16P) as an intermediate. Furthermore, pgmB plays a key role in the regulation of the flow of carbohydrate intermediates in glycolysis and the formation of the sugar nucleotide UDP-glucose. In this study, pgmB expression was upregulated (P < 0.05) in response to high salt stress but remained unaltered in response to low salt stress. Under high salt stress, L. plantarum FS5-5 may coordinate the supply of intracellular capacity and increase cell growth by upregulating the expression of pgmB.

The pentose phosphate pathway is also one of the major pathways for carbohydrate metabolism (Kovářová and Barrett 2016). In the present study, glucose 6-phosphate dehydrogenase (Gpd) and glucose 6-phosphate decarboxylase (Gnd) were both over-expressed in L. plantarum FS5-5 in response to 6.0, 7.0, and 8.0% (w/v) NaCl stress but remained unaltered in response to low salt stress. In the pentose phosphate pathway, Gpd catalyzes glucose 6-phosphate to produce phosphogluconate and generate NADPH; Gnd catalyzes glucose 6-phosphate to produce D-ribulose-5-phosphate, which is one of the major components of nucleotides and its important coenzymes, and NADPH to produce ribose-5-phosphate (Shi et al. 2009). These sugar phosphates are needed for the biosynthesis of nucleotides and coenzymes, and these compounds perhaps could be involved in the mechanism of L. plantarum FS5-5 response to salt stress.

Proteins involved in fatty acid metabolism

The adaptation of L. plantarum FS5-5 to high salinity is also accompanied by rearrangements in either the composition or structure of the cell envelope. In particular, lipid and fatty acid composition of the cytoplasmic membrane is affected. Malonyl CoA, an important precursor in the biosynthesis of fatty acids, is converted from acetyl-CoA, which is catalyzed by acetyl-CoA carboxylase (AccB/AccC) (Tao et al. 2016). In bacterial cells, enzymes that play a dominant role in catalyzing fatty acid production are primarily acyl carrier protein polymers (FabZ2, FabG2, FabD, and FabH). Acetyl-CoA is catalyzed by Fab to obtain long-chain fatty acyl-ACP, which then enters the phospholipid synthesis pathway. In the present study, AccB, FabZ2, FabG2, and FabD were repressed in L. plantarum FS5-5 in response to 6.0, 7.0, and 8.0% (w/v) NaCl stress but remained unaltered in response to low salt stress. This result indicated that high salt stress inhibited the synthesis of phospholipids in L. plantarum FS5-5 and damaged the formation of cell membranes. L. plantarum FS5-5 could not resist osmotic stress to maintain the normal growth of cells through this pathway. Similar results have been observed in a previous study by Heunis et al. (2014), who observed a marked decrease in proteins playing a role in fatty acid biosynthesis in L. plantarum 423 under acid stress. We speculate the reason for these results may be that fatty acid metabolism in L. plantarum was inefficient in resisting environmental stress.

Proteins involved in nucleotide metabolism

Purine and pyrimidine metabolism were also affected by salt stress; the expression of purine and pyrimidine metabolic enzymes was significantly downregulated under salt stress.

GuaC, PurH, PurA, and PurB are involved in purine metabolism. GuaC is a key enzyme that catalyzes the production of IMP (hypoxanthine nucleotide) by GMP (guanine nucleotide). PurH and PurB are key enzymes that catalyze the production of IMP by GAR (5′-phosphoribosyl-glycinamide). PurA is a key enzyme that catalyzes IMP to produce AMP (adenosine monophosphate); GMP, IMP, and AMP are precursors for cellular nucleic acid synthesis. In this study, the expressions of GuaC, purH, PurA, and PurB were downregulated under salt stress. This result indicated that under salt stress, the purine metabolic process of L. plantarum FS5-5 was disrupted, Moreover, the generation of precursors for nucleic acid synthesis was reduced, which blocked the synthesis of DNA and RNA in cells, and this may be a reason for the slow growth of cells under salt stress. Similar to our results, PurH and PurB of L. rhamnosus GG were downregulated in response to acid stress (Koponen et al. 2012). Conversely, it has been reported that the expressions of PurA and PurH are upregulated in L. sakei CRL1756 and in L. lactis SK11 under salt stress (Zhang et al. 2010; Belfiore et al. 2013). These contrasting results may indicate that modified purine metabolism may be strain-specific, and for L. plantarum FS5-5, purine metabolism may be unnecessary or inefficient in resisting salt stress.

CarA (carbamoyl-phosphate synthase small subunit), PyrE (orotate phosphoribosyltransferase), and PyrF (orotidine-5′-phosphate decarboxylase) were downregulated in response to all salt stress conditions, and PyrB (aspartate carbamoyltransferase catalytic subunit), PyrC (dihydroorotase), PyrD (dihydroorotate dehydrogenase (fumarate)), PyrG (CTP synthase), involved in pyrimidine metabolism, were downregulated in response to high salt stress conditions in the present study. CarA, PyrB, PyrC, PyrD, PyrE, and PyrF are key enzymes in the pathway that catalyzes the formation of UMP (uridine monophosphate) by L-glutamine. PyrG is a key enzyme that catalyzes the formation of CTP (cytidine triphosphate) by UTP (uridine triphosphate), and both UTP and CTP are precursors to RNA synthesis. In our study, downregulation of these enzymes indicated that the synthesis of pyrimidines in L. plantarum FS5-5 was blocked under salt stress, thus, tampering with RNA synthesis. A previous study has shown that the abundance of pyrimidine and purine biosynthesis enzymes in L. rhamnosus GG is highly reduced in response to lower pH condition (Koponen et al. 2012). These results may be due to the fact that the pyrimidine synthesis system is susceptible to damage under environmental stress, which impedes RNA synthesis in cells, and this may also be one of the reasons for the slow growth of cells under environmental stress.

Proteins involved in peptidoglycan biosynthesis

MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) and MurB (UDP-N-acetylmuramate dehydrogenase) are involved in the peptidoglycan biosynthesis pathway, wherein monomers are utilized for peptidoglycan biosynthesis. Previous reports have reported that peptidoglycan biosynthesis-related enzymes are inhibited in L. fermentum NCDC 400 under salt stress and in L. johnsonii PF01 under bile salt stress (Lee et al. 2013; Kaur et al. 2017). These enzymes are a part of the more complex amino–sugar metabolic pathway that consumes energy and may be unnecessary and inefficient for cells coping with a harsh environment.

Ribosomal, transporter, DNA repair, and stress proteins

Ribosomal proteins are the main components of the ribosome and play an important role in the biosynthesis of proteins in cells. In our study, the expressions of rpsL (30S ribosomal protein S12), rpmA (50S ribosomal protein L27), rpmI (50S ribosomal protein L35), rpsT (30S ribosomal protein S20), rpmB (50S ribosomal protein L28), rplO (50S ribosomal protein L15), and rplB (50S ribosomal protein L2) were downregulated by high salt stress but remained unaltered in response to low salt stress. These downregulated genes comprise regulatory genes involved in replication, transcription, and translation. Under high salt stress, the expression of these genes was significantly inhibited, indicating that the rate of protein synthesis in L. plantarum FS5-5 decreased under high salt stress, thus, inhibiting cell growth. Previous studies have shown that ribosomal protein L10 is downregulated by acid stress (Wu et al. 2011), and ribosomal proteins are sensitive to cold and heat shock (Jones et al. 1996). The results of the present study imply that ribosomal proteins in L. plantarum FS5-5 were also highly sensitive to salt stress and did not play an active role in response to salt stress.

OpuA (osmoprotectant transport system ATP-binding protein) is a compatible transporter belonging to GB (glycine betaine) transport systems. GB, as one of the most universal and effective osmoprotectants, is accumulated by bacteria (Considine et al. 2011). OpuA expression was highly upregulated under higher concentrations of NaCl, which may indicate that L. plantarum FS5-5 utilized the compatible solute regulatory system to sustain its growth. The change in OpuA expression confirmed that the compatible solute regulatory system is one of the mechanisms of L. plantarum FS5-5 in response to salt stress. Expression of ABC transporter proteins increased in L. plantarum FS5-5 under high salt stress but remained unaltered in response to low salt stress in this study, which may indicate that the cells require ABC transporter to maintain the balance of osmotic pressure under high salt stress. A previous study has reported that ABC transporter LmrCD is the major transporter responsible for bile acid resistance in L. lactis (Zaidi et al. 2008).

Damage repair seems to be the ultimate mechanism of resistance against oxidative and other stresses. High salt stress triggered upregulation of genes encoding DNA repair proteins, including uvrB and recA, in L. plantarum FS5-5. Similarly, the expression of DNA repair proteins has been reported to be upregulated in L. plantarum ST-III under salt stress (Zhao et al. 2014). This upregulation indicated that DNA repair is an essential strategy for L. plantarum to adapt to high salt environments.

Stress proteins play an important role in protein expression and repair. In this study, the expressions of universal stress protein, small heat shock protein, alkaline shock protein, and general stress protein were increased by high salt stress. In response to salt stress, osmotic pressure caused high plasmolysis and reduced water activity, leading to the accumulation of denatured proteins, which in turn induced a stress protein regulation system to protect proteins and macromolecules in the cells and to prevent cell damage caused by salt stress.

Conclusions

In present study, we reported the proteomics of salt-tolerant L. plantarum at different salt concentrations. The response of L. plantarum to increased salt concentration is likely complex, involving a combination of different metabolic pathways. Dramatic changes were observed in amino acid, carbohydrate, nucleotide, and lipid metabolism; ABC transporter; ribosomal protein; replication and repair; and PTS. The gene expression of N876_0118940, involved in peptidoglycan hydrolysis, and metS, involved in amino acid metabolism, were upregulated in cells at all NaCl levels. GshAB, GshR3, PepN, GshR4, and serA, involved in amino acid metabolism, and I526_2330, Gpd, and Gnd, involved in carbohydrate metabolism, were upregulated in cells at high NaCl levels. We found that L. plantarum FS5-5 needed to initiate more stress responses, including maintaining intracellular and extracellular osmotic pressure balance by increasing the concentration of similar compatible solutes, reducing cell damage under osmotic stress by increasing GSH content, repairing DNA damage by increasing DNA repair protein expression, and priming stress proteins to protect proteins and macromolecules in cells, to prevent cell damage caused by salt stress and to resist high salt stress compared with low salt stress. Moreover, we speculate that N876_0118940, metS, GshAB, GshR3, PepN, GshR4, serA, I526_2330, Gpd, and Gnd are closely related to the salt-tolerance mechanism of L. plantarum FS5-5 and need to be studied further. The results of the present study provide some new and relevant information on proteomic changes that occur in L. plantarum FS5-5 in response to salt stress and sheds light on the adaptive process for different salt concentrations. In later studies, specific molecular pathways involved in mediating the adaptive response to salt shock stress should be further identified.