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Lactobacillus plantarum J26, a significant probiotic isolated from Chinese traditional fermented dairy products, exerts a positive immunomodulatory effect by regulating the expression of immune-related genes. We investigated expression of the cytokines IL-1α, IL-1β, IL-6, and tumor necrosis factor-α in the intestinal tract of mice stimulated by L. plantarum J26. In vivo, these cytokines were upregulated, peaked on d 5, and then decreased to the control level, indicating that L. plantarum J26 could induce expression of the genes encoding these proinflammatory cytokines. Teichoic acids produced by L. plantarum are recognized as key immunomodulatory molecules involved in the regulation of the host immune response. To better understand the genetic basis of this immunomodulatory mechanism, we sequenced and analyzed the whole genome of L. plantarum J26. The genome of L. plantarum J26 contains a circular chromosome and 4 circular plasmids. Lactobacillus plantarum J26 was predicted to synthesize ribitol-type backbones of wall teichoic acid. Furthermore, orthologous average nucleotide identity (OrthoANI) values showed that the genome was highly similar (>98.00%) to other L. plantarum strains, especially to L. plantarum ST-III and JDM1. The genomic data of L. plantarum J26 provide a genetic basis to further elucidate its mechanism of immunoregulation and will facilitate its application in the functional dairy food industry.
) that inhabits relatively abundant ecological niches and plays a significant role in food microbiology and human nutrition because of its fermentative and probiotic functions (
The effect of Lactobacillus plantarum 299 v on the bacterial composition and metabolic activity in faeces of healthy volunteers: A placebo-controlled study on the onset and duration of effects.
). Among these properties, the potential of L. plantarum to inhibit inflammation has received extensive attention. Lactobacillus plantarum GB-LP2 has also shown antiviral effects against the influenza virus in mice (
Teichoic acid (TA), the major component of cell walls of gram-positive bacteria, is recognized as key immunomodulatory molecule involved in the regulation of host immune response. For example, heat-killed L. plantarum MYL26 and bacterial cell wall extracts are able to reduce LPS-induced inflammation by impairing toll-like receptor 4 (TLR4)-nuclear factor κB (NF-κB) signal transduction in vitro, suggesting that constituents of bacterial cell wall help attenuate inflammation (
Lactobacillus plantarum J26 is a common probiotic strain isolated from traditional fermented dairy products. Our recent investigation showed that strain J26 could alleviate oxidative stress by modulating the production of antioxidant enzymes significantly in vitro (
). In the current study, we confirmed that the strain exhibits potent immunomodulatory activity. To better understand the immunomodulatory mechanism of L. plantarum J26 and obtain detailed insight into the genetic basis, we analyzed the whole genome sequence of L. plantarum J26 combined with biochemical assays.
Lactobacillus plantarum J26 was isolated from Chinese traditional fermented dairy products in Inner Mongolia (China). It was grown in de Man, Rogosa, and Sharpe (Qingdao Hope Bio-Technology Co. Ltd., Qingdao, China) broth at 37°C for 16 h through 2 propagation steps. Genomic DNA was extracted using the Qiagen DNA Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.
Quantity and quality determinations of genomic DNA in L. plantarum J26 were conducted by Qubit 2.0 (Thermo Fisher Scientific, Waltham, MA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The genomic DNA was sequenced by Illumina Miseq (insert size of 400 bp; Illumina Inc., San Diego, CA) with paired-end sequencing mode and Pacbio sequencing (20,000 bp template library; Pacific Biosciences, Menlo Park, CA) with standard sequencing mode using next-generation sequencing technology and single molecule real-time (SMRT) technology. A total of 3,221,902 reads were obtained. Genome assembly was performed using A5-miseq v20150522 (
) software for the data obtained by second- and third-generation sequencing platforms. Then, the assembled results were confirmed by MUMmer software (http://mummer.sourceforge.net/) to determine the positional relationship between contigs and to fill gaps (
). Gene prediction was carried out by GeneMarkS (http://topaz.gatech.edu/). The predictions of transfer (t)RNA, rRNA, and small (s)RNA were identified using tRNAscan-SE (
) softwares, respectively. Genome annotation was accomplished by National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (
) and confirmed by BLAST analysis (www.ncbi.nlm.nih.gov/protein). Using the multiple sequence alignment software MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/) to align the core gene sequences of the L. plantarum J26 and other species, we constructed a phylogenetic tree based on the core genes (
). Furthermore, the average nucleotide identity of the genome sequence between the L. plantarum J26 and other reference strains was evaluated by orthologous average nucleotide identity (OrthoANI; http://www.ezbiocloud.net/tools/orthoani) tool.
The expression of immune-related genes was evaluated in vivo. All of the procedures used here were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University in China, and the animal experiments were performed following the Northeast Agricultural University guidelines for Laboratory Animals Care and Use.
Forty BALB/cl mice, 8 wk old and 18 to 22 g of BW, were provided by Heilongjiang Province Medical University Laboratory Animal Center (Harbin, China). Mice were randomly divided into 2 groups and acclimated in pathogen-free cages under controlled conditions (22°C ± 2°C, relative humidity 55% ± 5%, 12-h light/dark cycles) with free access to water and a standard diet for 7 d. After this period, the experimental group was given 0.3 mL of skim milk containing L. plantarum J26 (108 cfu/mL) each day. The mice in the control group were given skim milk without L. plantarum J26. The mice were killed at 1, 3, 5, or 7 d after supplementation of bacteria. The colon and ileum tissue were removed, washed with cold saline, and placed in liquid nitrogen (
Induction of cytokines via NF-κB and p38 MAP kinase signalling pathways associated with the immunomodulation by Lactobacillus plantarum NDC 75017 in vitro and in vivo..
). To evaluate the mRNA expression levels of IL1A, IL1B, IL6, and TNFA, RNA of colon and ileum was extracted using a commercial kit (RNAprep Tissue Kit, Tiangen Biotech, Shanghai, China) and converted to cDNA using a PrimeScript RT reagent kit (Takara, Dalian, China). The expression of immune-related genes was analyzed by using real-time quantitative PCR (ABI Prism 7500 system; Applied Biosystems, Foster City, CA). Approximately 2 μL of the RNA sample was added to the 20-μL total reaction volume according to the requirements of the SYBR PremicExTaqTMII (PerfectReal-time) kit (Takara). The GAPDH gene was used as the reference gene. To amplify the selected immune-related genes IL1A, IL1B, IL6, and TNFA and GAPDH, specific primers were designed using Primer 5.0 software (Premier Biosoft, USA; http://www.premierbiosoft.com/primerdesign/index.html) as shown in Table 1. The protocol for the amplification reactions was as follows: 95°C for 30 s (denaturation) and 40 cycles of 95°C for 5 s and 60°C for 34 s (amplification and quantification). The results were expressed as relative values after normalization to GAPDH mRNA (
Induction of cytokines via NF-κB and p38 MAP kinase signalling pathways associated with the immunomodulation by Lactobacillus plantarum NDC 75017 in vitro and in vivo..
The complete genome of L. plantarum J26 comprises a circular chromosome (3,096,468 bp) and 4 circular plasmids: pJ26p1 (83,889 bp), pJ26p2 (43,153 bp), pJ26p3 (35,191 bp), and pJ26p4 (8,686 bp), with GC contents of 44.79, 41.14, 40.30, 45.48, and 35.92%, respectively (Figure 1). Among the 3,289 identified protein-coding genes, 3,068 were in the chromosome and 105, 62, 44, and 10 in plasmids pJ26p1 to pJ26p4, respectively. Furthermore, the chromosome included 16 rRNA operons, 71 tRNA, and 1 sRNA (Table 2). Each gene was assigned to a functional category according to the Clusters of Orthologous Groups database (www.ncbi.nlm.nih.gov/COG; Table 3). Among 23 functional categories, the most abundant were assigned to transcription (248 genes); 236 genes were assigned to carbohydrate transport and metabolism; and 137 genes were responsible for cell wall, membrane, and envelope biogenesis.
Figure 1Circular genome map of Lactobacillus plantarum J26. From periphery to center: genome sequence (ring 1 and 2), Cluster of Orthologous Groups of proteins (COG) annotated coding sequences (ring 3 and 4), intact prophages in red and clustered regularly interspaced short palindromic repeats (CRISPR) in black (ring 5), noncoding (nc)RNA genes (ring 6), GC content (ring 7), and GC skew [(G+C)/(G+C)] (ring 8), where values >0 are in green, and values <0 are in purple. The smaller maps show the genome map of plasmids in L. plantarum J26. The image was created using the software Circos (http://circos.ca/software/download/circos/).
A phylogenetic tree was constructed based on homology of core genes of L. plantarum J26 with related strains. Phylogenetic tree analysis (Figure 2) showed that L. plantarum J26 was more closely related to L. plantarum JDM1 than to other L. plantarum strains and evolved from the common ancestor compared with ST-III (which contains TA synthesis protein B instead of TA synthesis proteins TagF1 and TagF2 (
). The genome of J26 was compared with 10 other L. plantarum strains. The OrthoANI values (Table 4) indicated that the genome of L. plantarum J26 was closest to L. plantarum JDM1 (99.30% OrthoANI), followed by L. plantarum 5-2 (98.73%) and L. plantarum WCFS1 (98.71%). Generally, two genomes are considered the same species when the ANI value is higher than 95 to 96% (
). Consequently, L. plantarum J26 was confirmed to belong to the species of L. plantarum.
Figure 2Phylogenetic tree based on core gene sequence showing the phylogenetic relationships of 11 Lactobacillus plantarum strains. Phylogenetic analysis was evaluated by MUSCLE software (https://www.ebi.ac.uk/Tools/msa/muscle/).
Intestinal epithelial cells stimulated by microbes can secrete cytokines and pattern recognition receptors to participate in appropriate immune regulation (
). As shown in Figure 3, we investigated mRNA expression of proinflammatory cytokines IL-1α, IL-1β, IL-6, and TNF-α in the intestinal tract of mice stimulated with L. plantarum J26. These cytokines were all upregulated, with expression peaking at d 5, and then decreasing to control levels. Moreover, the expression of IL1A, IL1B, IL6, and TNFA was significantly different from that in the control group (P < 0.01) on d 5, indicating that L. plantarum J26 was able to trigger the mRNA expression of the genes encoding these proinflammatory cytokines in vivo, results consistent with our previous study with another L. plantarum strain (
Induction of cytokines via NF-κB and p38 MAP kinase signalling pathways associated with the immunomodulation by Lactobacillus plantarum NDC 75017 in vitro and in vivo..
). Although IL-1α, IL-1β, IL-6, and TNF-α are considered proinflammatory cytokines, the specific mechanism by which L. plantarum J26 regulates their expression is unknown. We found no evidence that the immunomodulatory activity of L. plantarum J26 was directly related to certain host cells; our results indicate that the immune responses induced by J26 were likely linked to the type of immune-related cytokines, which is the focus of our further research.
Figure 3Expression of IL1A, IL1B, IL6, and TNFA genes induced by Lactobacillus plantarum J26 in mice. Mice were intragastrically administered 0.3 mL of L. plantarum J26 (gray bars) or an equal amount of sterilized skim milk (white bars) for 7 d, and the expression of IL1A (a), IL1B (b), IL6 (c), and TNFA (d) was determined. Results are expressed as mean ± SD of 3 independent determinations. *P < 0.05, **P < 0.01 compared with control.
The immunomodulatory mechanism of J26 was further investigated by analysis of the whole genome of L. plantarum J26. Teichoic acids consist of 2 types: wall teichoic acid (WTA) and lipoteichoic acid (LTA). For L. plantarum WCFS1, d-alanylated TA in the cell walls account for several host immunomodulatory effects (
). Because TA are recognized as immunoregulatory molecules, the TA-related genes of L. plantarum J26 have been searched by in silico analysis. The synthesis genes encoding the WTA backbones are tag and tar homologs, whereas those of LTA are ltaS. The genes encoding LTA synthase (ltaS), TA glycosylation proteins (gtcA1, gtcA2, gtcA3), and d-alanylation protein (dltX) were found in the L. plantarum J26 genome. Two gene regions, tagD1-tagF2 (tagD1, tagF1, and tagF2) and tarI-tarL (tarI, tarJ, tarK and tarL), were regarded as the synthesis genes of glycerol-type and ribitol-type backbones of WTA. In L. plantarum J26, only a glycerol-3-phosphate cytidylyltransferase gene tagD1 was detected. The genes encoding TA synthesis protein F (tagF1 and tagF2) were absent. However, there are 2 TA synthesis protein B genes (tagB1 and tagB2) instead, and tagB1 and tagB2 found in L. plantarum were supposed as tarK and tarL. The tarI and tarJ genes were annotated as d-ribitol-5-phosphate cytidylyltransferase and ribitol-5-phosphate 2-dehydrogenase. Hence, we propose that L. plantarum J26 contains tarI-tarL region and synthesizes ribitol-type backbones of WTA. Besides, 2 TA transporters (permease protein, tag; and ATP-binding protein, tagH), subunits of the ABC transporter complex, were identified and may be associated with the immunomodulatory effects of L. plantarum J26.
In the current study, we demonstrated that the L. plantarum J26 is a probiotic strain with immunomodulatory activity (regulating the expression of immune-related genes). The genome data of L. plantarum J26 provide substantial information for understanding the genetic basis of its probiotic properties. In addition, we discovered genes involved in key immunomodulatory molecules (teichoic acids) in the genome of J26. These results contribute to the development of immune-related functional dairy foods containing lactic acid bacteria. Further investigation into the molecular mechanism by which the TA-related gene products affect the host immune response are ongoing. The complete genomic sequence of L. plantarum J26 has been deposited in National Center for Biotechnology Information under GenBank accession nos. CP033616, CP033617, CP033618, CP033619, and CP033620.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (Beijing; project number 31871828) and the National High-Level Talents Special Support Program of China (810098).
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Beneficial effects of Lactobacillus plantarum on glycemia and homocysteine levels in postmenopausal women with metabolic syndrome.
The effect of Lactobacillus plantarum 299 v on the bacterial composition and metabolic activity in faeces of healthy volunteers: A placebo-controlled study on the onset and duration of effects.
Induction of cytokines via NF-κB and p38 MAP kinase signalling pathways associated with the immunomodulation by Lactobacillus plantarum NDC 75017 in vitro and in vivo..
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