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Research article
Transcriptional Response and Enhanced Intestinal Adhesion Ability of Lactobacillus rhamnosus GG after Acid Stress
1Division of Animal Science, Chonnam National University, Gwangju 61186, Republic of Korea, 2Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea, 3Food R&D Center, Samyang Corp., Seongnam 13488, Republic of Korea
J. Microbiol. Biotechnol. 2018; 28(10): 1604-1613
Published October 28, 2018 https://doi.org/10.4014/jmb.1807.07033
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
Fermented dairy products have been consumed for centuries, and yogurt is one of the most popular fermented milk products. Generally, the final pH of fermented dairy products is relatively low (approximately pH 4.5) owing to the accumulation of fermentation end products, especially lactic acid. This low-pH environment might affect the survival of lactic acid bacteria within fermented milk. The ability to withstand an acidic environment is crucial for the survival and commercial utilization of probiotic bacteria. For instance, a previous study reported that certain
During fermentation, the lactic acid produced by
After surviving acid stress in fermented dairy products and the gastrointestinal tract, it is essential for probiotic strains to adhere to the host mucosal surface, where they interact with the gut-associated lymphoid tissue and mediate local and systemic immune effects. As suggested by previous studies, only adherent probiotics can effectively induce immuno-modulatory effects and stabilize the host’s gut mucosal barrier [14-16].
In the present study, to decipher the metabolic responses in LGG under acid stress at pH 4.5, which is similar to the pH of most fermented milk-based food products, the genetic responses of LGG were examined using RNA-sequencing. Additionally, the adherent ability of LGG under acid stress was investigated via in vitro and in vivo approaches. Our findings provide important insights into the role of the pilus fiber of LGG in mediating adhesion to the intestinal mucosa during acid stress.
Materials and Methods
Bacteria Cultivation and Cell Harvesting
RNA Sequencing and Data Analyses
Total RNA was isolated from the collected cell pellets using RNeasy Mini Kits (Qiagen, USA), digested with RNase-free-DNase (Qiagen) to remove DNA, and depleted of ribosomal RNA using a Ribo-Zero rRNA Removal Kit (Illumina, USA) according to the manufacturer’s instructions. Ten micrograms of extracted RNA was used to prepare a sequencing library using a TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions. RNA sequencing was performed with an Illumina MiSeq platform. All data sets have been deposited in the Gene Expression Omnibus database under accession number GSE107337. Data analysis was performed using EdgeR, Bioconductor components in R packages with LGG as the reference genome.
Validating the Overexpression of Adhesion-Related Genes in Acid-Stressed LGG by Quantitative Reverse-Transcription PCR
Total RNA was extracted from the collected cell pellets using RNeasy Mini Kits (Qiagen) and DNA was removed with RNase-free-DNase (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of the extracted RNA with an RT Premix Oligo (dt) Kit (Intron, Korea). The primers used in this study were designed using Primer3plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi), based on the genome sequence of LGG (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control gene. Quantitative PCR (qPCR) was performed using KAPA SYBR FAST qPCR kit (KapaBiosystems, USA) in a thermal cycler (Bio-Rad Laboratories, USA) with the conditions of initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 10 sec, annealing at 54°C for 15 sec and extension at 72°C for 15 sec. The expression of each gene was determined via a comparative analysis based on Ct values as follows:
-
Table 1 . Oligonucleotide primers used for the qRT-PCR analysis of adhesion-related gene expression.
Gene Sequence of PCR primers (5’ to 3’) Target locus Product Reference spaF Forward : GCTGATGTTACTGCTGATGC
Reverse : ATCCGTATTTTGAACGGGTALRHM_2281 putative cell surface protein PMID 19820099 spaC Forward : CAACTTGATGGGACAACGTA
Reverse : TCTGGTGCTTTTGTTTCTGALRHM_0428 Cell surface protein PMID 19820099 pili Forward : GATTATCGGGTTGATTCTGG
Reverse : AAATCGCCTTCGTACATCTCLGG_02339 Pili_PIN/TRAM domain-containing protein PMID 19820099 Pilus444 Forward : CAACTTGATGGGACAACGTA
Reverse : TTTGCAGGATTGCTTTGATALGG_00444 Pilus protein PMID 19820099 Pilus443 Forward : CTAAATCCTTCCGTCCGTTA
Reverse : CTCAACGTCGTTTGTGCTACLGG_00443 Pilus protein PMID 19820099 Gene Sequence of PCR primers (5’ to 3’) Target locus Product Reference Pilus442 Forward : GATGGTTCTCGGGTTTAATG
Reverse : ACCCACGTCAATCGATAGTTLGG_00442 pilus protein PMID 19820099 Pilus2370 Forward : AACTATCCATTCGGGTTTGA
Reverse : GTTGTCGGATCAAGGATTTCLGG_02370 Pilus protein PMID 19820099 mbf Forward : TGAAGTTGGAAAAGCGTACA
Reverse : AGGAAAAGTTCCTGATGCTGAB968049 Mucus binding factor PMID 19820099 GAPDH Forward : GATCGTTTCTGCAGGTTCTT
Reverse : CCGTTCAATTCTGGGATAACLGG_00933 Type I glyceraldehyde-3-phosphate dehydrogenase PMID 19820099
Relative expressio
Mucin-Binding Property of Acid-Stressed LGG
A 96-well microtiter plate was inoculated with 100 µl of 10 mg/ml mucin solution (Sigma-Aldrich, USA) and kept overnight at 4°C. Wells were then washed twice with 0.85% NaCl prior to inoculation with LGG. LGG was grown in MRS broth until it reached an OD600nm of 0.6 and centrifuged to collect cell pellets. The cell pellets were re-suspended in acidic MRS broth (pH 4.5) for 1 or 24 h, or neutral MRS broth (pH 7.0; control) for 1 h. 100 µl of each culture (108 CFU/ml) was added to the microtiter wells and incubated for 2 h at 37°C. Thereafter, the wells were washed five times with 0.85% NaCl to remove unbound bacteria. Adherent bacteria were removed with 0.1% Triton X-100 solution, followed by serial 10-fold dilutions and then plated on MRS agar. Colonies were counted after incubation at 37°C for 48 h.
In Vivo Assessment of the Intestine-Binding Ability of Acid-Stressed LGG
LGG cultures were grown in MRS broth until reaching an OD600nm of 0.6 and centrifuged to collect cell pellets. The cell pellets were re-suspended in acidic MRS broth (pH 4.5) for 24 h or neutral MRS broth (pH 7.0; control) for 1 h. The control consisted of an LGG culture grown overnight without acidic or neutral MRS broth treatment. Then, cell pellets were collected via centrifugation and incubated with fluorescein isothiocyanate (FITC) solution (1 mg/ml in 50 mmol/l sodium carbonate buffer, pH 8.9) for 2 h at room temperature. The labeled cells were washed 5 times with 0.85% NaCl to remove the remaining FITC and then orally administered to male C57BL/6J mice. After 12 h, the mice were sacrificed, and their intestines were collected to detect the presence of adherent labeled cells using a confocal laser scanning microscope (FV500, Olympus, Japan). Animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Chonnam National University (project number: CNUIACUC-YB-2012-40).
The number of viable LGG adhered to the intestines was also determined. Fifty milligram portions of ileum, cecum, and colon were mixed with an equal volume of phosphate buffer saline and homogenized using a tissue grinder (Thomas Scientific, USA). Serial 10-fold dilutions were made and the number of viable LGG was counted on glucose blood liver (BL) agar supplemented with vancomycin after being incubated for 48 h at 37°C [21].
Statistical Analyses
All values were expressed as the mean ± standard deviation from triplicate runs (
Results
Transcriptional Response to LGG to Acid Stress
The effects of short-term (1 h) and long-term (24 h) acid stress condition (pH 4.5) on the global gene expression of LGG were investigated via RNA sequencing. Through normalization, analysis, and comparison of the global gene expression profiles of the acid-stressed LGG (pH 4.5 for 1 or 24 h) and the control LGG (pH 7.0 for 1 h), we identified 228 genes that were significantly (
-
Fig. 1. (A) Heat map showing the differentially expressed genes in
Lactobacillus rhamnosus GG (LGG) exposed to acid stress at pH 4.5 for 1 h or 24 h. Under acid stress condition, 128 genes were significantly upregulated and 100 genes were significantly downregulated. Results were expressed as log2 (fold change of acid stress LGG/fold change of control LGG). Red or blue blots indicated genes that were detected as differentially expressed withp < 0.05. Differentially expressed genes: (B) upregulated and (C) downregulated genes in acid-stressed LGG were categorized based on biological process using the Cluster of Orthologous Groups of Proteins (COG) database.
Among the upregulated genes, 47% (61/129) encoded for hypothetical proteins with unknown functions. Meanwhile, genes encoding phage-related proteins demonstrated the highest fold change upon acid stress exposure, followed by hypothetical proteins and DNA-related proteins (Fig. 1B). Based on the transcriptomic data, the stress response-related genes LGG_02240 and LGG_01072, which encode the co-chaperonin
Meanwhile, genes that were downregulated in response to acid stress conditions were mainly involved in the carbohydrate pyruvoyl-tetrahydropterin synthase (PTS) system, including those encoding the galactitol-, fructose-, mannitol-, and mannose-specific transporter subunit. Genes encoding amino acid and nucleotide transport systems, as well as various enzymes involved in biosynthesis, transfer, and proteolysis were also suppressed in response to acid stress, including the genes involved in L-lysine biosynthesis via the diaminopimelate (DAP) pathway (LGG_00113, LGG_00115, LGG_00108, and LGG_00109). The transcriptomics analysis also revealed drastic differences in the profiles of downregulated genes between the different exposure times (1 and 24 h) to acid at pH 4.5. The expression of these genes was greatly suppressed after exposure to acid stress for 1 h and gradually recovered towards normal levels after exposure to acid stress for 24 h (Fig. 1C).
The Expression Levels of Adhesion-Related Genes under Acid Stress
The gene expression analysis revealed that the expression of all the adhesion-related genes was affected upon exposure to acid at pH 4.5, and the exposure time played a key role in the regulation of these adhesion-related genes in LGG (Fig. 2). The gene expression of
-
Fig. 2. Gene expression of (A)
spaC , (B)spaF , (C)pili , (D)pilus444 , (E)pilus443 , (F)pillus442 , (G)pilus2370 , (H)internalin , and (I)mbf fromLactobacillus rhamnosus GG (LGG) upon exposure to acid stress at pH 4.5 for 1 h or 24 h. The mean values of samples with different lowercase letters were significantly different (p < 0.05).
Mucin-Binding Property of Acid-Stressed LGG
To assess the reliability of the transcriptomic and gene expression analyses, the mucin-binding ability of LGG upon acid stress was investigated. The results showed a positive correlation between the transcriptomic and gene expression data and the mucin-binding ability of LGG. The number of LGG cells attached to the mucin layer was 1logCFU/ml higher (
-
Fig. 3. The ability of acid-stressed
Lactobacillus rhamnosus GG (LGG) to adhere to mucin protein. Results are expressed as the means ± standard deviation (n = 3). The means values of LGG treatments with different lowercase letters were significantly different (p < 0.05).
In Vivo Assessment of the Intestine-Binding Ability of Acid-Stressed LGG
The intestine-binding ability of acid-stressed LGG was determined by detecting the fluorescence expression of FITC-labeled LGG cells that were attached to mice intestines. A stronger fluorescent signal was detected in the acid-stressed LGG as compared to the pH 7.0-treated LGG and the non-treated LGG control in the ileum, caecum and colon samples, indicating that the acid-stressed LGG displayed a greater ability to adhere to the intestinal wall (Fig. 4). The enhanced adherence capacity of the acid-stressed LGG was further illustrated by the finding of a significantly higher number of viable LGG in the acid-stressed samples as compared to pH 7.0-treated LGG and non-treated LGG control in the ileum, caecum, and colon of the mice (Fig. 5). The number of viable acid-stressed LGG cells was 1 log CFU/g greater (
-
Fig. 4. Fluorescence expression of orally administered acid-stressed
Lactobacillus rhamnosus GG (LGG) adhered to the (A) ileum, (B) caecum, and (C) colon of mice. The LGG cells were labeled with fluorescein isothiocyanate (FITC) solution (1 mg/ml in 50 mmol/l sodium carbonate buffer, pH 8.9) before being administered to the mice. Control, non-treated cells; pH 4.5, treated with pH 4.5 medium for 24 h; pH 7.0, treated with pH 7.0 medium.
-
Fig. 5. The viability of the orally administered acid-stressed
Lactobacillus rhamnosus GG (LGG) in the (A) ileum, (B) caecum, and (C) colon of the mice. Results are expressed as the means ± standard deviation (n = 3). The means values of LGG treatments with different lowercase letters were significantly different (p < 0.05).
Discussion
The ability of probiotic bacteria to withstand and survive the acidic environment of the mammalian gut is of paramount importance for them to reach and adhere to the intestinal walls as part of the gut microbiota. As a functional probiotic that has been extensively used in fermented foods and dietary supplements, LGG tolerates acid stress well and has several defense mechanisms to ensure its survivability. A previous study demonstrated the excellent acid tolerance ability of LGG, which had a high survival rate after 52 weeks of storage in yogurt (as a representative acidic fermented dairy products [1]. The mechanisms of acid resistance in lactic acid bacteria have been well documented, including the modulation of the proton-translocating ATPase to maintain the cytoplasmic pH homeostasis, the transition of pyruvate metabolism towards acetyl-CoA production, and the alteration of amino acid biosynthesis to supply energy for resisting acid stress [1, 8, 22].
In this study, differences in the gene expression of LGG after exposure to acidic conditions for 1 or 24h were investigated. Our transcriptomic data showed that the expression of general stress response genes was elevated in the acid-stressed LGG. Our results agree with those of other studies that have shown the importance of several stress response genes during acid stress. For instance, the activation of co-chaperonin ES (
In response to acid stress, the PTS carbohydrate transport genes of LGG were downregulated. Numerous studies have reported the alteration of carbohydrate metabolism under condition of acid stress, which enables bacteria to resist acid stress better by increasing their supply of energy [22, 25]. Similar findings were observed in this study, where several genes, including LGG_01064, which encodes 6-phospho-β-glucosidase that hydrolyzes 6-phospho-β-D-glucosyl-(1,4)-D-glucose into D-glucose and D-glucose-6-phosphate, the two substrates used in glycolysis for energy generation, were upregulated under acid stress conditions. On the contrary, genes whose products catalyze the reverse reaction of glycolysis, for instance, the fructose-1,6-bisphosphatase encoding gene (LGG-02032) and the transaldolase encoding gene (LGG_00418), were apparently downregulated, to conserve energy for resisting acid stress. This conserved energy and the ATP generated from glycolysis are used to induce the F1F0-ATPase proton pump and regulate the internal pH of LGG, as demonstrated in the previous study [1]. To maintain the anion pool within the cytoplasm, the ammonium transporter,
The amino acid metabolism of LGG was also affected by acid stress. Amino acid metabolism has several physiological roles in lactic acid bacteria, including the control of intracellular pH, the generation of metabolic energy or redox power, and the resistance to stress [26]. Previous studies have reported that aspartate could protect lactic acid bacteria against acid stress [26, 27]. Our transcriptomic analysis of the acid-stressed LGG demonstrated decreases in the gene expression of aspartate kinase (
Another important finding from the transcriptomic analysis of acid-stressed LGG was the upregulation of
The outcomes of the transcriptomic analysis of acid-stressed LGG were further confirmed via gene expression analysis. The common SpaCBA pili cluster gene,
Following the transcriptomic and gene expression analyses, the mucin and gastrointestinal tract-binding abilities of LGG under acid stress conditions were further justified via in vitro and in vivo analyses. A previous study showed that wild-type LGG displayed high adhesion capacities towards intestinal epithelial cells and mucin [31]. Considering that LGG is currently the only known probiotic strain with mucus-binding pili, an enhancement of the activities of the SpaCBA and SpaFED mucus-binding pili could further improve the competitiveness of LGG in the mucosal environment as compared to other probiotic strains [17]. The previous study also reported the importance of pili in facilitating the adherence ability of LGG to human intestinal tissue and thereby prolonging the retention of LGG during their transit through the gastrointestinal tract. This is important because only adherent probiotics can effectively exert immunomodulatory effects and stabilize the intestinal mucosal barrier [14, 18]. In this study, we found that the acid-stressed LGG exhibited a higher capacity for adhesion to mucin. Owing to the inability of in vitro assays to resemble the complex environmental conditions of cells within a living organism, an animal study was conducted to evaluate the effect of acid stress on the adherence and retention of LGG during transit through the gastrointestinal tract. The results of the in vivo study confirmed the enhanced adhesion of LGG in the gastrointestinal tract under acid stress conditions, indicating that acid stress could enhance the adhesion of LGG and thereby prolong its retention in the gastrointestinal tract, enabling it to exert its health-promoting properties.
The findings presented in this study provide a detailed understanding of the adaptation of LGG towards acid stress conditions, including the enhancement of its adhesion ability (Fig. 6). Our findings lead us to a novel discovery regarding the enhanced adhesion properties of LGG upon acid stress treatment, especially the expression of the
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Fig. 6. Interaction of
Lactobacillus rhamnosus GG (LGG) subjected to acid stress conditions with epithelial cells in the mucosa. Upon exposure to an acidic environment, pili genes of LGG such asspaF andspaC were highly expressed and the cells bound strongly to the gastrointestinal mucus layer of the host. These results indicated that the adhesion ability of LGG was enhanced after exposure to acid stress conditions.
Supplemental Materials
Acknowledgments
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (NRF-2016R1A2B4007519).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB
Article
Research article
J. Microbiol. Biotechnol. 2018; 28(10): 1604-1613
Published online October 28, 2018 https://doi.org/10.4014/jmb.1807.07033
Copyright © The Korean Society for Microbiology and Biotechnology.
Transcriptional Response and Enhanced Intestinal Adhesion Ability of Lactobacillus rhamnosus GG after Acid Stress
Miseon Bang 1, Cheng-Chung Yong 1, Hyeok-Jin Ko 2, 3, In-Geol Choi 2 and Sejong Oh 1*
1Division of Animal Science, Chonnam National University, Gwangju 61186, Republic of Korea, 2Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea, 3Food R&D Center, Samyang Corp., Seongnam 13488, Republic of Korea
Abstract
Lactobacillus rhamnosus GG (LGG) is a probiotic commonly used in fermented dairy products.
In this study, RNA-sequencing was performed to unravel the effects of acid stress on LGG.
The transcriptomic data revealed that the exposure of LGG to acid at pH 4.5 (resembling the
final pH of fermented dairy products) for 1 h or 24 h provoked a stringent-type transcriptomic
response wherein stress response- and glycolysis-related genes were upregulated, whereas
genes involved in gluconeogenesis, amino acid metabolism, and nucleotide metabolism were
suppressed. Notably, the pilus-specific adhesion genes, spaC, and spaF were significantly
upregulated upon exposure to acid-stress. The transcriptomic results were further confirmed
via quantitative polymerase chain reaction analysis. Moreover, acid-stressed LGG
demonstrated an enhanced mucin-binding ability in vitro, with 1 log more LGG cells (p < 0.05)
bound to a mucin layer in a 96-well culture plate as compared to the control. The enhanced
intestinal binding ability of acid-stressed LGG was confirmed in an animal study, wherein
significantly more viable LGG cells (≥ 2 log CFU/g) were observed in the ileum, caecum, and
colon of acid-stressed LGG-treated mice as compared with a non-acid-stressed LGG-treated
control group. To our knowledge, this is the first report showing that acid stress enhanced the
intestine-binding ability of LGG through the induction of pili-related genes.
Keywords: Stress response, Lactobacillus, transcriptomics, probiotics, microbial physiology, adhesion
Introduction
Fermented dairy products have been consumed for centuries, and yogurt is one of the most popular fermented milk products. Generally, the final pH of fermented dairy products is relatively low (approximately pH 4.5) owing to the accumulation of fermentation end products, especially lactic acid. This low-pH environment might affect the survival of lactic acid bacteria within fermented milk. The ability to withstand an acidic environment is crucial for the survival and commercial utilization of probiotic bacteria. For instance, a previous study reported that certain
During fermentation, the lactic acid produced by
After surviving acid stress in fermented dairy products and the gastrointestinal tract, it is essential for probiotic strains to adhere to the host mucosal surface, where they interact with the gut-associated lymphoid tissue and mediate local and systemic immune effects. As suggested by previous studies, only adherent probiotics can effectively induce immuno-modulatory effects and stabilize the host’s gut mucosal barrier [14-16].
In the present study, to decipher the metabolic responses in LGG under acid stress at pH 4.5, which is similar to the pH of most fermented milk-based food products, the genetic responses of LGG were examined using RNA-sequencing. Additionally, the adherent ability of LGG under acid stress was investigated via in vitro and in vivo approaches. Our findings provide important insights into the role of the pilus fiber of LGG in mediating adhesion to the intestinal mucosa during acid stress.
Materials and Methods
Bacteria Cultivation and Cell Harvesting
RNA Sequencing and Data Analyses
Total RNA was isolated from the collected cell pellets using RNeasy Mini Kits (Qiagen, USA), digested with RNase-free-DNase (Qiagen) to remove DNA, and depleted of ribosomal RNA using a Ribo-Zero rRNA Removal Kit (Illumina, USA) according to the manufacturer’s instructions. Ten micrograms of extracted RNA was used to prepare a sequencing library using a TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions. RNA sequencing was performed with an Illumina MiSeq platform. All data sets have been deposited in the Gene Expression Omnibus database under accession number GSE107337. Data analysis was performed using EdgeR, Bioconductor components in R packages with LGG as the reference genome.
Validating the Overexpression of Adhesion-Related Genes in Acid-Stressed LGG by Quantitative Reverse-Transcription PCR
Total RNA was extracted from the collected cell pellets using RNeasy Mini Kits (Qiagen) and DNA was removed with RNase-free-DNase (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of the extracted RNA with an RT Premix Oligo (dt) Kit (Intron, Korea). The primers used in this study were designed using Primer3plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi), based on the genome sequence of LGG (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control gene. Quantitative PCR (qPCR) was performed using KAPA SYBR FAST qPCR kit (KapaBiosystems, USA) in a thermal cycler (Bio-Rad Laboratories, USA) with the conditions of initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 10 sec, annealing at 54°C for 15 sec and extension at 72°C for 15 sec. The expression of each gene was determined via a comparative analysis based on Ct values as follows:
-
Table 1 . Oligonucleotide primers used for the qRT-PCR analysis of adhesion-related gene expression..
Gene Sequence of PCR primers (5’ to 3’) Target locus Product Reference spaF Forward : GCTGATGTTACTGCTGATGC
Reverse : ATCCGTATTTTGAACGGGTALRHM_2281 putative cell surface protein PMID 19820099 spaC Forward : CAACTTGATGGGACAACGTA
Reverse : TCTGGTGCTTTTGTTTCTGALRHM_0428 Cell surface protein PMID 19820099 pili Forward : GATTATCGGGTTGATTCTGG
Reverse : AAATCGCCTTCGTACATCTCLGG_02339 Pili_PIN/TRAM domain-containing protein PMID 19820099 Pilus444 Forward : CAACTTGATGGGACAACGTA
Reverse : TTTGCAGGATTGCTTTGATALGG_00444 Pilus protein PMID 19820099 Pilus443 Forward : CTAAATCCTTCCGTCCGTTA
Reverse : CTCAACGTCGTTTGTGCTACLGG_00443 Pilus protein PMID 19820099 Gene Sequence of PCR primers (5’ to 3’) Target locus Product Reference Pilus442 Forward : GATGGTTCTCGGGTTTAATG
Reverse : ACCCACGTCAATCGATAGTTLGG_00442 pilus protein PMID 19820099 Pilus2370 Forward : AACTATCCATTCGGGTTTGA
Reverse : GTTGTCGGATCAAGGATTTCLGG_02370 Pilus protein PMID 19820099 mbf Forward : TGAAGTTGGAAAAGCGTACA
Reverse : AGGAAAAGTTCCTGATGCTGAB968049 Mucus binding factor PMID 19820099 GAPDH Forward : GATCGTTTCTGCAGGTTCTT
Reverse : CCGTTCAATTCTGGGATAACLGG_00933 Type I glyceraldehyde-3-phosphate dehydrogenase PMID 19820099
Relative expressio
Mucin-Binding Property of Acid-Stressed LGG
A 96-well microtiter plate was inoculated with 100 µl of 10 mg/ml mucin solution (Sigma-Aldrich, USA) and kept overnight at 4°C. Wells were then washed twice with 0.85% NaCl prior to inoculation with LGG. LGG was grown in MRS broth until it reached an OD600nm of 0.6 and centrifuged to collect cell pellets. The cell pellets were re-suspended in acidic MRS broth (pH 4.5) for 1 or 24 h, or neutral MRS broth (pH 7.0; control) for 1 h. 100 µl of each culture (108 CFU/ml) was added to the microtiter wells and incubated for 2 h at 37°C. Thereafter, the wells were washed five times with 0.85% NaCl to remove unbound bacteria. Adherent bacteria were removed with 0.1% Triton X-100 solution, followed by serial 10-fold dilutions and then plated on MRS agar. Colonies were counted after incubation at 37°C for 48 h.
In Vivo Assessment of the Intestine-Binding Ability of Acid-Stressed LGG
LGG cultures were grown in MRS broth until reaching an OD600nm of 0.6 and centrifuged to collect cell pellets. The cell pellets were re-suspended in acidic MRS broth (pH 4.5) for 24 h or neutral MRS broth (pH 7.0; control) for 1 h. The control consisted of an LGG culture grown overnight without acidic or neutral MRS broth treatment. Then, cell pellets were collected via centrifugation and incubated with fluorescein isothiocyanate (FITC) solution (1 mg/ml in 50 mmol/l sodium carbonate buffer, pH 8.9) for 2 h at room temperature. The labeled cells were washed 5 times with 0.85% NaCl to remove the remaining FITC and then orally administered to male C57BL/6J mice. After 12 h, the mice were sacrificed, and their intestines were collected to detect the presence of adherent labeled cells using a confocal laser scanning microscope (FV500, Olympus, Japan). Animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Chonnam National University (project number: CNUIACUC-YB-2012-40).
The number of viable LGG adhered to the intestines was also determined. Fifty milligram portions of ileum, cecum, and colon were mixed with an equal volume of phosphate buffer saline and homogenized using a tissue grinder (Thomas Scientific, USA). Serial 10-fold dilutions were made and the number of viable LGG was counted on glucose blood liver (BL) agar supplemented with vancomycin after being incubated for 48 h at 37°C [21].
Statistical Analyses
All values were expressed as the mean ± standard deviation from triplicate runs (
Results
Transcriptional Response to LGG to Acid Stress
The effects of short-term (1 h) and long-term (24 h) acid stress condition (pH 4.5) on the global gene expression of LGG were investigated via RNA sequencing. Through normalization, analysis, and comparison of the global gene expression profiles of the acid-stressed LGG (pH 4.5 for 1 or 24 h) and the control LGG (pH 7.0 for 1 h), we identified 228 genes that were significantly (
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Figure 1. (A) Heat map showing the differentially expressed genes in
Lactobacillus rhamnosus GG (LGG) exposed to acid stress at pH 4.5 for 1 h or 24 h. Under acid stress condition, 128 genes were significantly upregulated and 100 genes were significantly downregulated. Results were expressed as log2 (fold change of acid stress LGG/fold change of control LGG). Red or blue blots indicated genes that were detected as differentially expressed withp < 0.05. Differentially expressed genes: (B) upregulated and (C) downregulated genes in acid-stressed LGG were categorized based on biological process using the Cluster of Orthologous Groups of Proteins (COG) database.
Among the upregulated genes, 47% (61/129) encoded for hypothetical proteins with unknown functions. Meanwhile, genes encoding phage-related proteins demonstrated the highest fold change upon acid stress exposure, followed by hypothetical proteins and DNA-related proteins (Fig. 1B). Based on the transcriptomic data, the stress response-related genes LGG_02240 and LGG_01072, which encode the co-chaperonin
Meanwhile, genes that were downregulated in response to acid stress conditions were mainly involved in the carbohydrate pyruvoyl-tetrahydropterin synthase (PTS) system, including those encoding the galactitol-, fructose-, mannitol-, and mannose-specific transporter subunit. Genes encoding amino acid and nucleotide transport systems, as well as various enzymes involved in biosynthesis, transfer, and proteolysis were also suppressed in response to acid stress, including the genes involved in L-lysine biosynthesis via the diaminopimelate (DAP) pathway (LGG_00113, LGG_00115, LGG_00108, and LGG_00109). The transcriptomics analysis also revealed drastic differences in the profiles of downregulated genes between the different exposure times (1 and 24 h) to acid at pH 4.5. The expression of these genes was greatly suppressed after exposure to acid stress for 1 h and gradually recovered towards normal levels after exposure to acid stress for 24 h (Fig. 1C).
The Expression Levels of Adhesion-Related Genes under Acid Stress
The gene expression analysis revealed that the expression of all the adhesion-related genes was affected upon exposure to acid at pH 4.5, and the exposure time played a key role in the regulation of these adhesion-related genes in LGG (Fig. 2). The gene expression of
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Figure 2. Gene expression of (A)
spaC , (B)spaF , (C)pili , (D)pilus444 , (E)pilus443 , (F)pillus442 , (G)pilus2370 , (H)internalin , and (I)mbf fromLactobacillus rhamnosus GG (LGG) upon exposure to acid stress at pH 4.5 for 1 h or 24 h. The mean values of samples with different lowercase letters were significantly different (p < 0.05).
Mucin-Binding Property of Acid-Stressed LGG
To assess the reliability of the transcriptomic and gene expression analyses, the mucin-binding ability of LGG upon acid stress was investigated. The results showed a positive correlation between the transcriptomic and gene expression data and the mucin-binding ability of LGG. The number of LGG cells attached to the mucin layer was 1logCFU/ml higher (
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Figure 3. The ability of acid-stressed
Lactobacillus rhamnosus GG (LGG) to adhere to mucin protein. Results are expressed as the means ± standard deviation (n = 3). The means values of LGG treatments with different lowercase letters were significantly different (p < 0.05).
In Vivo Assessment of the Intestine-Binding Ability of Acid-Stressed LGG
The intestine-binding ability of acid-stressed LGG was determined by detecting the fluorescence expression of FITC-labeled LGG cells that were attached to mice intestines. A stronger fluorescent signal was detected in the acid-stressed LGG as compared to the pH 7.0-treated LGG and the non-treated LGG control in the ileum, caecum and colon samples, indicating that the acid-stressed LGG displayed a greater ability to adhere to the intestinal wall (Fig. 4). The enhanced adherence capacity of the acid-stressed LGG was further illustrated by the finding of a significantly higher number of viable LGG in the acid-stressed samples as compared to pH 7.0-treated LGG and non-treated LGG control in the ileum, caecum, and colon of the mice (Fig. 5). The number of viable acid-stressed LGG cells was 1 log CFU/g greater (
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Figure 4. Fluorescence expression of orally administered acid-stressed
Lactobacillus rhamnosus GG (LGG) adhered to the (A) ileum, (B) caecum, and (C) colon of mice. The LGG cells were labeled with fluorescein isothiocyanate (FITC) solution (1 mg/ml in 50 mmol/l sodium carbonate buffer, pH 8.9) before being administered to the mice. Control, non-treated cells; pH 4.5, treated with pH 4.5 medium for 24 h; pH 7.0, treated with pH 7.0 medium.
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Figure 5. The viability of the orally administered acid-stressed
Lactobacillus rhamnosus GG (LGG) in the (A) ileum, (B) caecum, and (C) colon of the mice. Results are expressed as the means ± standard deviation (n = 3). The means values of LGG treatments with different lowercase letters were significantly different (p < 0.05).
Discussion
The ability of probiotic bacteria to withstand and survive the acidic environment of the mammalian gut is of paramount importance for them to reach and adhere to the intestinal walls as part of the gut microbiota. As a functional probiotic that has been extensively used in fermented foods and dietary supplements, LGG tolerates acid stress well and has several defense mechanisms to ensure its survivability. A previous study demonstrated the excellent acid tolerance ability of LGG, which had a high survival rate after 52 weeks of storage in yogurt (as a representative acidic fermented dairy products [1]. The mechanisms of acid resistance in lactic acid bacteria have been well documented, including the modulation of the proton-translocating ATPase to maintain the cytoplasmic pH homeostasis, the transition of pyruvate metabolism towards acetyl-CoA production, and the alteration of amino acid biosynthesis to supply energy for resisting acid stress [1, 8, 22].
In this study, differences in the gene expression of LGG after exposure to acidic conditions for 1 or 24h were investigated. Our transcriptomic data showed that the expression of general stress response genes was elevated in the acid-stressed LGG. Our results agree with those of other studies that have shown the importance of several stress response genes during acid stress. For instance, the activation of co-chaperonin ES (
In response to acid stress, the PTS carbohydrate transport genes of LGG were downregulated. Numerous studies have reported the alteration of carbohydrate metabolism under condition of acid stress, which enables bacteria to resist acid stress better by increasing their supply of energy [22, 25]. Similar findings were observed in this study, where several genes, including LGG_01064, which encodes 6-phospho-β-glucosidase that hydrolyzes 6-phospho-β-D-glucosyl-(1,4)-D-glucose into D-glucose and D-glucose-6-phosphate, the two substrates used in glycolysis for energy generation, were upregulated under acid stress conditions. On the contrary, genes whose products catalyze the reverse reaction of glycolysis, for instance, the fructose-1,6-bisphosphatase encoding gene (LGG-02032) and the transaldolase encoding gene (LGG_00418), were apparently downregulated, to conserve energy for resisting acid stress. This conserved energy and the ATP generated from glycolysis are used to induce the F1F0-ATPase proton pump and regulate the internal pH of LGG, as demonstrated in the previous study [1]. To maintain the anion pool within the cytoplasm, the ammonium transporter,
The amino acid metabolism of LGG was also affected by acid stress. Amino acid metabolism has several physiological roles in lactic acid bacteria, including the control of intracellular pH, the generation of metabolic energy or redox power, and the resistance to stress [26]. Previous studies have reported that aspartate could protect lactic acid bacteria against acid stress [26, 27]. Our transcriptomic analysis of the acid-stressed LGG demonstrated decreases in the gene expression of aspartate kinase (
Another important finding from the transcriptomic analysis of acid-stressed LGG was the upregulation of
The outcomes of the transcriptomic analysis of acid-stressed LGG were further confirmed via gene expression analysis. The common SpaCBA pili cluster gene,
Following the transcriptomic and gene expression analyses, the mucin and gastrointestinal tract-binding abilities of LGG under acid stress conditions were further justified via in vitro and in vivo analyses. A previous study showed that wild-type LGG displayed high adhesion capacities towards intestinal epithelial cells and mucin [31]. Considering that LGG is currently the only known probiotic strain with mucus-binding pili, an enhancement of the activities of the SpaCBA and SpaFED mucus-binding pili could further improve the competitiveness of LGG in the mucosal environment as compared to other probiotic strains [17]. The previous study also reported the importance of pili in facilitating the adherence ability of LGG to human intestinal tissue and thereby prolonging the retention of LGG during their transit through the gastrointestinal tract. This is important because only adherent probiotics can effectively exert immunomodulatory effects and stabilize the intestinal mucosal barrier [14, 18]. In this study, we found that the acid-stressed LGG exhibited a higher capacity for adhesion to mucin. Owing to the inability of in vitro assays to resemble the complex environmental conditions of cells within a living organism, an animal study was conducted to evaluate the effect of acid stress on the adherence and retention of LGG during transit through the gastrointestinal tract. The results of the in vivo study confirmed the enhanced adhesion of LGG in the gastrointestinal tract under acid stress conditions, indicating that acid stress could enhance the adhesion of LGG and thereby prolong its retention in the gastrointestinal tract, enabling it to exert its health-promoting properties.
The findings presented in this study provide a detailed understanding of the adaptation of LGG towards acid stress conditions, including the enhancement of its adhesion ability (Fig. 6). Our findings lead us to a novel discovery regarding the enhanced adhesion properties of LGG upon acid stress treatment, especially the expression of the
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Figure 6. Interaction of
Lactobacillus rhamnosus GG (LGG) subjected to acid stress conditions with epithelial cells in the mucosa. Upon exposure to an acidic environment, pili genes of LGG such asspaF andspaC were highly expressed and the cells bound strongly to the gastrointestinal mucus layer of the host. These results indicated that the adhesion ability of LGG was enhanced after exposure to acid stress conditions.
Supplemental Materials
Acknowledgments
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (NRF-2016R1A2B4007519).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.
Fig 2.
Fig 3.
Fig 4.
Fig 5.
Fig 6.
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Table 1 . Oligonucleotide primers used for the qRT-PCR analysis of adhesion-related gene expression..
Gene Sequence of PCR primers (5’ to 3’) Target locus Product Reference spaF Forward : GCTGATGTTACTGCTGATGC
Reverse : ATCCGTATTTTGAACGGGTALRHM_2281 putative cell surface protein PMID 19820099 spaC Forward : CAACTTGATGGGACAACGTA
Reverse : TCTGGTGCTTTTGTTTCTGALRHM_0428 Cell surface protein PMID 19820099 pili Forward : GATTATCGGGTTGATTCTGG
Reverse : AAATCGCCTTCGTACATCTCLGG_02339 Pili_PIN/TRAM domain-containing protein PMID 19820099 Pilus444 Forward : CAACTTGATGGGACAACGTA
Reverse : TTTGCAGGATTGCTTTGATALGG_00444 Pilus protein PMID 19820099 Pilus443 Forward : CTAAATCCTTCCGTCCGTTA
Reverse : CTCAACGTCGTTTGTGCTACLGG_00443 Pilus protein PMID 19820099 Gene Sequence of PCR primers (5’ to 3’) Target locus Product Reference Pilus442 Forward : GATGGTTCTCGGGTTTAATG
Reverse : ACCCACGTCAATCGATAGTTLGG_00442 pilus protein PMID 19820099 Pilus2370 Forward : AACTATCCATTCGGGTTTGA
Reverse : GTTGTCGGATCAAGGATTTCLGG_02370 Pilus protein PMID 19820099 mbf Forward : TGAAGTTGGAAAAGCGTACA
Reverse : AGGAAAAGTTCCTGATGCTGAB968049 Mucus binding factor PMID 19820099 GAPDH Forward : GATCGTTTCTGCAGGTTCTT
Reverse : CCGTTCAATTCTGGGATAACLGG_00933 Type I glyceraldehyde-3-phosphate dehydrogenase PMID 19820099
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