Integrated transcriptomic and proteomic analysis of the bile stress response in probiotic Lactobacillus salivarius LI01

    Integrated transcriptomic and proteomic analysis of the bile stress response in probiotic Lactobacillus salivarius LI01 Long-Xian Lv, Ren Yan, Hai-Yan Shi, Ding Shi, Dai-Qiong Fang, HuiYong Jiang, Wen-Rui Wu, Fei-Fei Guo, Xia-Wei Jiang, Si-Lan Gu, Yun-Bo Chen, Jian Yao, Lan-Juan Li PII: DOI: Reference:

S1874-3919(16)30398-0 doi: 10.1016/j.jprot.2016.08.021 JPROT 2667

To appear in:

Journal of Proteomics

Received date: Revised date: Accepted date:

7 March 2016 24 June 2016 25 August 2016

Please cite this article as: Lv Long-Xian, Yan Ren, Shi Hai-Yan, Shi Ding, Fang DaiQiong, Jiang Hui-Yong, Wu Wen-Rui, Guo Fei-Fei, Jiang Xia-Wei, Gu Si-Lan, Chen Yun-Bo, Yao Jian, Li Lan-Juan, Integrated transcriptomic and proteomic analysis of the bile stress response in probiotic Lactobacillus salivarius LI01, Journal of Proteomics (2016), doi: 10.1016/j.jprot.2016.08.021

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Integrated Transcriptomic and Proteomic Analysis of the Bile Stress Response in

RI P

T

Probiotic Lactobacillus salivarius LI01

Long-Xian Lv #, Ren Yan #, Hai-Yan Shi #, Ding Shi #, Dai-Qiong Fang #, Hui-Yong

SC

Jiang #, Wen-Rui Wu, Fei-Fei Guo, Xia-Wei Jiang, Si-Lan Gu, Yun-Bo Chen, Jian Yao,

MA

NU

and Lan-Juan Li*

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases,

PT ED

Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, College of Medicine, Zhejiang University, 310003 Hangzhou,

CE

China.

AC

# Long-Xian Lv, Ren Yan, Hai-Yan Shi, Ding Shi, Dai-Qiong Fang, and Hui-Yong Jiang contributed equally to this work.

* Correspondence should be addressed to Lan-Juan Li ([email protected]); Tel.: +86-571-8723-6458; Fax: +86-571-8723-6459.

1

ACCEPTED MANUSCRIPT

ABBREVIATIONS

T

LGG: Lactobacillus rhamnosus GG; DGE: digital gene expression; iTRAQ: isobaric

RI P

tags for relative and absolute quantitation; RT: room temperature; RPKM: reads per kilobase of exon model per million mapped reads; DEGs: differentially expressed genes; false

discovery

rate;

GO:

Gene

SC

FDR:

sulfonate;

CHAPS: PMSF:

NU

3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane

Ontology;

MA

phenylmethanesulfonyl fluoride; EDTA: ethylene diamine tetraacetic acid; DTT: dithiothreitol; IAM: iodacetamide; TEAB: tetraethylammonium bromide; SCX: strong

PT ED

cation exchange; HPLC: high-performance liquid chromatography; ACN: acetonitrile; FA: formic acid; FWHM: full width at half maxima; TOF: time of flight; MS: mass spectrometry; IDA: information dependent acquisition; COG: Cluster of Orthologous Groups; BSH: bile

CE

salt hydrolase; TCRS: two-component regulatory system; RP: ribosomal protein; LSU:

AC

large subunit; SSU: small subunit; rRNA: ribosomal RNA; USP: universal stress protein; HSP: heat shock protein; LTA: lipoteichoic acid; FAs: fatty acids; SFAs: saturated fatty acids; UFAs: unsaturated fatty acids; MDR: multidrug resistance; MFS: major facilitator superfamily; ABC: ATP-binding cassette; AAAs: aromatic amino acids; BCAAs: branched-chain amino acids; DAG: diacylglycerol; UMP: uridine monophosphate; GMP: guanosine monophosphate; ATP: adenosine triphosphate; HE: hepatic encephalopathy.

2

ACCEPTED MANUSCRIPT

ABSTRACT

T

Lactobacillus salivarius LI01, isolated from healthy humans, has demonstrated

RI P

probiotic properties in the prevention and treatment of liver failure. Tolerance to bile stress is crucial to allow lactobacilli to survive in the gastrointestinal tract and exert their

SC

benefits. In this work, we used a Digital Gene Expression transcriptomic and iTRAQ

NU

LC-MS/MS proteomic approach to examine the characteristics of LI01 in response to bile

MA

stress. Using culture medium with or without 0.15% ox bile, 591 differentially transcribed genes and 347 differentially expressed proteins were detected in LI01. Overall, we found

PT ED

the bile resistance of LI01 to be based on a highly remodeled cell envelope and a reinforced bile efflux system rather than on the activity of bile salt hydrolases. Additionally, some differentially expressed genes related to regulatory systems, the general stress

CE

response and central metabolism processes, also play roles in stress sensing,

AC

bile-induced damage prevention and energy efficiency. Moreover, bile salts appear to enhance proteolysis and amino acid uptake (especially aromatic amino acids) by LI01, which may support the liver protection properties of this strain. Altogether, this study establishes a model of global response mechanisms to bile stress in L. salivarius LI01. KEYWORDS: transcriptomics; proteomics; Lactobacillus salivarius; probiotic; bile stress response; iTRAQ.

3

ACCEPTED MANUSCRIPT

1. INTRODUCTION

T

The genus Lactobacillus contains a large group of lactic acid bacteria that are widely

RI P

used in food product fermentation. Some well-characterized Lactobacillus strains, especially L. acidophilus, L. casei, L. johnsonii, L. paracasei, L. plantarum, and L.

SC

rhamnosus, have proven health-beneficial effects [1-4]. Recently, several strains of L.

NU

salivarius, a promising probiotic species, have gained increasing attention because they

MA

exhibit good probiotic properties such as antimicrobial activity [5, 6], inflammation modulation [7, 8], and even neoplastic lesions reduction [9].

PT ED

When utilized as orally administered probiotics, lactobacilli are exposed to conditions of stress in the gastrointestinal tract. Bile in particular is a major challenge and is toxic to bacterial cells because it can cause membrane damage and protein misfolding

CE

or denaturation and can induce RNA and DNA damage, low pH conditions, and oxidative

AC

and osmotic stresses [10]. Consequently, the ability to survive and colonize a host during bile exposure is critical and commonly used as a criterion when evaluating potential probiotic strains. Lactobacillus strains vary in their resistance to bile [11]. And a number of transcriptomic and proteomic studies have revealed that bile-resistant lactobacilli possess complex regulatory networks to cope with bile exposure. For example, the bile stress response of L. casei BL23 involves up to 52 proteins and the transcription of 67 genes [12]. Analogously, bile exposure was found to result in the up- or down-regulation of 316 genes and 42 proteins in L. rhamnosus GG (LGG) [13], whereas 215 proteins were found to be differentially expressed in L. johnsonii PF01 [14]. Furthermore, a 4

ACCEPTED MANUSCRIPT

comparative proteomic study conducted on six L. casei strains revealed significant

T

differences in their responses to bile stress, with alterations in expression for up to 80%

RI P

of proteins, depending on the strain [15]. These findings suggest that the bile response of one Lactobacillus strain is of limited use when attempting to explain that of another

SC

strain.

NU

In a previous study, we reported that L. salivarius strain LI01 not only exhibits

MA

antibacterial and antifungal properties but also significantly improves acute liver injury induced by d-galactosamine in rats [16]. Strain LI01 also displayed a bile-tolerance trait,

PT ED

whereby it could survive in MRS broth containing 0.4% ox bile. However, the cellular response mechanism has not yet been explored in any strain of L. salivarius. Digital Gene Expression (DGE) is a tag-based transcriptome deep-sequencing approach that is

CE

mainly used to study comparative gene expression at a genome-wide level. This

AC

approach permits direct transcript profiling without potential bias, thus allowing for a more sensitive and accurate profile of the transcriptome that more closely resembles the biology of the cell [17]. As an efficient, accurate and widely accepted technique in proteomics research, Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) enables the quantification and comparison of protein levels directly from samples [18, 19]. In this study, we investigated the bile stress response and resistance mechanisms of L. salivarius LI01 based on profile analysis by combining RNA-Sequencing with iTRAQ LC-MS/MS. To the best of our knowledge, this work represents the first combined transcriptomic and proteomic analysis of the bile stress response in L. salivarius. 5

ACCEPTED MANUSCRIPT

2. MATERIALS AND METHODS

T

2.1 Growth conditions and bile treatment

RI P

L. salivarius LI01 was grown anaerobically at 37°C in MRS broth supplemented with 0.05% (w/v) L-cysteine (MRSc); three biological replicates were performed. After 12 h of

SC

cultivation, the cultures were used to inoculate 50 mL of MRSc broth (1% (v/v) inoculum)

NU

and monitored spectrophotometrically at 600 nm. The cells were grown to

MA

mid-logarithmic phase (OD600 of 0.6), and then challenged with ox bile solution (Sigma, St. Louis, US) to a final concentration of 0.15%. For transcriptomic analysis, cell samples

PT ED

were harvested before (time point 0 min) and after (time points 10, 30, 120 min) the addition of bile; samples for proteomics were collected at 0 and 60 min. Samples for RNA isolation and protein preparation were centrifuged (6,000 × g, 4°C for 5 min and 5000 × g,

CE

4°C for 10 min, respectively) and washed twice with chilled phosphate-buffered saline

AC

(PBS). All cell pellets were then stored at -80°C until use. 2.2 Transcriptomic analysis RNA isolation ‒ Total RNA at each time point was extracted using TRIzol Max Bacterial RNA Isolation Kit (Invitrogen, Waltham, US) following the manufacturer’s procedure. Cell samples from three independent biological replicates (up to 1 × 108 cells) were mixed, resuspended and lysed with 200 μL pre-heated Max Bacterial Enhancement Reagent at 95°C for 4 min. The lysate was mixed with 1 mL TRIzol reagent at room temperature (RT) for 5 min, and the mixture was extracted with 200 μL cold chloroform with vortexing for 15 s. After incubation for 3 min at RT, the phases were separated by 6

ACCEPTED MANUSCRIPT centrifugation at 12,000 × g, 4°C for 15 min. The aqueous phase was mixed with 500 μL

T

cold isopropanol and placed for 10 min at RT to precipitate total RNA. After centrifugation

RI P

at 15,000 × g, 4°C for 10 min, the precipitate was washed with 1 mL 75% ethanol and dissolved in 50 μL RNase-free H2O.

SC

mRNA library construction and sequencing ‒ The quantity and purity of total

NU

RNA were assessed using Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent,

MA

Santa Clara, US), with an RNA integrity number (RIN) >7.0. Approximately 5 μg of total RNA were used to deplete ribosomal RNA according to the Ribo-Zero Gold rRNA

PT ED

Removal Kit manual (Illumina, San Diego, US). Following purification, the fraction was fragmented into small pieces using divalent cations under elevated temperature. The mRNA was incubated with Fragmentation Buffer (Illumina) in a pre-heated tube for 5 min

CE

at 94°C. And the cleaved RNA fragments were reverse-transcribed to create the final

AC

cDNA library, in accordance with the mRNA-Seq sample preparation kit protocol (Illumina). The average insert size for the single-end libraries was 250 bp (±50 bp). We then performed single-read sequencing using an Illumina Hiseq 2500 following the vendor's recommended protocol to obtain raw read of 50 nt in length. Data processing and analysis ‒ Prior to alignment, the raw reads were filtered to obtain clean reads with 36 nt in length by removing the adaptor sequences, low-quality sequences with more than 2 N nucleotides (i.e., nucleotides that could not be sequenced), and low-quality sequences with Q-values ≤30. The clean reads were further evaluated based on the distribution of the phred-like quality score at each cycle 7

ACCEPTED MANUSCRIPT

and then subjected to quality assessment, including sequencing saturation and read

T

classification, expression and distribution analyses. All clean reads were mapped to

RI P

transcript sequences using Bowtie, with only 1 bp mismatch allowed. For monitoring mapping events on both strands, both sense and complementary antisense sequences

SC

were included in the data collection. The number of perfect clean reads corresponding to

NU

each gene was calculated and normalized to the number of reads per kilobase of exon

MA

model per million mapped reads (RPKM). For mRNA identification, genome information was downloaded from NCBI (NCBI reference sequence: GCA 000008925.1). The DEG

PT ED

analysis in this study is depicted using the software RStudio 0.99.467 (R version 3.3.0) with package “DESeq” [20]. P values were corrected using the Benjamini-Hochberg false discovery rate (FDR) adjustment [21]. Based on the obtained expression levels,

CE

significant differentially expressed genes (DEGs) among different samples were

AC

identified with a FDR-adjusted p-value ≤ 0.05 and a change of at least 2-fold. Gene Ontology (GO) analysis was conducted for the functional classification of DEGs, and pathway analysis was carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG). The results are illustrated using RStudio with packages “ggplot2” and “pheatmap”. 2.3 Proteomic analysis Protein preparation and digestion ‒ Protein extracts were obtained from three independent biological replicates for each time point (0 and 60 min). Homogenized cell samples were ground into powder in liquid nitrogen and extracted with lysis buffer (7 M 8

ACCEPTED MANUSCRIPT

urea, 2 M thiourea, 4% CHAPS, 40 mM Tris-HCl, pH 8.5) containing 1 mM PMSF and 2

T

mM EDTA (final concentration). After 5 min, DTT was added to a final concentration of 10

RI P

mM. After mixing, the suspension was sonicated at 0°C, 200 W for 15 min and then centrifuged at 25,000 × g, 4°C for 20 min. The supernatant was mixed well with chilled

SC

acetone (1:5, v/v) containing 10 mM DTT and incubated at -20°C overnight. After

NU

centrifugation at 25,000 × g, 4°C, the precipitate was washed three times with chilled

MA

acetone. To reduce disulfide bonds in the proteins, the precipitate was dissolved in lysis buffer with 10 mM DTT and incubated at 56°C for 1 h. To block the cysteines, 55 mM IAM

PT ED

was added (final concentration) and incubated for 1 h in the dark. The supernatant was mixed well with 5-fold volume of chilled acetone and incubated at -20°C overnight for protein precipitation. After centrifugation at 25,000 × g, 4°C, the precipitate was

CE

dissolved in 0.5 M TEAB and sonicated at 0°C, 200 W for 15 min. Finally, the proteins

AC

were collected by centrifugation at 30,000 × g, 4°C for 15 min. For digestion, 100 μg of total protein quantified by the Bradford method was incubated overnight with Trypsin Gold (Promega, Fitchburg, US; 1:30, w/w, added at 0 and 4 h) at 37°C. iTRAQ labeling and SCX fractionation ‒ After trypsin digestion, the peptides were dried by vacuum centrifugation, and iTRAQ labeling was performed using an iTRAQ reagent 8-plex kit (Applied Biosystems, Waltham, US) according to the manufacturer’s protocol. Briefly, one unit of iTRAQ reagent was thawed and reconstituted in 24 μL isopropanol. Two samples were labeled with iTRAQ tags as Sample 0 min (118- tag) and Sample 60 min (119- tag). The peptides were labeled with respective isobaric tags, 9

ACCEPTED MANUSCRIPT

incubated at RT for 2 h and vacuum centrifuged to dryness. For the SCX HPLC

T

fractionation procedure, iTRAQ-labeled samples were loaded onto an Ultremex SCX

RI P

column (4.6 × 250 mm, 5 μm particle size, Phenomenex, Torrance, US) and eluted by the following step linear elution program: 10 min equilibration in Buffer A (25 mM

SC

NaH2PO4 in 25% ACN, pH 2.7), 7 min fast elution in 5% of Buffer B (25 mM NaH 2PO4, 1

NU

M KCl in 25% ACN, pH 2.7), 20 min linear elution from 5-60% of B, 2 min washing elution

MA

from 60-100% of B. The system was then maintained at 100% B for 1 min before equilibrating with A for 10 min prior to the next injection. All fractionation procedures were

PT ED

manipulated using an LC-20AB HPLC Pump system (Shimadzu, Tokyo, Japan) at a flow rate of 1.0 mL/min; the peptides were monitored by absorbance measurement at 214 nm. The eluted peptides were collected and pooled into 20 fractions, desalted with a Strata X

CE

C18 column (Phenomenex) and vacuum-dried.

AC

LC-ESI-MS/MS analysis ‒ Each fraction was resuspended in Solvent A (95% water/5% ACN, 0.1% FA) and centrifuged at 20,000 × g for 10 min; the final concentration of peptide was approximately 0.5 μg/μL, on average. The peptides were separated using a C18 trap column (350 μm × 0.5 mm, 3 μm particle size, 120A, AB SCIEX, Framingham, US) and a C18 analytical column (75 μm × 150 mm, 3 μm particle size, 120A, Welch Materials, Annapolis, US) with an LC-20AD nano HPLC (Shimadzu) equipped with an autosampler. First, 5 μL supernatant was loaded onto the trap column for 4 min at a flow rate of 8 μL/min; the peptides were then eluted onto the analytical column (packed in-house) at a flow rate of 300 nL/min. The elution gradient was 5% 10

ACCEPTED MANUSCRIPT

Solvent B (5% water/95% ACN, 0.1% FA) for 5 min, 5-35% B for 35 min, 35-60% B for 5

T

min, 60-80% B for 2 min, maintenance at 80% B for 4 min, and finally a return to 5% B in

RI P

1 min. Data acquisition was performed with a Triple TOF 5600 System (AB SCIEX) equipped with a Nanospray Ⅲ source (AB SCIEX) and a pulled quartz tip (New

SC

Objectives, Woburn, US) as the emitter. The mass spectrometer was operated with a

NU

mass resolution of ≥ 30,000 FWHM for the TOF-MS scan to detect precursor ions. For

MA

IDA, survey scans were acquired in 250 ms, and as many as 30 production scans exceeding 120 counts per second with a charge-state of 2+ to 5+ were collected. A

PT ED

sweeping collision energy setting of 35 ± 5 eV coupled with iTRAQ adjust rolling collision energy was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set at 1/2 of peak width (15 s), and the precursor was then refreshed off

CE

the exclusion list.

AC

Proteomic data processing and analysis ‒ The raw data files were converted into MGF format using Proteome Discoverer 1.2 (Thermo Fisher, Scientific, Waltham, US). Protein identification and quantification were performed using Mascot 2.3.02 (Matrix Science,

Boston,

US)

against

the

Ensemble

database

(ftp://ftp.ensemblgenomes.org/pub/bacteria/release-30/fasta/bacteria_18_collection/lact obacillus_salivarius_ucc118/pep/). For protein identification, a mass tolerance of 0.05 Da was permitted for intact peptide masses and 0.1 Da for fragmented masses, with allowance for one missed cleavage upon trypsin digest. Several parameters in Mascot were set for peptide searching, including Gln→pyro-Glu (N-term Q), oxidation (M) and 11

ACCEPTED MANUSCRIPT

deamidated (NQ) as potential variable modifications and carbamidomethyl (C), iTRAQ

T

8-plex (N-term) and iTRAQ 8-plex (K) as fixed modifications. A protein containing at least

RI P

two unique peptides was required for quantitation. The quantitative protein ratios were weighted and normalized by the median ratio in Mascot. The peptide FDR was estimated

SC

by searching against a decoy database in Mascot. Proteins with at least 1.2-fold change

NU

between two samples at FDR-adjusted p ≤ 0.05 were considered to be significant

analysis

was

conducted

according

AC

CE

PT ED

(http://www.ncbi.nlm.nih.gov/COG/).

MA

differentially expressed proteins. COG (Cluster of Orthologous Groups of proteins)

12

to

the

NCBI

database

ACCEPTED MANUSCRIPT

3. RESULTS

T

3.1 Transcriptomic data for L. salivarius LI01 under bile stress

RI P

The effect of bile stress on the LI01 transcriptome was first investigated using DGE. Differentially transcribed genes at the mRNA level were evaluated by comparing

SC

bile-challenged samples harvested at three time points (10, 30, and 120 min) with the

NU

0-min time point (set as the control). The mapping data analysis revealed that of the total 2014 coding genes covered in the genome, 1782 genes at 10 min, 1780 at 30 min, 1786

MA

at 120 min and 1789 at 0 min were transcribed. After filtering (≥ 2-fold change, adjusted p

PT ED

≤ 0.05), 591 genes were detected as being significantly differentially expressed (342 upand 249 down-regulated) under the bile stress condition (supplemental Table S-1): including 449 at 10 min (256 up- and 193 down-regulated), 408 at 30 min (203 up- and

CE

205 down-regulated), and 417 at 120 min (263 up- and 154 down-regulated) (Fig. 1A).

AC

Among the 342 up-regulated genes, most could be observed at 10 min or 120 min, with only one gene (glmM, LSL_1176) being unique at 30 min (Fig. 1A). However, among the 249 down-regulated genes, 205 were observed at 30 min, with 10 being unique at 120 min (Fig. 1A). Overall, 115 genes were consistently down-regulated, and 164 consistently up-regulated. Table 1 lists the genes of LI01 highly regulated (≥ 4-fold change, adjusted p ≤ 0.05) at the transcriptional level under bile stress. Among these, LSL_1885, encoding a hypothetical protein, was the most up-regulated (12.78-fold at 10 min, 13.28-fold at 30 min, 15.63-fold at 120 min); the protease-encoding gene clpL (LSL_0059) was the most down-regulated (57.97-fold at 10 min, 44.23-fold at 30 min, 13

ACCEPTED MANUSCRIPT

and 17.24-fold at 120 min). Furthermore, we classified these genes into different

T

categories using GO (Fig. 2) and KEGG analyses (Fig. 3). The differentially expressed

RI P

genes are involved in such categories as stress response, glycolysis and transport and such pathways as carbohydrate metabolism and amino acid biosynthesis.

SC

3.2 Proteomic data for L. salivarius LI01 under bile stress

NU

Because proteins represent the actual functional molecules in a cell, we conducted

MA

iTRAQ LC-MS/MS to investigate the responses of strain LI01 to bile stress at the proteomic level at 0 min (set as control) and at 60 min after the addition of bile. In total,

PT ED

1260 proteins were identified and quantified. After data filtering (≥ 1.2-fold change, adjusted p ≤ 0.05), 347 significant differentially expressed proteins (214 up- and 133 down-regulated) were observed (supplemental Table S-2 and Fig. 1B). Of these, a

CE

hypothetical protein (LSL_1100) was the most highly bile induced, increasing by nearly

AC

18-fold (Table 2). In contrast, a ribosomal protein (RpsU, LSL_0700) and a stress-responsive

transcriptional

regulator

(PspC,

LSL_0108)

were

the

most

down-regulated, decreasing nearly 3-fold (Table 2). As observed in the GO (Fig. 2) and KEGG analyses (Fig. 3), proteins with altered abundance under bile stress are largely involved in DNA modification, regulation of gene expression and cellular component disassembly and metabolic processes. Overall, 114 genes were regulated at both the transcriptional and translational levels under bile stress (Fig. 1C). Among these, 50 were altered with matching results (48 were up/up-regulated and 2 down/down-regulated); of these 48 genes, LSL_1643, encoding a 14

ACCEPTED MANUSCRIPT

nucleoside deoxyribosyltransferase was the only gene up-regulated by more than 3-fold

T

at all four time points and both levels (3.91 to 5.12-fold increase in transcription and

RI P

3.60-fold increase in protein abundance (Table 2 and Fig. 4F). In contrast, both treB (LSL_1512) and treR (LSL_1513) were down-regulated nearly 2 to 3 folds at the

SC

transcriptional level and showed an approximately 1.5-fold decrease in protein

NU

abundance (Fig. 4F). Interestingly, these 3 genes are all involved in metabolic

MA

processes.

3.3 Bile stress response of L. salivarius LI01

PT ED

It is generally considered that the activities of bile salts hydrolases (BSHs) in lactobacilli contribute to bile tolerance via detoxification of conjugated bile salts [22]. When strain LI01 was exposed to 0.15% bile, transcription of a BSH gene (LSL_1801)

CE

was found to be up-regulated at all three time points (3.14-fold at 10 min, 2.95-fold at 30

AC

min, and 3.04-fold at 120 min), even though corresponding changes were not detected at the translational level. This is in line with a previous report that some Lactobacillus salivarius strains are non-BSH-producing bacteria [23]. Complementarily, the expression of other genes involved in general stress responses and cell surface molecules were strongly up- or down-regulated to cope with bile stress (Fig. 4). Sensing systems ‒ As the most commonly used stimulus-response coupling systems in bacteria [24], two-component regulatory systems (TCRSs) are highly important for bile salt sensing in LI01. The TCRS YycG-YycF (LSL_0036 and LSL_0035), which enhances the biosynthesis of murein and exopolysaccharide, cell division and 15

ACCEPTED MANUSCRIPT

biofilm formation [25, 26], was up-regulated in strain LI01 under bile salt stress (Fig. 4A),

of

genes

(LSL_0079

and

LSL_1581)

encoding

MerR

family

RI P

up-regulation

T

thus helping the cells to maintain surface homeostasis. Moreover, we also observed

metalloregulators, which alleviate toxicity caused by an excess of metal ions by

SC

triggering the expression of specific efflux or detoxification factors [27], in both the

NU

transcriptome and proteome of LI01 under bile stress.

MA

Cell surface ‒ The cell surface is the first line of defense against bile stress for LI01. First, gene expression related to peptidoglycan autolysis (LSL_1310, LSL_1034 and

PT ED

LSL_1371) and peptidoglycan biosynthesis, including murI (LSL_0836), dacC (LSL_1532), and murE (LSL_0588), varied strongly (Fig. 4B and Fig. 5). This result indicates that the peptidoglycan layer of LI01 was restructured under bile stress. Second,

genes

related

to

phosphatidate

(1,

2-diacyl-sn-glycerol-3-phosphate)

AC

example,

CE

biosynthesis of lipids and fatty acids (FAs) was altered to cope with bile influx. For

biosynthesis (LSL_0028, LSL_1601, LSL_1710, LSL_0110, LSL_0508 and LSL_0896) were generally up-regulated (Fig. 4B), enhancing the hydrophobicity of the cell surface. In type II FA synthesis, down-regulation of FabB (LSL_0455), AccC (LSL_0458), AccD (LSL_0459) and FabD (LSL_0453) may lead to a decrease in total FA synthesis. Up-regulation of fabA (LSL_0449) and fabG (LSL_1133) and down-regulation of fabK (LSL_1900) are thought to modulate the FA composition to achieve a higher ratio of unsaturated/saturated fatty acids (UFAs/SFAs) (Fig. 4B and Fig. 6). Third, more than 30 genes annotated as “hypothetical membrane spanning/associated protein” were 16

ACCEPTED MANUSCRIPT

up-regulated at the transcriptional level when LI01 was exposed to bile (Table S-1).

T

Because bile can denature and dissociate integral membrane proteins, up-regulation of

RI P

such membrane protein-related genes may be helpful for tolerating bile exposure. For example, the LSL_1575 gene contains a sequence of putative metal-dependent

SC

membrane proteases belonging to the CAAX family; therefore, up-regulated expression

NU

potentially improves protein and/or peptide modification and secretion [28]. Indeed,

MA

transcription of an annotated CAAX protease gene, LSL_0126, was up-regulated 3.90 to 5.76-fold. Finally, bile stress also induced up-regulation of lytR (LSL_0180 and

PT ED

LSL_1477), which belongs to the LCP (LytR-CpsA-Psr) family and is related to drug resistance, biofilm formation, and stress tolerance in most gram-positive bacteria. This increased expression may help to promote attachment of secondary cell wall polymers

AC

cell wall.

CE

with discrete linkage units to peptidoglycan [29-31], thus maintaining the structure of the

Transmembrane transport ‒ Transcriptomic analysis revealed modulation of 44 annotated genes encoding transporters (as well as permeases, pumps, antiporters) when LI01 was exposed to bile (Table S-1). Remarkably, gene expression of a multidrug resistance (MDR) efflux system in LI01 was selectively promoted: expression of genes encoding secondary transporters, such as major facilitator superfamily (MFS) permeases (LSL_1515 and LSL_1544), a Na+-driven multidrug efflux pump (LSL_0052) and several related antiporters (napA, LSL_1533 and nhaC, LSL_0855), was up-regulated (Fig. 4C). In contrast, only two ATP-binding cassette (ABC) MDR 17

ACCEPTED MANUSCRIPT

transporter-related genes (LSL_1616 and LSL_0032) were up-regulated at the

T

transcriptional level; even the translation of two ATPase components (LSL_0724 and

RI P

LSL_1805) of the ABC-type MDR transporter was down-regulated (Fig. 4C). Similarly, expression of secondary transporters was preferably enhanced with regard to amino

SC

acid transport as well: only four (glnP (LSL_0062), azlC (LSL_1644), azlD (LSL_1645)

NU

and LSL_1541) of the eleven up-regulated genes are of the ABC-type, whereas the

MA

remaining seven are permeases (LSL_0168, LSL_0433, potE, etc.) (Fig. 4C), which are driven by favorable ion gradients. These findings suggest a low energy-consumption

PT ED

strategy in LI01 under bile stress.

Ribosomal formation and function ‒ In this study, bile stress appeared to significantly regulate ribosomal formation and functions. On the one hand, expression of

CE

nearly all ribosomal protein (RP)-related genes, including 32 of 34 encoding the 50S

AC

large subunit (LSU) and 21 of 22 encoding the 30S small subunit (SSU), were down-regulated (Table S-2); this finding indicates that RP synthesis was repressed in LI01 under bile stress. On the other hand, increases in several factors involved in promoting protein biosynthesis were observed in LI01 when exposed to bile (Fig. 4D). Ribosome-associated proteins are key factors of newly synthesized proteins [32], and the relative abundance of IF-1 (InfA, LSL_1413) and EF-P (Efp, LSL_0530), which are required for translation initiation and elongation, were both increased. IF-1 enhances the binding of IF-2 and IF-3 to the 30S subunit, to which N-formylmethionyl-tRNA (fMet) subsequently binds, and contributes to mRNA selection, thus yielding the 30S 18

ACCEPTED MANUSCRIPT

pre-initiation complex (PIC) [33]. The enrichment of EF-P may help to stimulate the

T

peptidyltransferase activity of 70S prokaryotic ribosomes and to enhance the synthesis

RI P

of certain dipeptides initiated by N-formylmethionine, thus promoting protein synthesis and cell viability [34].

SC

General stress response ‒ The expression of many stress-related genes was

NU

altered in LI01 to cope with bile stress. First, transcription of genes encoding chaperones,

MA

including the dnaK operon (hrcA-grpE-dnaK-dnaJ, LSL_0576-0579) and groE operon (groEL-groES, LSL_1211 and 1212), and proteases, such as clpL, clpE and clpP, were

PT ED

generally down-regulated; however, the corresponding stress proteins were overall enriched (Fig. 4E). This situation is not rare in bacteria in response to specific environments, and the reasons will be discussed in the next section. In brief, increases in

CE

molecular chaperones are helpful for preventing newly synthesized polypeptide chains

AC

and assembled subunits from aggregating and misfolding into nonfunctional structures due to stress [35]. Simultaneously, increased Clp protease abundance will contribute to protein quality control [36], together with molecular chaperones, during stress conditions [37]. Second, genes encoding universal stress proteins (Usp, LSL_0060, _1081, _1120 and _1317), a ferric uptake regulator (Fur, LSL1367) and a heat shock protein (HtpX, LSL_0215) were all highly expressed in LI01 under bile stress (Fig. 4E). Such up-regulation may be helpful for protecting cells against stress conditions and toxicants [38-40] as well as controlling protein quality [41, 42]. Metabolic processes ‒ Carbohydrate utilization by LI01 was more efficient in the 19

ACCEPTED MANUSCRIPT

presence of bile. Indeed, transcription of genes encoding a carbohydrate metabolism

T

regulator (glpR, LSL_0163) and a sugar transporter (LSL_1282) was markedly

RI P

decreased (6.47 to 7.60 folds and 4.01 to 4.29-fold, respectively). GlpR is reported to be an indispensable factor in carbohydrate and glycerol metabolism: it represses

SC

expression of fructose and glucose metabolic enzymes and promotes glycerol utilization

NU

[43, 44]. Moreover, genes involved in metabolizing of fructose (fruA, LSL_0165; fruK,

MA

LSL_0164), glucose (galM, LSL_1281; nagC, LSL_1937), maltose (LSL_1279; malR, LSL_1283; mapA, LSL_1280), sucrose (scrR, LSL_0064; scrA, LSL_0066), trehalose

PT ED

(treR, LSL_1513; treB, LSL_1512; treC, LSL_1514), mannose (manX, LSL_0086, LSL_0654; LSL_0087, LSL_0088; LSL_655, LSL_656), mannitol (LSL_1619; mtlA, LSL_1620; mtlD, LSL_1618), sorbose/sorbitol (LSL_1531, LSL_1143, LSL_1894), and

CE

galactose (galM, LSL_1281) as well as glycolysis (LSL_1903; gpmA, LSL_1255,

AC

LSL_1498; gpmB, LSL_0125, LSL_1511) were also regulated (Fig. 4F and Fig. 5). Therefore, under bile exposure, LI01 primarily metabolized fructose and sorbose/sorbitol rather than other carbohydrates, such as sucrose, trehalose, and galactose. Hydrophobic amino acids, such as aromatic amino acids (AAAs, phenylalanine, tyrosine and tryptophan) and branched-chain amino acids (BCAAs, isoleucine, leucine and valine), can protect proteins against bile attack by creating hydrophobic areas [45]. In this study, a clear up-regulation in pheST (phenylalanyl-tRNA synthetase alpha/beta chain, LSL_0813 and LSL_0814), tyrS (tyrosyl-tRNA synthetase, LSL_1366), ileS (isoleucyl-tRNA synthetase, LSL_1042) and leuS (leucyl-tRNA synthetase, LSL_0442) 20

ACCEPTED MANUSCRIPT

transcription was observed, indicating that more hydrophobic amino acids are needed

T

during peptide and protein synthesis in LI01 under bile stress. According to our results,

RI P

the gene LSL_0186, encoding acetolactate synthase, which catalyzes the first step in BCAA synthesis, was up-regulated at both the transcriptional and translational levels.

SC

However, transcription of aroD (LSL_1796) and aroE (LSL_1795), encoding

NU

3-dehydroquinate dehydratase and shikimate 5-dehydrogenase, which are involved in

AAAs from other pathways.

MA

AAA synthesis, were down-regulated (Fig. 4F), suggesting that LI01 cells may obtain

PT ED

Regarding pyrimidine metabolism, clear down-regulation of related genes was observed at the transcriptional level, including those all genes encoding enzymes functioning in de novo biosynthesis of UMP (carAB and pyrBCDEF), the bifunctional

CE

regulator pyrR (LSL_0827) and the uracil permease gene uraA (LSL_0828) (Fig. 4F).

AC

Because pyrimidine derivatives are toxic under conditions of oxidative stress, down-regulation of pyrimidine biosynthesis is a common response to stress conditions, including bile exposure, in lactobacilli [13, 46]. In contrast, opposing regulation was observed for genes involved in purine nucleotide metabolism; for example, genes related to GMP, guanine and adenine biosynthesis (e.g., guaA, LSL_1452; guaB, LSL_1537; uRH, LSL_0819, 1528; hpt, LSL_1354) were generally up-regulated (Fig. 4F). Furthermore, a large number of ATP-dependent proteins were induced in LI01 in the presence of bile, thus the increase in adenine may contribute to the accumulation of ATP. Overall, as shown in Fig. 7, the bile-induced defense mechanism of LI01 appears to 21

ACCEPTED MANUSCRIPT

be primarily based on a highly remodeled cell envelope and a reinforced bile efflux

T

system. Regulatory systems, the general stress response and metabolic processes also

RI P

play important roles in sensing stress and reducing damage as well as energy efficiency

AC

CE

PT ED

MA

NU

SC

in strain LI01.

22

ACCEPTED MANUSCRIPT

4. DISCUSSION

T

Bile in the small intestine is a major stress for bacteria after ingestion by a host,

RI P

and bacteria such as lactobacilli have developed different strategies to protect against bile stress. Alterations in the microbes will not only determine their survival but also affect

SC

their interaction with the host. L. salivarius, which harbors the largest plasmid with many

NU

functional genes among lactobacilli, is currently one of the most popular probiotics;

MA

however, its performance under bile stress remains unclear. In this study, we reveal for the first time the bile-induced defense mechanism of L. salivarius LI01, a probiotic with

PT ED

potential application for the prevention and treatment of liver failure. The defense mechanism is primarily based on a highly remodeled cell envelope and a strengthened bile efflux system. Altered regulatory systems, general stress response systems as well

CE

as metabolic processes also play important roles in bile stress sensing, damage

AC

reduction and energy efficiency. Furthermore, we found some alterations such as the production and membrane transport of branched-chain and aromatic amino acids, which would be helpful to liver protection. Some of the observed changes in gene expression would be expected to enhance the cell surface hydrophobicity of LI01 against bile stress. For instance, genes glpF (LSL_1630)

and

glpT

(LSL_0048),

encoding

glycerol

uptake-related

facilitators/transporters and aquaporins, were strongly down-regulated (up to 22.41-fold), whereas genes related to the biosynthesis of phosphatidate and phosphatidate-based diacylglycerol (DAG), which containing hydrophobic diglyceride structure, were generally 23

ACCEPTED MANUSCRIPT

up-regulated (e.g., cardiolipin synthetase, cls, LSL_0673). Moreover, the demand for

T

hydrophobic amino acids (BCAAs and AAAs) was found to be increased under bile

RI P

stress. Accompanying the down-regulated expression of genes involved in AAA biosynthesis, genes related to amino acid transporters and proteolysis (e.g.,

SC

oligoendopeptidase F, LSL_0025; pepQ, LSL_0419; pepD, LSL_0848) (Fig. 4F) were

NU

more highly expressed in LI01 under bile stress, indicating that LI01 may acquire certain

MA

hydrophobic amino acids, such as AAAs, from the external environment. The decrease in BCAAs and the increase in AAAs in humans play a central role in the pathogenesis of

PT ED

liver diseases; therefore, the BCAA/AAA ratio is a good index of liver impairment [47]. Thus, the potential of LI01 to remove AAAs from the external environment (host) under bile stress may contribute to its good performance in the prevention and treatment of

CE

liver diseases [16].

AC

The autolysis-resynthesis process of the cell wall, which mainly consists of peptidoglycan and lipoteichoic acid (LTA), was also found to be important for the bile resistance of LI01. On the one hand, to promote peptidoglycan autolysis and catabolism, genes encoding “peptidoglycan binding lysin motif (LysM) domain” proteins (LSL_1034 and LSL_1371) and muramidase (LSL_1310) were more strongly expressed. On the other hand, MurI (LSL_0836) and DacC (LSL_1532) of peptidoglycan biosynthesis were up-regulated to increase total peptidoglycan production, and MurE (LSL_0588) was down-regulated to decrease the proportion of peptidoglycan cross-linking by meso-diaminopimeloyl-meso-diaminopimelic acid peptide bridges (Fig. 5). This type of 24

ACCEPTED MANUSCRIPT

peptidoglycan remodeling was helpful for reducing the uptake of bile salts, which has

T

also been found in S. enterica exposed to bile [48]. Moreover, expression of genes (rfaG

RI P

and alr) related to lipoteichoic acid (LTA) biosynthesis was down-regulated; a similar tendency was observed for the dltABCD operon, which is responsible for LTA

SC

D-alanylation. Because D-alanylated LTAs can inhibit cell wall autolysis in lactobacilli by

NU

altering surface charge and affecting the abundance of bivalent cations during autolysis

remodeling.

In

addition,

(GlcNAc)

amino and

sugar

metabolism

N-acetylmuramate

pathways (MurNAc)

from to

PT ED

N-acetylglucosamine

two

MA

[49-51], their down-regulation will contribute to the autolysis process and cell wall

β-D-fructose-6-phosphate were promoted (Fig. 5). As GlcNAc and MurNAc are the main components of peptidoglycan, this finding further proves our suggestion that autolysis

AC

stress.

CE

and restructuring of the peptidoglycan layer play a key role in LI01 when exposed to bile

Decreases in FA biosynthesis and increases in the UFA/SFA ratio may help in the protection against bile stress in strain LI01. In addition to catalyzing SFA elongation, proteins such as FabA, FabK, and FabG play unique roles in UFA biosynthesis. As reported, up-regulation of FabA can potentially promote isomerization of trans-2- to cis-3-decenoyl-ACP, an essential step in UFA formation [52]; deletion of fabK in E. faecalis significantly promotes UFA synthesis [53], and up-regulation of FabG contributes to elongation of long-chain polyeneacyl-CoA in UFA biosynthesis. Bile-induced changes in FA biosynthesis have also been observed in L. reuteri CRL1098 [54]. Notably, L. casei 25

ACCEPTED MANUSCRIPT

ATCC393 cells with a lower UFA/SFA ratio were more sensitive to bile salts [55].

T

The up-regulated expression of secondary MDR transporter-related genes was

RI P

probably helpful for compensating for the inadequate MDR capacity of LI01 cell by strengthening bile efflux. As illustrated in Fig. 7, the differences in electrochemical

SC

potential between the internal and external portions of the cell membrane caused by

NU

ATPase, Na+/H+ antiporters (NapA and NhaC) and Na+ efflux pumps (LSL_0881 and

MA

LSL_0478) contributes to driving H+/drug and Na+/drug exchangers to pump bile salts out of the cell. The advantage of this type of bile efflux mechanism is the simultaneous

PT ED

maintenance of cytoplasmic pH homeostasis [56, 57] and with lower energy consumption compared to ABC-type. In L. lactis, the ABC-type MDR transporter LmrCD seemed to play a central role in the efflux-based defense mechanism invoked for bile

CE

acid resistance [58]. However, interestingly, the mutant strain L. lactis NZ9000 ΔlmrCD

[59].

AC

no longer depends on this extrusion mechanism and exhibits improved bile resistance

Some differences were observed between the transcription and translation of genes related to the general stress response in LI01 exposed to bile. Expression of chaperones including GrpE, DnaJ and GroES and proteases including ClpE, ClpP, ClpB and HslV (ClpQ) was increased, whereas the transcription of genes such as the dnaK operon, groE operon and clp family was generally down-regulated. In many other Lactobacillus strains, such as L. casei [15, 60], L. delbrueckii [61], L. plantarum [62] and LGG [13], these stress proteins, especially DnaK, GroESL, ClpL, ClpP and ClpE, were also 26

ACCEPTED MANUSCRIPT

commonly found to be up-regulated in response to bile, however, the related regulation

T

mechanisms have not yet been clearly defined. HrcA is a transcriptional repressor of the

RI P

dnaK and groE operons [63], whereas Clp proteases can be repressed by CtsR [64]; HrcA is also involved in the transcriptional control of clp genes in some low-GC

SC

gram-positive bacteria. For instance, in L. gasseri ATCC33323, HrcA instead of CtsR

NU

regulates the clpL gene [65]; in S. salivarius, the expression of clpP is controlled by both

MA

HrcA and CtsR [66]. Therefore, because the change in CtsR (LSL_0195) was insignificant, the 1.63-fold higher abundance of HrcA (LSL_0576) in LI01 may be one

PT ED

cause of the down-regulated transcription of the dnaK and groE operons. Regardless, this result does not exclude the possibility that expression of stress response proteins in LI01 may also be controlled by other mechanisms independent of the HrcA or CtsR

CE

regulon.

AC

Transcription of nearly all RP genes was down-regulated, whereas corresponding decreases in protein were detected for only in 8 RPs in LI01 exposed to bile. A similar phenomenon was observed in LGG under bile stress [13]. RP synthesis is primarily controlled at the translational level by the amount of rRNA available for RP binding, therefore, transcription of rRNA is the rate-limiting step of ribosome synthesis [67]. Given that rRNA has a much longer half-life than mRNA due to RP binding, repression of rRNA transcription may have the benefit of saving resources for the transcription of mRNAs encoding genes that protect against bile stress. Two pieces of evidence from our study support the above hypothesis. One is the 4.90-fold increase in ribosome recycling factor 27

ACCEPTED MANUSCRIPT

(Rrf, LSL_0562), which is responsible for the dissociation of ribosomes from mRNA after

T

termination of translation and thus allows the released ribosomes to more quickly

RI P

participate in next round of protein synthesis [68]. The other piece of evidence is the fact that two of the subunits of DNA-directed RNA polymerase, RpoE (LSL_0341) and RpoZ

SC

(LSL_0612), were 1.65- and 2.54-fold more abundant, respectively, after exposure to bile.

NU

RpoZ functions to promote RNA polymerase assembly and stability. RpoE can increase

MA

transcript specificity by blocking the binding of RNA polymerases at weak promoter sites [69]; moreover, RpoE is required for rapid changes in gene expression for cell survival

PT ED

under diverse stress conditions [70, 71]. Therefore, LI01 may alter gene expression by selectively promoting or repressing the transcription of certain RNAs. In extreme environments, shutting down some less important processes is beneficial for the

CE

effective utilization of resources and thus cell survival.

AC

Strategies employed by in LI01 to improve energy efficiency in response to bile stress were observed with regard to RNA synthesis, MDR and amino acid transport and carbohydrate utilization. Under normal conditions, L. salivarius can utilize D-glucose, D-fructose, D-mannose, ribose, lactose, mannitol, sorbitol, cellobiose, and maltose as energy resources [72]. However, in the presence of bile, the energy generation process of LI01 became more direct. As shown in Fig. 5, several genes controlling incorporation of carbohydrates from other pathways into glycolysis were strongly regulated under bile stress. For example, down-regulation of fruA and fruK may repress the metabolic pathway

from

fructose

to

D-fructose-1-phosphate 28

and

further

to

ACCEPTED MANUSCRIPT β-D-fructose-1,6-bisphosphate

but

does

not

affect

fructose

metabolism

via

T

β-D-fructose-6-phosphate. As a result, fructose and sorbose/sorbitol, which can be more

RI P

easily converted to β-D-fructose-1,6-bisphosphate, may be primarily metabolized during bile exposure. In contrast, complex metabolic pathways such as those for sucrose,

SC

trehalose and galactose may not be preferably invoked by LI01 under bile stress; one

NU

exception is the repression of mannose/mannitol metabolism, which is convenient for

MA

energy acquisition but is also involved in bacteriocin-related defense mechanisms. This repression has also been observed in LGG [13] and L. johnsonii PF01 [14].

PT ED

The often low correlation of the abundance of mRNAs and proteins is a real challenge among studies using integrative transcriptomic and proteomic analysis, as observed in ours studies as well as the studies of others [12, 13, 73]. First, this is likely

CE

because the transcriptional and translational processes are uncoupled. The fate of

AC

mRNAs is tightly regulated by a complex interplay of modification, processing, storage, decay, and translation; some mRNAs are directed toward translation, whereas others are diverted towards storage and translational repression. Furthermore, although proteins are translated from mRNA, some proteins may be transported out of the cell or some proteins may be promptly degraded [74, 75]. Second, the low correlation between mRNAs and proteins abundance may result from differences or limitations during the process of transcriptomic or proteomic analysis, including the methods used for sample preparations, molecular detections, and bioinformatics. Third, some factors, such as environmental stress, are prone to cause a low correlation in mRNAs and proteins 29

ACCEPTED MANUSCRIPT

abundance. Nonetheless, although some levels of altered mRNAs and proteins

T

abundance do not directly correlate with each other, such alterations are complementary

RI P

and necessary for a complete understanding of how the cell functions under various stimuli.

SC

Another issue encountered in transcriptomic and proteomic studies is the large

NU

degree of highly induced “hypothetical protein” encoding genes; indeed, hypothetical

MA

proteins account for 20% to 40% of proteins encoded in newly sequenced genomes [76]. By domain homology searching or structure homology modelling, several highly altered

PT ED

hypothetical proteins, such as LSL_0422 and LSL_1886 in this study, can be predicted as containing DNA-binding or transmembrane domain and thus potentially classified as a

AC

CE

regulator or transporter, suggesting promising future research directions.

30

ACCEPTED MANUSCRIPT

5. CONCLUSION

T

In this study, we explored the response of a potential probiotic strain, L. salivarius

RI P

LI01, to 0.15% ox bile using complementary profiling of transcriptome- and proteome-level changes. Based on this approach, 591 differentially transcribed genes

SC

and 347 differentially expressed proteins were detected. As BSHs were not significantly

NU

induced in LI01 in response to bile stress, these enzymes may not be involved in

MA

protection. Conversely, expression of several two-component systems, regulators and translation factors was altered, and these factors potentially act as sensors or first

PT ED

responders to bile challenge. In addition, the cell envelope was modified through remodeling of the structure of the peptidoglycan layer and by increasing the ratio of hydrophobic lipids and unsaturated fatty acids, with key roles in preventing bile salt influx

CE

and in tolerance to bile salt. Up-regulation of secondary MDR transporters, rather than

AC

ABC-type MDR transporters, potentially contributes to pumping bile salts out of the cell and to maintaining cytoplasmic pH homeostasis. Furthermore, various proteins related to the general stress response, such as chaperones GrpE, DnaJ, and GroES, proteases Clp, and universal stress proteins, were overexpressed to protect LI01 cells against bile damage. And to conserve resources, low energy consumption strategies were established for processes such as RNA synthesis and carbohydrate utilization. Lastly, bile stress may enhance proteolysis and amino acid (especially aromatic amino acids) uptake by LI01, which is favorable for the control and treatment of liver diseases. Our findings allow us to propose a bile stress response mechanism model for L. salivarius 31

ACCEPTED MANUSCRIPT

AC

CE

PT ED

MA

NU

SC

RI P

T

LI01 (Fig. 7).

32

ACCEPTED MANUSCRIPT

ASSOCIATED CONTENT

T

Supporting Information

RI P

Table S-1A: List of differentially transcribed genes at 10min after bile addition compared to the control. Table S-1B: List of differentially transcribed genes at 30min after bile

SC

addition compared to the control. Table S-1C: List of differentially transcribed genes at

NU

120min after bile addition compared to the control. Table S-2A: List of significantly

MA

up-regulated proteins at 60min after bile addition compared to the control. Table S-2B: List of significantly down-regulated proteins at 60min after bile addition compared to the

PT ED

control. Table S-2C: List of all identified proteins in this research.

AUTHOR INFORMATION

CE

Corresponding Author

AC

*Lanjuan Li; E-mail: [email protected]; Tel.: +86-571-8723-6458; Fax: +86-571-8723-6459 The First Affiliated Hospital, College of Medicine, Zhejiang University, 310003 Hangzhou, China.

33

ACCEPTED MANUSCRIPT

ACKNOWLEDGEMENTS

T

This study was supported by the general program (No. 81570512) of the National

RI P

Natural Science Foundation of China (NSFC), the National Basic Research Program of China (973 program) (No. 2013CB531401), the key program (No. 81330011) of NSFC,

AC

CE

PT ED

MA

NU

SC

and the Fundamental Research Funds for the Central Universities (No. 2015FZA7014).

34

ACCEPTED MANUSCRIPT

REFERENCES [1] X.W. Gao, M. Mubasher, C.Y. Fang, C. Reifer, L.E. Miller, Dose-Response Efficacy of a Proprietary Probiotic Formula of Lactobacillus acidophilus CL1285 and Lactobacillus casei LBC80R for Antibiotic-Associated Diarrhea and

T

Clostridium difficile-Associated Diarrhea Prophylaxis in Adult Patients, The American journal of gastroenterology

RI P

105(7) (2010) 1636-1641.

[2] M. Hickson, A.L. Souza, N. Muthu, T.R. Rogers, S. Want, C. Rajkumar, C.J. Bulpitt, Use of probiotic Lactobacillus preparation to prevent diarrhoea associated with antibiotics: randomised double blind placebo controlled trial,

SC

BMJ 335(7610) (2007) 80.

[3] P.S. Hsieh, Y.C. Tsai, Y.C. Chen, S.F. Teh, C.M. Ou, V.A. King, Eradication of Helicobacter pylori infection by the probiotic strains Lactobacillus johnsonii MH-68 and L. salivarius ssp. salicinius AP-32, Helicobacter 17(6) (2012)

NU

466-77.

[4] K. Tsilingiri, T. Barbosa, G. Penna, F. Caprioli, A. Sonzogni, G. Viale, M. Rescigno, Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model, Gut 61(7) (2012) 1007-15.

MA

[5] S.C. Corr, Y. Li, C.U. Riedel, P.W. O'Toole, C. Hill, C.G. Gahan, Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118, Proceedings of the National Academy of Sciences of the United States of America 104(18) (2007) 7617-21.

PT ED

[6] E. Riboulet-Bisson, M.H. Sturme, I.B. Jeffery, M.M. O'Donnell, B.A. Neville, B.M. Forde, M.J. Claesson, H. Harris, G.E. Gardiner, P.G. Casey, P.G. Lawlor, P.W. O'Toole, R.P. Ross, Effect of Lactobacillus salivarius bacteriocin Abp118 on the mouse and pig intestinal microbiota, PloS one 7(2) (2012) e31113. [7] C. Daniel, S. Poiret, D. Goudercourt, V. Dennin, G. Leyer, B. Pot, Selecting lactic acid bacteria for their safety and functionality by use of a mouse colitis model, Appl Environ Microb 72(9) (2006) 5799-5805. [8] J. McCarthy, L. O'Mahony, L. O'Callaghan, B. Sheil, E.E. Vaughan, N. Fitzsimons, J. Fitzgibbon, G.C. O'Sullivan, B.

CE

Kiely, J.K. Collins, F. Shanahan, Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance, Gut 52(7) (2003) 975-80.

AC

[9] L. O'Mahony, M. Feeney, S. O'Halloran, L. Murphy, B. Kiely, J. Fitzgibbon, G. Lee, G. O'Sullivan, F. Shanahan, J.K. Collins, Probiotic impact on microbial flora, inflammation and tumour development in IL-10 knockout mice, Aliment Pharmacol Ther 15(8) (2001) 1219-25. [10] M. Begley, C.G. Gahan, C. Hill, The interaction between bacteria and bile, FEMS microbiology reviews 29(4) (2005) 625-51. [11] C.N. Jacobsen, V. Rosenfeldt Nielsen, A.E. Hayford, P.L. Moller, K.F. Michaelsen, A. Paerregaard, B. Sandstrom, M. Tvede, M. Jakobsen, Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans, Appl Environ Microbiol 65(11) (1999) 4949-56. [12] C. Alcantara, M. Zuniga, Proteomic and transcriptomic analysis of the response to bile stress of Lactobacillus casei BL23, Microbiology+ 158(Pt 5) (2012) 1206-18. [13] K. Koskenniemi, K. Laakso, J. Koponen, M. Kankainen, D. Greco, P. Auvinen, K. Savijoki, T.A. Nyman, A. Surakka, T. Salusjarvi, W.M. de Vos, S. Tynkkynen, N. Kalkkinen, P. Varmanen, Proteomics and transcriptomics characterization of bile stress response in probiotic Lactobacillus rhamnosus GG, Molecular & cellular proteomics : MCP 10(2) (2011) M110 002741. [14] J.Y. Lee, E.A. Pajarillo, M.J. Kim, J.P. Chae, D.K. Kang, Proteomic and transcriptional analysis of Lactobacillus 35

ACCEPTED MANUSCRIPT johnsonii PF01 during bile salt exposure by iTRAQ shotgun proteomics and quantitative RT-PCR, Journal of proteome research 12(1) (2013) 432-43. [15] E. Hamon, P. Horvatovich, M. Bisch, F. Bringel, E. Marchioni, D. Aoude-Werner, S. Ennahar, Investigation of biomarkers of bile tolerance in Lactobacillus casei using comparative proteomics, Journal of proteome research

T

11(1) (2012) 109-18.

RI P

[16] L.X. Lv, X.J. Hu, G.R. Qian, H. Zhang, H.F. Lu, B.W. Zheng, L. Jiang, L.J. Li, Administration of Lactobacillus salivarius LI01 or Pediococcus pentosaceus LI05 improves acute liver injury induced by D-galactosamine in rats, Applied microbiology and biotechnology 98(12) (2014) 5619-32.

SC

[17] L.X. Xiang, D. He, W.R. Dong, Y.W. Zhang, J.Z. Shao, Deep sequencing-based transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish, BMC genomics 11 (2010) 472.

NU

[18] C.S. Gan, P.K. Chong, T.K. Pham, P.C. Wright, Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ), Journal of proteome research 6(2) (2007) 821-7. [19] P.K. Chong, C.S. Gan, T.K. Pham, P.C. Wright, Isobaric tags for relative and absolute quantitation (iTRAQ)

MA

reproducibility: Implication of multiple injections, Journal of proteome research 5(5) (2006) 1232-40. [20] L. Wang, Z. Feng, X. Wang, X. Wang, X. Zhang, DEGseq: an R package for identifying differentially expressed genes from RNA-seq data, Bioinformatics 26(1) (2010) 136-8.

PT ED

[21] Y. Benjamini, D. Drai, G. Elmer, N. Kafkafi, I. Golani, Controlling the false discovery rate in behavior genetics research, Behav Brain Res 125(1-2) (2001) 279-84.

[22] M. Begley, C. Hill, C.G. Gahan, Bile salt hydrolase activity in probiotics, Appl Environ Microbiol 72(3) (2006) 1729-38.

[23] F. Fang, Y. Li, M. Bumann, E.J. Raftis, P.G. Casey, J.C. Cooney, M.A. Walsh, P.W. O'Toole, Allelic variation of bile 191(18) (2009) 5743-57.

CE

salt hydrolase genes in Lactobacillus salivarius does not determine bile resistance levels, Journal of bacteriology [24] A.M. Stock, V.L. Robinson, P.N. Goudreau, Two-component signal transduction, Annu Rev Biochem 69 (2000)

AC

183-215.

[25] K.J. Wayne, S. Li, K.M. Kazmierczak, H.C. Tsui, M.E. Winkler, Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and limiting cross-talk in Streptococcus pneumoniae D39 cells, Molecular microbiology 86(3) (2012) 645-60. [26] M.E. Winkler, J.A. Hoch, Essentiality, bypass, and targeting of the YycFG (VicRK) two-component regulatory system in gram-positive bacteria, Journal of bacteriology 190(8) (2008) 2645-8. [27] N.L. Brown, J.V. Stoyanov, S.P. Kidd, J.L. Hobman, The MerR family of transcriptional regulators, FEMS microbiology reviews 27(2-3) (2003) 145-63. [28] J. Pei, N.V. Grishin, Type II CAAX prenyl endopeptidases belong to a novel superfamily of putative membrane-bound metalloproteases, Trends Biochem Sci 26(5) (2001) 275-7. [29] J. Hubscher, L. Luthy, B. Berger-Bachi, P. Stutzmann Meier, Phylogenetic distribution and membrane topology of the LytR-CpsA-Psr protein family, BMC genomics 9 (2008) 617. [30] Y.G. Chan, H.K. Kim, O. Schneewind, D. Missiakas, The capsular polysaccharide of Staphylococcus aureus is attached to peptidoglycan by the LytR-CpsA-Psr (LCP) family of enzymes, The Journal of biological chemistry 289(22) (2014) 15680-90. [31] M. Liszewski Zilla, Y.G. Chan, J.M. Lunderberg, O. Schneewind, D. Missiakas, LytR-CpsA-Psr enzymes as 36

ACCEPTED MANUSCRIPT determinants of Bacillus anthracis secondary cell wall polysaccharide assembly, Journal of bacteriology 197(2) (2015) 343-53. [32] R.D. Wegrzyn, E. Deuerling, Molecular guardians for newborn proteins: ribosome-associated chaperones and their role in protein folding, Cell Mol Life Sci 62(23) (2005) 2727-38.

T

[33] S. Brock, K. Szkaradkiewicz, M. Sprinzl, Initiation factors of protein biosynthesis in bacteria and their structural

RI P

relationship to elongation and termination factors, Molecular microbiology 29(2) (1998) 409-17. [34] H. Aoki, K. Dekany, S.L. Adams, M.C. Ganoza, The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis, The Journal of biological chemistry 272(51) (1997) 32254-9.

SC

[35] J. Frydman, Folding of newly translated proteins in vivo: the role of molecular chaperones, Annu Rev Biochem 70 (2001) 603-47.

[36] D. Frees, K. Savijoki, P. Varmanen, H. Ingmer, Clp ATPases and ClpP proteolytic complexes regulate vital

NU

biological processes in low GC, Gram-positive bacteria, Molecular microbiology 63(5) (2007) 1285-95. [37] M. Zolkiewski, A camel passes through the eye of a needle: protein unfolding activity of Clp ATPases, Molecular microbiology 61(5) (2006) 1094-100.

MA

[38] A. Diez, N. Gustavsson, T. Nystrom, The universal stress protein A of Escherichia coli is required for resistance to DNA damaging agents and is regulated by a RecA/FtsK-dependent regulatory pathway, Molecular microbiology 36(6) (2000) 1494-1503.

PT ED

[39] C. Seifart Gomes, B. Izar, F. Pazan, W. Mohamed, M.A. Mraheil, K. Mukherjee, A. Billion, Y. Aharonowitz, T. Chakraborty, T. Hain, Universal stress proteins are important for oxidative and acid stress resistance and growth of Listeria monocytogenes EGD-e in vitro and in vivo, Plos One 6(9) (2011) e24965. [40] S. Lopez-Gomollon, E. Sevilla, M.T. Bes, M.L. Peleato, M.F. Fillat, New insights into the role of Fur proteins: FurB (All2473) from Anabaena protects DNA and increases cell survival under oxidative stress, The Biochemical journal 418(1) (2009) 201-7.

CE

[41] M. Sakoh, K. Ito, Y. Akiyama, Proteolytic activity of HtpX, a membrane-bound and stress-controlled protease from Escherichia coli, The Journal of biological chemistry 280(39) (2005) 33305-10.

AC

[42] Y. Akiyama, Quality control of cytoplasmic membrane proteins in Escherichia coli, Journal of biochemistry 146(4) (2009) 449-54.

[43] L. Cai, S. Cai, D. Zhao, J. Wu, L. Wang, X. Liu, M. Li, J. Hou, J. Zhou, J. Liu, J. Han, H. Xiang, Analysis of the transcriptional regulator GlpR, promoter elements, and posttranscriptional processing involved in fructose-induced activation of the phosphoenolpyruvate-dependent sugar phosphotransferase system in Haloferax mediterranei, Appl Environ Microbiol 80(4) (2014) 1430-40. [44] I.F. Escapa, C. del Cerro, J.L. Garcia, M.A. Prieto, The role of GlpR repressor in Pseudomonas putida KT2440 growth and PHA production from glycerol, Environ Microbiol 15(1) (2013) 93-110. [45] B. Sanchez, M.C. Champomier-Verges, P. Anglade, F. Baraige, C.G. de Los Reyes-Gavilan, A. Margolles, M. Zagorec, Proteomic analysis of global changes in protein expression during bile salt exposure of Bifidobacterium longum NCIMB 8809, Journal of bacteriology 187(16) (2005) 5799-808. [46] M. Stetina, J. Behr, R.F. Vogel, The transcriptional response of Lactobacillus sanfranciscensis DSM 20451T and its tcyB mutant lacking a functional cystine transporter to diamide stress, Applied and environmental microbiology 80(14) (2014) 4114-25. [47] M. Holecek, Ammonia and amino acid profiles in liver cirrhosis: effects of variables leading to hepatic encephalopathy, Nutrition 31(1) (2015) 14-20. 37

ACCEPTED MANUSCRIPT [48] S.B. Hernandez, F. Cava, M.G. Pucciarelli, F. Garcia-Del Portillo, M.A. de Pedro, J. Casadesus, Bile-induced peptidoglycan remodelling in Salmonella enterica, Environ Microbiol 17(4) (2015) 1081-9. [49] C. Weidenmaier, A. Peschel, Teichoic acids and related cell-wall glycopolymers in Gram-positive physiology and host interactions, Nat Rev Microbiol 6(4) (2008) 276-87.

T

[50] A. Steen, E. Palumbo, M. Deghorain, P.S. Cocconcelli, J. Delcour, O.P. Kuipers, J. Kok, G. Buist, P. Hols, Autolysis

RI P

of Lactococcus lactis is increased upon D-alanine depletion of peptidoglycan and lipoteichoic acids, Journal of bacteriology 187(1) (2005) 114-24.

[51] M. Perea Velez, T.L. Verhoeven, C. Draing, S. Von Aulock, M. Pfitzenmaier, A. Geyer, I. Lambrichts, C. Grangette,

SC

B. Pot, J. Vanderleyden, S.C. De Keersmaecker, Functional analysis of D-alanylation of lipoteichoic acid in the probiotic strain Lactobacillus rhamnosus GG, Appl Environ Microbiol 73(11) (2007) 3595-604. [52] M.S. Kimber, F. Martin, Y. Lu, S. Houston, M. Vedadi, A. Dharamsi, K.M. Fiebig, M. Schmid, C.O. Rock, The

NU

structure of (3R)-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Pseudomonas aeruginosa, The Journal of biological chemistry 279(50) (2004) 52593-602.

[53] L. Zhu, H. Bi, J. Ma, Z. Hu, W. Zhang, J.E. Cronan, H. Wang, The two functional enoyl-acyl carrier protein

MA

reductases of Enterococcus faecalis do not mediate triclosan resistance, mBio 4(5) (2013) e00613-13. [54] M.P. Taranto, M.L. Fernandez Murga, G. Lorca, G.F. de Valdez, Bile salts and cholesterol induce changes in the lipid cell membrane of Lactobacillus reuteri, J Appl Microbiol 95(1) (2003) 86-91.

PT ED

[55] M.C. Machado, C.S. Lopez, H. Heras, E.A. Rivas, Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane, Archives of biochemistry and biophysics 422(1) (2004) 61-70. [56] O. Lewinson, E. Padan, E. Bibi, Alkalitolerance: a biological function for a multidrug transporter in pH homeostasis, Proceedings of the National Academy of Sciences of the United States of America 101(39) (2004) 14073-8.

[57] S.R. Holdsworth, C.J. Law, Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in

CE

alkaline pH homeostasis in Escherichia coli, Bmc Microbiol 13 (2013) 113. [58] J. Lubelski, A. De Jong, R. Van Merkerk, H. Agustiandari, O.P. Kuipers, J. Kok, A.J.M. Driessen, LmrCD is a major

AC

multidrug resistance transporter in Lactococcus lactis, Molecular microbiology 61(3) (2006) 771-781. [59] A.H. Zaidi, P.J. Bakkes, J. Lubelski, H. Agustiandari, O.P. Kuipers, A.J. Driessen, The ABC-type multidrug resistance transporter LmrCD is responsible for an extrusion-based mechanism of bile acid resistance in Lactococcus lactis, Journal of bacteriology 190(22) (2008) 7357-66. [60] R. Wu, Z. Sun, J. Wu, H. Meng, H. Zhang, Effect of bile salts stress on protein synthesis of Lactobacillus casei Zhang revealed by 2-dimensional gel electrophoresis, J Dairy Sci 93(8) (2010) 3858-68. [61] P. Burns, B. Sanchez, G. Vinderola, P. Ruas-Madiedo, L. Ruiz, A. Margolles, J. Reinheimer, C.G. de los Reyes-Gavilan, Inside the adaptation process of Lactobacillus delbrueckii subsp. lactis to bile, International journal of food microbiology 142(1-2) (2010) 132-41. [62] E. Hamon, P. Horvatovich, E. Izquierdo, F. Bringel, E. Marchioni, D. Aoude-Werner, S. Ennahar, Comparative proteomic analysis of Lactobacillus plantarum for the identification of key proteins in bile tolerance, BMC microbiology 11 (2011) 63. [63] M. Hecker, W. Schumann, U. Volker, Heat-shock and general stress response in Bacillus subtilis, Molecular microbiology 19(3) (1996) 417-28. [64] I. Derre, G. Rapoport, T. Msadek, CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria, Mol Microbiol 31(1) (1999) 117-31. 38

ACCEPTED MANUSCRIPT [65] A. Suokko, M. Poutanen, K. Savijoki, N. Kalkkinen, P. Varmanen, ClpL is essential for induction of thermotolerance and is potentially part of the HrcA regulon in Lactobacillus gasseri, Proteomics 8(5) (2008) 1029-41. [66] A. Chastanet, T. Msadek, clpP of Streptococcus salivarius is a novel member of the dually regulated class of

T

stress response genes in gram-positive bacteria, Journal of bacteriology 185(2) (2003) 683-687.

RI P

[67] B.J. Paul, W. Ross, T. Gaal, R.L. Gourse, rRNA transcription in Escherichia coli, Annual review of genetics 38 (2004) 749-70.

[68] L. Janosi, I. Shimizu, A. Kaji, Ribosome recycling factor (ribosome releasing factor) is essential for bacterial

SC

growth, Proceedings of the National Academy of Sciences of the United States of America 91(10) (1994) 4249-53. [69] E.C. Achberger, H.R. Whiteley, The role of the delta peptide of the Bacillus subtilis RNA polymerase in promoter selection, The Journal of biological chemistry 256(14) (1981) 7424-32.

NU

[70] X. Xue, J. Tomasch, H. Sztajer, I. Wagner-Dobler, The delta subunit of RNA polymerase, RpoE, is a global modulator of Streptococcus mutans environmental adaptation, Journal of bacteriology 192(19) (2010) 5081-92. [71] A. Rabatinova, H. Sanderova, J. Jirat Matejckova, J. Korelusova, L. Sojka, I. Barvik, V. Papouskova, V. Sklenar, L.

MA

Zidek, L. Krasny, The delta subunit of RNA polymerase is required for rapid changes in gene expression and competitive fitness of the cell, Journal of bacteriology 195(11) (2013) 2603-11. [72] R. Martin, E. Jimenez, M. Olivares, M.L. Marin, L. Fernandez, J. Xaus, J.M. Rodriguez, Lactobacillus salivarius Microbiol 112(1) (2006) 35-43.

PT ED

CECT 5713, a potential probiotic strain isolated from infant feces and breast milk of a mother-child pair, Int J Food [73] H. An, F.P. Douillard, G. Wang, Z. Zhai, J. Yang, S. Song, J. Cui, F. Ren, Y. Luo, B. Zhang, Y. Hao, Integrated transcriptomic and proteomic analysis of the bile stress response in a centenarian-originated probiotic Bifidobacterium longum BBMN68, Molecular & cellular proteomics : MCP 13(10) (2014) 2558-72. [74] D. Greenbaum, C. Colangelo, K. Williams, M. Gerstein, Comparing protein abundance and mRNA expression

CE

levels on a genomic scale, Genome biology 4(9) (2003) 1-8. [75] T. Maier, M. Güell, L. Serrano, Correlation of mRNA and protein in complex biological samples, Febs Lett 583(24)

AC

(2009) 3966-3973.

[76] M.Y. Galperin, Conserved 'hypothetical' proteins: new hints and new puzzles, Comp Funct Genomics 2(1) (2001) 14-8.

39

ACCEPTED MANUSCRIPT Figure Legends

T

Figure 1. The profile of differentially transcribed genes and differentially expressed

RI P

proteins of L. salivarius LI01 in response to bile stress. A, The number of differentially transcribed genes at 10, 30, and 120 min after bile challenge. B, The number of

SC

differentially expressed proteins at 60 min after bile challenge. C, Comparison of the

NU

number of differentially transcribed genes and differentially expressed proteins. Proteins with at least 1.2-fold change or genes with mRNA showing at least 2-fold change are

PT ED

MA

shown; adjusted p ≤ 0.05 for all data selected.

Figure 2. Functional classification based on GO enrichment analysis of differentially transcribed genes and differentially expressed proteins of L. salivarius LI01 in response

CE

to bile stress. A, GO function classification of differentially transcribed genes. B, GO

AC

function classification of differentially expressed proteins. Up-regulated ones (genes or proteins) are shown at the top of the intersection and down-regulated ones at the bottom. Proteins with at least 1.2-fold change or genes with mRNA showing at least 2-fold change are shown; adjusted p ≤ 0.05 for all data selected.

Figure 3. Pathway classification based on KEGG enrichment analysis of differentially transcribed genes and differentially expressed proteins of L. salivarius LI01 in response to bile stress. A, Pathway classification of differentially transcribed genes. B. Pathway classification of differentially expressed proteins. Rich factor, the ratio of the number of 40

ACCEPTED MANUSCRIPT

differentially expressed genes (translated proteins) to the number of total genes (proteins)

T

in this pathway. Proteins with at least 1.2-fold change or genes with mRNA showing at

RI P

least 2-fold change are shown; adjusted p ≤ 0.05 for all data selected.

SC

Figure 4. Heat map of important changes in gene transcription and translation in L.

NU

salivarius LI01 exposed to bile stress. A, Changes in sensing systems. B, Changes in

MA

cell surface genes. C, Changes in transport systems. D, Changes in genes involved in translation. E, Changes in general stress response genes. F, Changes in metabolic

PT ED

processes. Proteins with at least 1.2-fold change or genes with mRNA showing at least 2-fold change are shown; adjusted p ≤ 0.05 for all data selected.

CE

Figure 5. Proposed simplified pathways of carbohydrate metabolism and peptidoglycan

AC

biosynthesis under bile stress in L. salivarius LI01. The annotations were taken from Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.kegg.jp/).

Figure 6. Proposed simplified fatty acid biosynthesis pathways under bile stress in L. salivarius LI01. The annotations were taken from Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.kegg.jp/).

Figure 7. The proposed bile stress response mechanism model of L. salivarius LI01.

41

ACCEPTED MANUSCRIPT

Tables

Fold Change

RI P

corresponding proteins in response to bile stress.

Proteo

Location

Gene ID

mic

120

10 min

30 min

LSL_0161

Chromosome

5.32

ABE00140

LSL_1335

Chromosome

4.39

ABE00143

LSL_1338

Chromosome

4.50

ABE00315

LSL_1511

Chromosome

4.75

NU

ABE00447

LSL_1645

Chromosome

5.72

ABE00691

LSL_1885

ABE00708

LSL_1903

ABE00711

LSL_1907

ABE00712

LSL_1908

ABD98880

LSL_0059

ABD99637

AC

ABD99005

LSL_0190

LSL_0827

4.55

PT ED

LSL_1884

4.16

2.16

4.37

‒

Hypothetical protein

4.73

5.28

‒

4.18

4.11

‒

GmpB

4.29

4.19

‒

AzlD2

Mbp3

Mucus binding protein DNA-binding protein Phosphoglycerate mutase Branched-chain amino acid transport protein

4.19

4.19

1.20

PepN

Aminopeptidase N

pMP 118

6.26

6.26

6.29

‒

Hypothetical protein

pMP 118

12.78

13.28

15.63

‒

Hypothetical protein

pMP 118

5.17

5.45

5.82

‒

pMP 118

7.35

7.67

7.90

-1.91

Hypothetical protein

pMP 118

8.60

5.82

5.74

‒

Hypothetical protein

Chromosome

-58.00

-44.23

-17.24

‒

CE

ABE00690

pMP 118

Function symbol

4.38

MA

ABD98976

LSL_1745

4.36

Gene

60 min

min

ABE00547

Protein

b

SC

Accession

Transcriptomic

a

in Li01 and their

T

Table 1. Highly transcribed genes (more than 4-fold changed)

"Fructose-1,6-bisphosphat ase"

Clp protease ATP-binding ClpL subunit Aspartate

Chromosome

-12.30

-13.10

-13.98

‒

PyrB

carbamoyltransferase catalytic

Chromosome

-12.01

-15.88

-13.32

‒

Bifunctional pyrimidine PyrR regulatory protein

ABD99638 ABE00420

LSL_0828 LSL_1618

Chromosome Chromosome

-10.99 -26.86

-13.28 -33.11

-12.87 -30.99

‒ ‒

UraA

Uracil permease Mannitol-1-phosphate

MtlD 5-dehydrogenase

ABE00421

LSL_1619

Chromosome

-28.29

-30.89

-28.72

PTS, mannitol-specific IIA

‒

component ABE00422

LSL_1620

Chromosome

-23.46

-29.40

-23.69

‒

PTS, mannitol-specific MtlA IIBC component

ABE00432

LSL_1630

Chromosome

-19.63

-22.41

-19.78

‒

ABE00433

LSL_1631

Chromosome

-17.76

-22.93

-16.38

‒

ABE00434

LSL_1632

Chromosome

-16.30

-19.11

-14.05

2.39

42

GlpF

Glycerol uptake facilitator Hypothetical protein

DAK

Dihydroxyacetone kinase

ACCEPTED MANUSCRIPT Dihydroxyacetone kinase ABE00435

LSL_1633

Chromosome

-13.79

-14.25

-11.65

3.67

DhaK subunit

Chromosome

-4.86

-6.61

-5.07

1.33

ABD98969

LSL_0154

Chromosome

-5.53

-5.04

-4.00

‒

ABD98971

LSL_0155

LSL_0156

Chromosome

Chromosome

-8.47

-9.08

-6.74

-7.48

LSL_0157

Chromosome

-7.48

-6.92

ABD98978

LSL_0163

Chromosome

-6.47

-7.84

acetyltransferase

‒

-5.58

‒

-7.60

component Dihydrolipoamide

Lpd dehydrogenase

LplA

Lipoate-protein ligase A Transcription regulator of

‒

FruK

1-Phosphofructokinase

-6.01

-5.83

‒

PyrC

Dihydroorotase

-4.61

-4.66

-4.30

‒

CarA

-4.11

-6.24

-4.49

‒

-4.30

-5.46

-4.71

1.50

Chromosome

-4.01

-4.29

-4.19

‒

Chromosome

-5.38

-6.79

-5.96

‒

ABD99006

LSL_0191

Chromosome

-6.48

ABD99007

LSL_0192

Chromosome

ABD99083

LSL_0268

Chromosome

ABD99922

LSL_1114

Chromosome

ABE00089

LSL_1282

ABE00316

LSL_1512

PT ED

MA

-6.53

Chromosome

CE

LSL_1872

AceF

-7.70

Chromosome

ABE00679

Dihydrolipoamide

-7.02

LSL_0164

LSL_1668

1.92

E1 component

‒

ABD98979

ABE00470

-6.19

Pyruvate dehydrogenase AcoB

NU

ABD98972

-5.30

SC

ABD98970

Hypothetical protein

T

LSL_0058

RI P

ABD98879

pMP 118

fructose operon

Carbamoyl phosphate synthase small subunit Hypothetical protein AckA

Acetate kinase Sugar transporter PTS, trehalose-specific

TreB IIBC component

-5.68

-7.42

-5.14

‒

-5.21

-4.82

-4.40

1.92

Hypothetical protein Pyruvate formate-lyase PflA activating enzyme

AC

a, adjusted p ≤ 0.05 for all data selected. b, “‒ ” indicates the corresponding alteration in protein expression was not detected.

43

ACCEPTED MANUSCRIPT

Table 2. Highly expressed proteins (more than 3-fold changed)

in LI01 and their

Fold Change

Location

Gene ID

RI P

Accession

Proteo

Transcriptomicb

a

T

corresponding genes in response to bile stress.

Protein

mic

120 10 min

30 min

LSL_0080

Chromosome

‒

‒

ABD98975

LSL_0160

Chromosome

2.79

2.43

ABD99156

LSL_0343

Chromosome

2.19

‒

ABD99179

LSL_0366

Chromosome

‒

ABD99371

LSL_0562

Chromosome

‒

ABD99386

LSL_0577

Chromosome

-3.56

ABD99434

LSL_0624

Chromosome

ABD99687

LSL_0877

ABD99892

Function

symbol

SC

min ABD98901

Gene

60 min

‒

5.09

2.23

5.08

Hypothetical protein

GloA

Glyoxalase

14.87

Hypothetical protein

‒

3.92

Hypothetical protein

‒

‒

4.90

Rrf

Ribosome recycling factor

-2.98

-2.48

3.66

GrpE

Molecular chaperone

‒

‒

‒

3.83

AcpP

Acyl carrier protein

Chromosome

2.34

2.58

‒

3.44

‒

Hypothetical protein

LSL_1084

Chromosome

‒

‒

‒

3.13

PepQ

Xaa-Pro dipeptidase

ABD99908

LSL_1100

Chromosome

3.09

‒

2.05

18.24

ABD99912

LSL_1104

Chromosome

‒

‒

‒

4.93

ABD99918

LSL_1110

Chromosome

‒

‒

‒

7.13

ABD99944

LSL_1136

Chromosome

‒

‒

‒

3.59

PT ED

MA

NU

‒

‒

Hypothetical protein TrxA

Thioredoxin Hypothetical protein 23S rRNA methyltransferase

LSL_1212

Chromosome

‒

‒

‒

3.71

GroS

Molecular chaperone

ABE00059

LSL_1252

Chromosome

‒

‒

‒

3.60

WecD

Acetyltransferase

ABE00067

LSL_1260

Chromosome

2.05

2.14

2.20

3.32

NadE

NAD synthetase

ABE00105

LSL_1298

Chromosome

-2.36

-2.59

-2.60

5.91

AC

CE

ABE00020

Succinate-semialdehyde dehydrogenase

ABE00107

LSL_1300

Chromosome

‒

‒

‒

3.56

ABE00248

LSL_1444

Chromosome

‒

‒

‒

3.26

Hypothetical protein Glutamine transport GlnM system permease

ABE00273

LSL_1469

Chromosome

‒

-2.73

-2.65

4.43

ABE00330

LSL_1526

Chromosome

‒

‒

‒

3.01

Hypothetical protein Polysachharide biosynthesis protein

ABE00387

LSL_1583

Chromosome

‒

‒

‒

Phosphomethylpyrimidine 5.65

ThiD kinase Dihydroxyacetone kinase

ABE00435

LSL_1633

Chromosome

-13.79

-14.25

-11.65

3.67

DhaK subunit Nucleoside

ABE00445

LSL_1643

Chromosome

5.12

4.36

3.91

3.60 deoxyribosyltransferase

ABE00548

LSL_1746

pMP 118

‒

-2.02

44

-2.06

4.95

Oxidoreductase

ACCEPTED MANUSCRIPT

ABE00752

LSL_1948

pMP 118

‒

‒

‒

Ribulose-phosphate 3.18

Rpe 3-epimerase

ABD98925

LSL_0108

Chromosome

‒

‒

‒

Stress-responsive -3.52

PspC transcriptional regulator

LSL_0700

Chromosome

‒

‒

‒

RpsU

RI P

a, adjusted p ≤ 0.05 for all data selected.

-3.86

T

ABD99510

AC

CE

PT ED

MA

NU

SC

b, “‒ ” indicates the corresponding alteration in gene transcription was not detected

45

30S ribosomal protein S21

MA

NU

SC

RI P

T

ACCEPTED MANUSCRIPT

AC

CE

PT ED

Figure 1

46

AC

CE

PT ED

MA

NU

SC

RI P

T

ACCEPTED MANUSCRIPT

Figure 2 47

Figure 3

AC

CE

PT ED

MA

NU

SC

RI P

T

ACCEPTED MANUSCRIPT

48

AC

CE

PT ED

MA

NU

SC

RI P

T

ACCEPTED MANUSCRIPT

Figure 4 49

PT ED

MA

NU

SC

RI P

T

ACCEPTED MANUSCRIPT

AC

CE

Figure 5

50

MA

NU

SC

RI P

T

ACCEPTED MANUSCRIPT

AC

CE

PT ED

Figure 6

51

MA

NU

SC

RI P

T

ACCEPTED MANUSCRIPT

AC

CE

PT ED

Figure 7

52

ACCEPTED MANUSCRIPT

Conflict of Interest

AC

CE

PT ED

MA

NU

SC

RI P

T

All authors declare no competing financial interest.

53

ACCEPTED MANUSCRIPT

SIGNIFICANCE

T

L. salivarius strain LI01 exhibits not only antibacterial and antifungal properties but also

RI P

exerts a remarkable good health-promoting effect in acute liver failure. As a potential probiotic strain, its the bile-tolerance trait of strain LI01 is important, though this yet has

SC

not yet been explored. In this study, an analysis based on DGE and iTRAQ was

NU

performed to investigate the gene expression of in strain LI01 strain under bile stress at

MA

the mRNA and protein levels, respectively. To our knowledge, this work also represents the first combined transcriptomic and proteomic analysis of the bile stress response

AC

CE

PT ED

mechanism in L. salivarius.

54

AC

CE

Graphical abstract

PT ED

MA

NU

SC

RI P

T

ACCEPTED MANUSCRIPT

55

ACCEPTED MANUSCRIPT HIGHLIGHTS 1. An integrated transcriptomic and proteomic investigation of bile response

T

mechanism was first performed conducted in L. salivarius by using strain LI01.

RI P

2. L. salivarius LI01 is a non-BSH-producing strain. Its bile resistance was is mainly

3.

SC

based on a highly remodeled cell envelopes and reinforced bile efflux systems. Bile-induced alterations in regulatory systems, cell surface, general stress

NU

response and central metabolism processes were observed and also to contributed

MA

to the bile resistance of LI01.

4. Bile-induced potential alterations of LI 01 such as the uptake of aromatic amino

AC

CE

PT ED

acids may be helpful forin liver protection.

56