Exopolysaccharide, Isolated From a Novel Strain Bifidobacterium breve lw01 Possess an Anticancer Effect on Head and Neck Cancer – Genetic and Biochemical Evidences

frontiers RIGINAL RESEARC in Microbiology published: 09 May 201 d:10.338g/micb.2019.0104 Exopolysaccharide, Isolated From a Novel strain bifidobacterium breve Iw01 Possess an Anticancer Effect on head and neck cancer- Genetic and biochemical evidences OPEN ACCESS Lin Wang"t, Yifei Wang't, Qingxiang Li,, Kaiyue Tian, Le Xut, Guorong Liu3* and Edited by: Chuanbin Guo 1 Dame Drder, Lille University of Science ' Department of Oral and Maxilofacial Surgery, Peking University School and Hospital of Stomatology, Bejing, China and Technology France Department of Oral and Maxiofacial Plastic and Trauma Surgery, Beuing Stomatological Hospital, School of Stomatology Reviewed by: Capital Medical University, Beijing, China,Beijing Engineering and Technology Research Center of Food Additives, Beying Technology and Business University Bejing, China Maranta Kambourova Institute of Microbiology (BAS Zhi-oia Bulgaria Probiotic bacteria exopolysaccharides(EPS)have been recognized as molecules that iversity of Shanghai for Science regulate immune development and have anti-inflammation and anticancer effects. Yet and Technology, China these bioactivities are of interspecies diversity; thus, examining the gene clusters of EPS Correspondence: and biosynthesis pathways are essential for selecting the better application of specific Auguoongzo Guorong Lu EPS. In this study, we isolated a new Bifidobacterium strain, named B. breve lw01 83@126c0m; liuguorong@thbtbu.edu.cnAcompletegenomeofB.brevelw01wassequencedrevealingacircular2,313,172bp Chuanbin Guo chromosome. Furthermore, a deep excavation of genome sequence from different database based on the comparison-selected results was performed to explore the gene fThese authors have contributed equally to this work cluster responsible for EPS synthesis. We found that B. breve wo1 harbors a new EPS encoding cluster with 14 predicted genes, which could be divided into three groups Specialty section: according to the biosynthesis pathway hypothesis Using tertiary purification, high purity This article was submitted to Food Microbiology EPS were obtained. EPS is composed of rhamnose(Rha), arabinose(Ara), galactose a section of the joumal(Gal), glucose(Glc), and mannose(Man) in a molar ratio of 0.35: 0. 44: 1. 38: 0.67: 1 Frontiers in Microbiology With reference to its bioactivity, it showed to possess anticancer activity against Head Received: 29 December 2018 and Neck Squamous Cell Carcinoma cell line by regulating cell cycle arrest and cell Published:09 May 2019 apoptosis promotion. To sum up, this study examined the biosynthesis and bioactivity citation: of EPS using a new isolated B. breve strain, which could be used to clarify its further Wang L Wang Yu, Tran K, application in functional food or drug industry. Exopolysacchande, Isolated From Keywords: Bifidobacterium, exopolysaccharide, genome, biosynthesis, anticancer, probiotic, functional food a Nove strain bifidobacterium breve Mof Possess an Anticancer Effect Head and Neck Cancer- Genetic Abbreviations: bifido-EPS, Bifidobacteriumm-EPS: COG, clusters of orthologous groups: EPS, exopolysaccharide: HAPI and Biochemical evidences PAD, High PH anion exchange chromatography with pulsed amperometric detection; HePS, heteropolysaccharides HNSCC, head and neck squamous cell carcinoma; HoPS, homopolysaccharide; HPLC, high-performance liquid Front. Microbiol. 10: 7044. chromatography: MCM2, mini-chromosome maintenance 2: P-gt Priming-GTF: SEM, scanning electron microscope; TCA. do:13389mcb.2019.01044 FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044

fmicb-10-01044 May 8, 2019 Time: 14:36 # 1 ORIGINAL RESEARCH published: 09 May 2019 doi: 10.3389/fmicb.2019.01044 Edited by: Djamel Drider, Lille University of Science and Technology, France Reviewed by: Margarita Kambourova, Institute of Microbiology (BAS), Bulgaria Zhi-Qiang Xiong, University of Shanghai for Science and Technology, China *Correspondence: Guorong Liu liuguorong1983@126.com; liuguorong@th.btbu.edu.cn Chuanbin Guo guodazuo@sina.com †These authors have contributed equally to this work Specialty section: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology Received: 29 December 2018 Accepted: 25 April 2019 Published: 09 May 2019 Citation: Wang L, Wang Y, Li Q, Tian K, Xu L, Liu G and Guo C (2019) Exopolysaccharide, Isolated From a Novel Strain Bifidobacterium breve lw01 Possess an Anticancer Effect on Head and Neck Cancer – Genetic and Biochemical Evidences. Front. Microbiol. 10:1044. doi: 10.3389/fmicb.2019.01044 Exopolysaccharide, Isolated From a Novel Strain Bifidobacterium breve lw01 Possess an Anticancer Effect on Head and Neck Cancer – Genetic and Biochemical Evidences Lin Wang1† , Yifei Wang1† , Qingxiang Li1 , Kaiyue Tian2 , Le Xu1 , Guorong Liu3 * and Chuanbin Guo1 * 1 Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing, China, 2 Department of Oral and Maxillofacial Plastic and Trauma Surgery, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Beijing, China, 3 Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, China Probiotic bacteria exopolysaccharides (EPS) have been recognized as molecules that regulate immune development and have anti-inflammation and anticancer effects. Yet, these bioactivities are of interspecies diversity; thus, examining the gene clusters of EPS and biosynthesis pathways are essential for selecting the better application of specific EPS. In this study, we isolated a new Bifidobacterium strain, named B. breve lw01. A complete genome of B. breve lw01 was sequenced revealing a circular 2,313,172 bp chromosome. Furthermore, a deep excavation of genome sequence from different database based on the comparison-selected results was performed to explore the gene cluster responsible for EPS synthesis. We found that B. breve lw01 harbors a new EPS￾encoding cluster with 14 predicted genes, which could be divided into three groups according to the biosynthesis pathway hypothesis. Using tertiary purification, high purity EPS were obtained. EPS is composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), and mannose (Man) in a molar ratio of 0.35:0.44:1.38:0.67:1.65. With reference to its bioactivity, it showed to possess anticancer activity against Head and Neck Squamous Cell Carcinoma cell line by regulating cell cycle arrest and cell apoptosis promotion. To sum up, this study examined the biosynthesis and bioactivity of EPS using a new isolated B. breve strain, which could be used to clarify its further application in functional food or drug industry. Keywords: Bifidobacterium, exopolysaccharide, genome, biosynthesis, anticancer, probiotic, functional food Abbreviations: bifido-EPS, Bifidobacterium-EPS; COG, clusters of orthologous groups; EPS, exopolysaccharide; HAPEC￾PAD, High pH anion exchange chromatography with pulsed amperometric detection; HePS, heteropolysaccharides; HNSCC, head and neck squamous cell carcinoma; HoPS, homopolysaccharides; HPLC, high-performance liquid chromatography; MCM2, mini-chromosome maintenance 2; p-gtf, priming-GTF; SEM, scanning electron microscope; TCA, trichloroacetic acid. Frontiers in Microbiology | www.frontiersin.org 1 May 2019 | Volume 10 | Article 1044

Wang et al Anticancer Exopolysaccharide From B breve Iwo1 INTRODUCTION MATERIALS AND METHODS Microbial EPS are carbohydrate polymers surrounding the Isolation of Bacteria Stains envelope of most bacteria. The bacterial EPS has initially gained Fresh infant fecal sample collected and incubated in a lots of attention from researchers for its effect on biofilm or modified MRS-C liquid medium(MRS medium plus 0.59 capsular polysaccharides formation that act as virulence factors cysteine HCl)for 24 h. The medium was then transferred into in infectious diseases( Gonzalez-Escobedo et al, 2011: Yamanaka a modified MRS-C agar plate and cultured at 37 C in anaerobic et al, 2011).Recently, bacterial EPS have re-gained attention conditions for 48-72 h. Next, the different morphologies were for their implication on human health, especially since bacteria picked up, and viscous colonies were separately inoculated with some probiotic traits and their EPS could contribute to for pure culture. Staining and morphological characteristics the host health maintenance. Among these, Lactobacillus and of the isolated bacteria were tested by Bifidobacterium are mostly used in the formulation of probiotic protocol was approved by the Biomedical Ethics Committee of foods or supplements for human consumption Peking University School and Hospital of Stomatology. Written Bifidobacterium, which is one of the dominant early informed consent was obtained from parents of the infant. colonization bacteria in human intestinal tract, was proven to regulate immune development (Arboleya et al, 2012), Cell Lines and Culture Conditions immune modulation(Fanning et al, 2012), and to possess an Human Head and Neck Squamous Cell Carcinoma cell line ti-inflammation(Strisciuglio et al, 2015) and anticancer SCc15(ATCCS CRL-1623TM), CAL 27(ATCCe CRL-2095M (Wang et al, 2017) activity. The immune response is stain- and wSU-HN6(obtained from Central Laboratory of Peking pecific (Lopez et al, 2010)and one of the mechanisms University School and Hospital of Stomatology) were used driving the immune functions is regulated by bifido-EPS Bifido-EPS may facilitate Bifidobacterial-host interaction in this study. SCC15 were cultured in a 1: 1 mixture of DMEM and Ham's F12 medium(Life technology, Carlsbad, pathogen protection through a physical barrier, also known CA, United States)containing 10% FBS (Invitrogen, Waltham through immune modulation( Fanning et al, 2012)and provide as"Bifidobacterial biofilm"(Ruas-Madiedo et al., 2010) addition, bifido-EPS has shown to possess an antioxidant Houston, TX, United States)and 1% Penicillin/Streptomycin activity, which may further reduce tissue damage (Li et al, (Gibco, Waltham, MA, United States). CAL27 and WSU- 2014). In addition, EPS can inhibit DNA synthesis(You et al HN6 were cultured in DMEM medium containing 10% FBS and 1% Penicillin/Streptomycin. All the cells were kept in 2004)and the expression of gene involved in angiogenesis. Yet, these bioactivities, which rely on the related physicochemical a humidified atmosphere containing 95% air/5%CO2 at characteristic, are of interspecies diversity. Thus, for better 37oC. For the anaerobic culture, we used the chamber with 93%N2/5%CO2/2%O2 application of specific bifido-EPS, gene clusters, chemical omposition and its hypothetical biosynthesis pathways Genomic DNA lsolation, Sequencing and prediction need to be carefully examined With regards to chemical composition and its synthesis Analysis of EPs Cluster method, HePS, which are type of the EpS polymers reported to Bifidobacterium be the unique form in Bifidobacterium, compared to the HoPs Genome DNA was isolated using QIAamp DNA Mini Kit and Heps types in Lactobacillus strain(Ruas-Madiedo, 2014).(Cat. 51304, QIAGEN, MD, United States) according the Leivers et al.(2011) have reported the hypothetical biosynthesis manufacturer's instructions. 16s rRNA PCR was used to verify pathway based on one kind of EPS-unit of HMW-EPS in strain the purified strain type. The whole genome was sequenced on B animalis subsp. lactis IPLA-RI, which mainly included three Illumina Hiseq 2000 platform (2x 100 bp) second-generation steps: activated precursor's synthesis and EPS-unit formation, sequencing platform. Sequence blast searches were performed export-polymerization process and chain length determination. on National Center for Biotechnology Information website!.The In this study, we isolated a new probiotic and EPS-producing high-quality reads were assembled by SOAPdenovo. Based on the Bifidobaccterium, named B breve lwol After genome sequencing comparison-selected results, the relative abundance of different d in silico analysis, a new EPS cluster and the hypothetical functional levels and EPS cluster searchers were investigated synthesis pathway were discovered. The extraction of EPS, purity using KEGG(Kyoto Encyclopedia of genes and genomes), RAST determination and scanning electron microscopy observation (Rapid Annotation Subsystem Technology) and COG database. confirmed its morphology, thus determining the composition The genomes and EPS cluster information available in the of this EPS. Finally, we focused on its anti-tumor properties Gen Bank database were used for the comparative analysis among and preliminary investigated its mechanism. This study provided B breve lwOl and several Bifidobacterium strains genetic information on EPS cluster from new isolated EPS producing Bifidobacterium, connecting it with biosynthesis Extraction and Purification of EPs pathway and exploring its anti-tumor activities. Biochemical For EPS extraction, purified bacteria were anaerobically cultured evidence was shown to explain its anti-tumor activity. Our in 1l of 10% skimmed milk at 370C for 48 h. To isolate the research provided a theoretical basis for further application of this bifido-EPS in the probiotic field. http://www.ncbi.nlm.nih.gov FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044

fmicb-10-01044 May 8, 2019 Time: 14:36 # 2 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 INTRODUCTION Microbial EPS are carbohydrate polymers surrounding the envelope of most bacteria. The bacterial EPS has initially gained lots of attention from researchers for its effect on biofilm or capsular polysaccharides formation that act as virulence factors in infectious diseases (Gonzalez-Escobedo et al., 2011; Yamanaka et al., 2011). Recently, bacterial EPS have re-gained attention for their implication on human health, especially since bacteria with some probiotic traits and their EPS could contribute to the host health maintenance. Among these, Lactobacillus and Bifidobacterium are mostly used in the formulation of probiotic foods or supplements for human consumption. Bifidobacterium, which is one of the dominant early colonization bacteria in human intestinal tract, was proven to regulate immune development (Arboleya et al., 2012), immune modulation (Fanning et al., 2012), and to possess an anti-inflammation (Strisciuglio et al., 2015) and anticancer (Wang et al., 2017) activity. The immune response is stain￾specific (Lopez et al., 2010) and one of the mechanisms driving the immune functions is regulated by bifido-EPS. Bifido-EPS may facilitate Bifidobacterial-host interaction through immune modulation (Fanning et al., 2012) and provide pathogen protection through a physical barrier, also known as “Bifidobacterial biofilm” (Ruas-Madiedo et al., 2010). In addition, bifido-EPS has shown to possess an antioxidant activity, which may further reduce tissue damage (Li et al., 2014). In addition, EPS can inhibit DNA synthesis (You et al., 2004) and the expression of gene involved in angiogenesis. Yet, these bioactivities, which rely on the related physicochemical characteristic, are of interspecies diversity. Thus, for better application of specific bifido-EPS, gene clusters, chemical composition and its hypothetical biosynthesis pathways prediction need to be carefully examined. With regards to chemical composition and its synthesis method, HePS, which are type of the EPS polymers reported to be the unique form in Bifidobacterium, compared to the HoPS and HePS types in Lactobacillus strain (Ruas-Madiedo, 2014). Leivers et al. (2011) have reported the hypothetical biosynthesis pathway based on one kind of EPS-unit of HMW-EPS in strain B. animalis subsp. lactis IPLA-R1, which mainly included three steps: activated precursor’s synthesis and EPS-unit formation, export-polymerization process and chain length determination. In this study, we isolated a new probiotic and EPS-producing Bifidobaccterium, named B. brevelw01. After genome sequencing and in silico analysis, a new EPS cluster and the hypothetical synthesis pathway were discovered. The extraction of EPS, purity determination and scanning electron microscopy observation confirmed its morphology, thus determining the composition of this EPS. Finally, we focused on its anti-tumor properties and preliminary investigated its mechanism. This study provided genetic information on EPS cluster from new isolated EPS￾producing Bifidobacterium, connecting it with biosynthesis pathway and exploring its anti-tumor activities. Biochemical evidence was shown to explain its anti-tumor activity. Our research provided a theoretical basis for further application of this bifido-EPS in the probiotic field. MATERIALS AND METHODS Isolation of Bacteria Stains Fresh infant fecal sample was collected and incubated in a modified MRS-C liquid medium (MRS medium plus 0.5% cysteine • HCl) for 24 h. The medium was then transferred into a modified MRS-C agar plate and cultured at 37◦C in anaerobic conditions for 48–72 h. Next, the different morphologies were picked up, and viscous colonies were separately inoculated for pure culture. Staining and morphological characteristics of the isolated bacteria were tested by microscopy. The protocol was approved by the Biomedical Ethics Committee of Peking University School and Hospital of Stomatology. Written informed consent was obtained from parents of the infant. Cell Lines and Culture Conditions Human Head and Neck Squamous Cell Carcinoma cell line SCC15 (ATCC R CRL-1623TM), CAL 27 (ATCC R CRL-2095TM) and WSU-HN6 (obtained from Central Laboratory of Peking University School and Hospital of Stomatology) were used in this study. SCC15 were cultured in a 1:1 mixture of DMEM and Ham’s F12 medium (Life technology, Carlsbad, CA, United States) containing 10% FBS (Invitrogen, Waltham, MA, United States), 0.2% hydrocortisone (Selleck Chemicals, Houston, TX, United States) and 1% Penicillin/Streptomycin (Gibco, Waltham, MA, United States). CAL27 and WSU￾HN6 were cultured in DMEM medium containing 10% FBS and 1% Penicillin/Streptomycin. All the cells were kept in a humidified atmosphere containing 95% air/5% CO2 at 37◦C. For the anaerobic culture, we used the chamber with 93% N2/5% CO2/2% O2. Genomic DNA Isolation, Sequencing and Analysis of EPS Cluster in Bifidobacterium Genome DNA was isolated using QIAamp DNA Mini Kit (Cat. 51304, QIAGEN, MD, United States) according the manufacturer’s instructions. 16s rRNA PCR was used to verify the purified strain type. The whole genome was sequenced on Illumina Hiseq 2000 platform (2 × 100 bp) second-generation sequencing platform. Sequence blast searches were performed on National Center for Biotechnology Information website1 . The high-quality reads were assembled by SOAPdenovo. Based on the comparison-selected results, the relative abundance of different functional levels and EPS cluster searchers were investigated using KEGG (Kyoto Encyclopedia of genes and genomes), RAST (Rapid Annotation Subsystem Technology) and COG database. The genomes and EPS cluster information available in the GenBank database were used for the comparative analysis among B. breve lw01 and several Bifidobacterium strains. Extraction and Purification of EPS For EPS extraction, purified bacteria were anaerobically cultured in 1 L of 10% skimmed milk at 37◦C for 48 h. To isolate the 1http://www.ncbi.nlm.nih.gov Frontiers in Microbiology | www.frontiersin.org 2 May 2019 | Volume 10 | Article 1044

Wang et al Anticancer Exopolysaccharide From B breve Iwo1 rude EPS from the culture broth, 1 L of culture samples were was added to 5 mg EPS and was left to stand still. Then 800 centrifuged at 12,000 x g for 15 min at 4@C. After obtaining distilled water was added and hydrolyzed at 100 C for 2.5 h. After the supernatant, TCA was added to a final concentration of cooling, add to 2 mL and neutralize with solid BaCO3. Everything 10% for 12 h at 4oC Precipitated proteins were then removed was centrifuged at 5000 r/min for 10 min to remove impurities, by centrifugation at 4000 x g for 20 min at 4oC. The EPs and remove the supernatant. The supernatant was diluted 20 was precipitated from the supernatant with 3 volumes of times, filtered through a 0. 20 um microporous membrane and cold ethanol followed by an overnight incubation at 4C. subjected to test. After centrifugation at 6000 x g for 30 min at 4oC, the High pH anion exchange chromatography with pulsed pellet containing EPS was resuspended in 2 mL of distilled amperometric detection (HAPEC-PAD) was usedto water and dialyzed(molecular weight cut-off: 6000-8000 Da) determine monosaccharide composition on a DIONEX gainst 1 L of distilled water for 2 days with three water 2500 ion chromatograph system, equipped with on-line changes per 8h. automatic degassing GS50 quaternary gradient pump, Ion exchange chromatography of DEAE Sepharose Fast Flow ED50A electrochemical detector (pulse amperometry (16x 25 mm, GE Healthcare, Amersham, Uppsala, Sweden)was detection), Au working electrode and Ag/AgCl reference used for further EPS purification. Distilled water was applied electrode. Chromatographic conditions were set as follows: to keep the ph of the ion exchange column neutral, eluted sugar column(CarboPac PAl0, 4 x 250 mm) with 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl. The loading volume sugar n column(CarboPac PAl0, 4 x 50 mm) was 1 mL and the flow rate was 1 mL/min. Stepwise collection mobile A. H2O, B 200 mmol/L NaOH, A: B= 91: 9 of 5 mL was applied for each tube. Then, the purified EPs injection volume: 25 uL; flow rate: 1.0 mL/min; column from ion exchange chromatography was further purified by gel temperature: 30C. Chromeleon 6.5 chromatography was filtration chromatography of Sepharose CL-6B(GE Healthcare, used as workstation Amersham, Uppsala, Sweden). Elution was performed with distilled water at a flow rate of 1 mL/min. The final eluted EPs Cell Proliferation and Cytotoxic Analysis was freeze-dried and stored at 4C until analysis A HPLC (Agilent, Santa Clara, CA, United States)equipped examined using CCK-8 assay and DAPI staining. Appropriate ith a NH2 column(Agilent, Santa Clara, CA, United States)was number of cells in 100 HL suspension were plated into 96 used for EPS further purification. The mobile phase consisted well plate and incubated in normoxic or hypoxic environment of 80% ammonium acetate solution and 20% acetonitrile at descripted above. After each time point, 10 uL of the CCK-8 a flow rate of 1.0 mL/min, and the column temperature was solution(Bimake, Houston, TX, United States )was added to each kept on 30 C. The refractive index detector(RID) was used well and incubated for another 4 h at 37 C. The absorbance to detect the eps at 450 nm was determined using a microplate reader. Cell The amount of EPS was determined using the phenol/sulfuric nuclear morphology was stained by DAPI(Zhongshan Golden acid method. Glucose was used as the standard sugar. After Bridge Biotechnology Co., Beijing, China). Each sample had three the color reaction of the glucose standard solution with replicates and each experiment was run in triplicate. phenol/sulfuric acid, its absorbance was measured at 490 nm d the OD490-standard sugar concentration curve was drawn. Western Blot The diluted sample of EPS solution was examined using the After treating cells with EPS, cells were harvested and same color reaction. The 490 nm absorbance value was measured, lysed in RIPA buffer(Applygen Technology, Beijing, China) d the corresponding sugar concentration was found on the containing proteinase inhibitors and phosphatase inhibitors standard curve. The sugar concentration of the sample to be (ROCHe, Basel, Switzerland). BCA kit(Thermo Fisher Scientific, tested was calculated Rockford, IL, United States) was used to measure the protein concentration. Total of 70 ug amounts of protein samples were Scanning Electron Microscopy separated by 10% sodium dodecyl sulfate-polyacrylamide gel Scanning electron microscope was used to examine the electrophoresis(SDS-PAGE) and transferred to polyvinylider morphology of the EPS. Briefly, the freeze-dried EPS powder difluoride(PVDF)membranes by wet blotting. The membranes was mounted on specimens slide, sputter coated with gold and were blocked in 10% non-fat dry milk for I h and then probed examined using SEM(SEM; Hitachi, S-4700, Hitachi Ltd, Tokyo, with antibodies against MCM2 (1: 1,000, ABclonal, Boston, Japan)at accelerating voltages of 10 and 15 kV, respectively MA, United States), Caspases-3(1: 1,000, CST, Boston, MA, United States), PARP(1: 1, 000, CST, Boston, MA, United States), Monomer composition of EPs cleaved-PARP (1: 1, 000, CST, Boston, MA, United States) The monosaccharide standards dried to constant weight were and RPS18(1: 1, 000, ABclonal, Boston, MA, United States) weighed separately, and L-Rha, L-Ara, D-Gal, D-Glc, D-Man, separately at 4C overnight. Followed by incubation with d D-Fru prepared at different concentrations(0.2, 0.5, 1.0, peroxidase-linked secondary antibodies(1: 10,000, CST, Boston, 4.0, and 10.0 ug/mL) were mixed to standard solution. Standard MA, United States) for 1 h at room temperature, the curve for each monosaccharide was drew with monosaccharide enhanced chemiluminescent(ECL) reagent(Thermo Fisher concentration (ug/mL)as the abscissa (x) and peak area Scientific, Rockford, IL, United States)was used to visualize the (nC x min) as the ordinate (y). 200 uL concentrated H2S04 immunoreactive proteins. FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044

fmicb-10-01044 May 8, 2019 Time: 14:36 # 3 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 crude EPS from the culture broth, 1 L of culture samples were centrifuged at 12,000 × g for 15 min at 4◦C. After obtaining the supernatant, TCA was added to a final concentration of 10% for 12 h at 4◦C. Precipitated proteins were then removed by centrifugation at 4000 × g for 20 min at 4◦C. The EPS was precipitated from the supernatant with 3 volumes of cold ethanol followed by an overnight incubation at 4◦C. After centrifugation at 6000 × g for 30 min at 4◦C, the pellet containing EPS was resuspended in 2 mL of distilled water and dialyzed (molecular weight cut-off: 6000–8000 Da) against 1 L of distilled water for 2 days with three water changes per 8 h. Ion exchange chromatography of DEAE Sepharose Fast Flow (16 × 25 mm, GE Healthcare, Amersham, Uppsala, Sweden) was used for further EPS purification. Distilled water was applied to keep the pH of the ion exchange column neutral, eluted with 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl. The loading volume was 1 mL and the flow rate was 1 mL/min. Stepwise collection of 5 mL was applied for each tube. Then, the purified EPS from ion exchange chromatography was further purified by gel filtration chromatography of Sepharose CL-6B (GE Healthcare, Amersham, Uppsala, Sweden). Elution was performed with distilled water at a flow rate of 1 mL/min. The final eluted EPS was freeze-dried and stored at 4◦C until analysis. A HPLC (Agilent, Santa Clara, CA, United States) equipped with a NH2 column (Agilent, Santa Clara, CA, United States) was used for EPS further purification. The mobile phase consisted of 80% ammonium acetate solution and 20% acetonitrile at a flow rate of 1.0 mL/min, and the column temperature was kept on 30◦C. The refractive index detector (RID) was used to detect the EPS. The amount of EPS was determined using the phenol/sulfuric acid method. Glucose was used as the standard sugar. After the color reaction of the glucose standard solution with phenol/sulfuric acid, its absorbance was measured at 490 nm, and the OD490-standard sugar concentration curve was drawn. The diluted sample of EPS solution was examined using the same color reaction. The 490 nm absorbance value was measured, and the corresponding sugar concentration was found on the standard curve. The sugar concentration of the sample to be tested was calculated. Scanning Electron Microscopy Scanning electron microscope was used to examine the morphology of the EPS. Briefly, the freeze-dried EPS powder was mounted on specimens slide, sputter coated with gold and examined using SEM (SEM; Hitachi, S-4700, Hitachi Ltd., Tokyo, Japan) at accelerating voltages of 10 and 15 kV, respectively. Monomer Composition of EPS The monosaccharide standards dried to constant weight were weighed separately, and L-Rha, L-Ara, D-Gal, D-Glc, D-Man, and D-Fru prepared at different concentrations (0.2, 0.5, 1.0, 4.0, and 10.0 µg/mL) were mixed to standard solution. Standard curve for each monosaccharide was drew with monosaccharide concentration (µg/mL) as the abscissa (x) and peak area (nC × min) as the ordinate (y). 200 µL concentrated H2SO4 was added to 5 mg EPS and was left to stand still. Then 800 distilled water was added and hydrolyzed at 100◦C for 2.5 h. After cooling, add to 2 mL and neutralize with solid BaCO3. Everything was centrifuged at 5000 r/min for 10 min to remove impurities, and remove the supernatant. The supernatant was diluted 20 times, filtered through a 0.20 µm microporous membrane and subjected to test. High pH anion exchange chromatography with pulsed amperometric detection (HAPEC-PAD) was used to determine monosaccharide composition on a DIONEX- 2500 ion chromatograph system, equipped with on-line automatic degassing GS50 quaternary gradient pump, ED50A electrochemical detector (pulse amperometry detection), Au working electrode and Ag/AgCl reference electrode. Chromatographic conditions were set as follows: sugar analysis column (CarboPacTM PA10, 4 × 250 mm); sugar protection column (CarboPac PA10, 4 × 50 mm); mobile phase: A. H2O, B. 200 mmol/L NaOH, A:B = 91:9; injection volume: 25 µL; flow rate: 1.0 mL/min; column temperature: 30◦C. Chromeleon 6.5 chromatography was used as workstation. Cell Proliferation and Cytotoxic Analysis The EPS effect on cell proliferation and cytotoxicity were examined using CCK-8 assay and DAPI staining. Appropriate number of cells in 100 µL suspension were plated into 96- well plate and incubated in normoxic or hypoxic environment descripted above. After each time point, 10 µL of the CCK-8 solution (Bimake, Houston, TX, United States) was added to each well and incubated for another 4 h at 37◦C. The absorbance at 450 nm was determined using a microplate reader. Cell nuclear morphology was stained by DAPI (Zhongshan Golden Bridge Biotechnology Co., Beijing, China). Each sample had three replicates and each experiment was run in triplicate. Western Blot After treating cells with EPS, cells were harvested and lysed in RIPA buffer (Applygen Technology, Beijing, China) containing proteinase inhibitors and phosphatase inhibitors (ROCHE, Basel, Switzerland). BCA kit (Thermo Fisher Scientific, Rockford, IL, United States) was used to measure the protein concentration. Total of 70 µg amounts of protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes by wet blotting. The membranes were blocked in 10% non-fat dry milk for 1 h and then probed with antibodies against MCM2 (1:1,000, ABclonal, Boston, MA, United States), Caspases-3 (1:1,000, CST, Boston, MA, United States), PARP (1:1,000, CST, Boston, MA, United States), cleaved-PARP (1:1,000, CST, Boston, MA, United States) and RPS18 (1:1,000, ABclonal, Boston, MA, United States) separately at 4◦C overnight. Followed by incubation with peroxidase-linked secondary antibodies (1:10,000, CST, Boston, MA, United States) for 1 h at room temperature, the enhanced chemiluminescent (ECL) reagent (Thermo Fisher Scientific, Rockford, IL, United States) was used to visualize the immunoreactive proteins. Frontiers in Microbiology | www.frontiersin.org 3 May 2019 | Volume 10 | Article 1044

Wang et al Anticancer Exopolysaccharide From B breve Iwo1 TABLE 1 I Features of B breve lol genome TABLE 2 COG categories of coding proteins in B. breve lwo1 genome Features CoG Name Count Percent (p) GC content (%6 INA processing and modification RNAS Energy production and conversion TRNAS 2,2,25s,168,23s)D Cell cycle control, cell avision tRNAs Amino acid transport and 10.69 metabolism CDSs ( with protein 4.09 metabolism Statistical Analysis G carbohydrate transport an 253 1294 The absorbance value in CCK-8 assay and western blot results were expressed as means standard deviation(SD). One-way enzyme transport and ANOVa was used for comparison among different concentration group and Fishers least significant difference(LSD)method was d transport and metabolism Translation ribosomal structure and used for multiple comparisons in CCK-8 assay. Independent biogenesis sample t-test was used to compare the treatment group andK Transcription control group in western blot assay. All calculations and L replication, recombination and analyses were performed using SPSS 20.0 software(SPSS Inc Chicago, IL, United States). A P-value 0.05 was considered M statistically significant. Cell motility 061 Posttranslational modification RESULTS protein tumover, chaperones Inorganic ion transport and 4.81 metabolism Isolation of bifidobacterium Secondary metabolites a total of 653 strains bacteria were isolated from the infant fecal samples. Among those, 23 viscous strains were isolated catabolism d transferred in a modified mrs-C medium. one strain R General function prediction only was typically rod-shaped or bifurcated, non-spore-forming. S Function unknown Consequently, this clone was analyzed using 16S rRNA PCR (16S T Signal transduction mechanisms rRNA-F: 5-AGAGTTTGATCMTGGCTCAG-3' and 16S rRNA- U Intracellular traficking, secretion, R: 5'-TACGGYTACCTTGTTACGACTT-3). The 16S RNA and vesicular transport sequence analyses showed 99% identity with B breve DSM20213 V 67 3.43 (Supplementary Figure S1) Extrace ular structures Mobilome: prophages, transposons Genome Information of b, breve lwo1 The complete genome sequence of our new isolated B breve strain was submitted to GenBank under the accession Number genes. Priming-GTF (P-atf) is the enzyme that catalyzes CP034192. The complete genome of this strain is composed of the initial step of EPS-unit synthesis; consequently,we one circular chromosome of 2, 313, 172 bp with a GC content of first found this enzyme: UDP-galactosephosphotransferase 58.7%, which is similar to other reported B breve strains(GC (EH245_02110)coding for proteins of 576 amino acids ntent of 58.5-62.8%). In addition, the new strain contains Five genes were coding for the GTFs(EH245_02130, total of 63 RNAs including 54 tRNA genes, 2 rRNA, and 3 EH245_02135, EH24502140, EH245_02145, EH245 02155 ncRNAs. A total of 1862 coding genes out of 2077 total genes Two types of genes could be responsible for transporting were classified into 23 COG categories(Tables 1, 2 and Figure 1). of the repeat EPS-units across the cytoplasmic membrane The genome comparative analyses among B breve lwol and other flippase(EH245_02150) and membrane spanning protein seven Bifidobacterium strains are presented in Supplementary (EH245-_02120, EH245-_02180). The chain length regulation Table S1. Consequently, this strain, named B. breve Iwol was and polymerization system were also found in this EPS deposited in China Center of Industrial Culture Collection unit, which included the polymerase(EH245-_02190), chain (CICC)under the number CICC 24633 length regulator(EH245-_02200)and the protein tyrosine phosphatase(EH245-_02115). Another discovered feature was In silico Analysis of Bifido-EPS Cluster the presence of transposase (EH245-_02125, EH245-02165 Bifidobacterium breve lwol harbors an EPS-encoding EH245-_02170, EH245-_02175, EH245-_02185), an enzyme cluster with 14 predicted genes and 5 transposase coding participated putative horizontal transfer of EPS among FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044

fmicb-10-01044 May 8, 2019 Time: 14:36 # 4 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 TABLE 1 | Features of B. breve lw01 genome. Features Chromosome Genome size (bp) 2,313,172 GC content (%) 58.7 RNAs 63 rRNAs 2, 2, 2 (5S, 16S, 23S) tRNAs 54 ncRNAs 3 CDSs (with protein) 1862 Statistical Analysis The absorbance value in CCK-8 assay and western blot results were expressed as means ± standard deviation (SD). One-way ANOVA was used for comparison among different concentration group and Fisher’s least significant difference (LSD) method was used for multiple comparisons in CCK-8 assay. Independent sample t-test was used to compare the treatment group and control group in western blot assay. All calculations and analyses were performed using SPSS 20.0 software (SPSS Inc., Chicago, IL, United States). A P-value < 0.05 was considered statistically significant. RESULTS Isolation of Bifidobacterium A total of 653 strains bacteria were isolated from the infant fecal samples. Among those, 23 viscous strains were isolated and transferred in a modified MRS-C medium. One strain was typically rod-shaped or bifurcated, non-spore-forming. Consequently, this clone was analyzed using 16S rRNA PCR (16S rRNA-F: 50 -AGAGTTTGATCMTGGCTCAG-30 and 16S rRNA￾R: 50 -TACGGYTACCTTGTTACGACTT-30 ). The 16S rRNA sequence analyses showed 99% identity with B. breve DSM20213 (Supplementary Figure S1). Genome Information of B. breve lw01 The complete genome sequence of our new isolated B. breve strain was submitted to GenBank under the accession Number CP034192. The complete genome of this strain is composed of one circular chromosome of 2,313,172 bp with a GC content of 58.7%, which is similar to other reported B. breve strains (GC content of 58.5–62.8%). In addition, the new strain contains a total of 63 RNAs including 54 tRNA genes, 2 rRNA, and 3 ncRNAs. A total of 1862 coding genes out of 2077 total genes were classified into 23 COG categories (Tables 1, 2 and Figure 1). The genome comparative analyses among B. breve lw01 and other seven Bifidobacterium strains are presented in Supplementary Table S1. Consequently, this strain, named B. breve lw01 was deposited in China Center of Industrial Culture Collection (CICC) under the number CICC 24633. In silico Analysis of Bifido-EPS Cluster Bifidobacterium breve lw01 harbors an EPS-encoding cluster with 14 predicted genes and 5 transposase coding TABLE 2 | COG categories of coding proteins in B. breve lw01 genome. COG Name Count Percent (%) A RNA processing and modification 2 0.10 C Energy production and conversion 53 2.71 D Cell cycle control, cell division, chromosome partitioning 31 1.59 E Amino acid transport and metabolism 209 10.69 F Nucleotide transport and metabolism 80 4.09 G carbohydrate transport and metabolism 253 12.94 H Coenzyme transport and metabolism 77 3.94 I Lipid transport and metabolism 54 2.76 J Translation, ribosomal structure and biogenesis 167 8.54 K Transcription 153 7.83 L Replication, recombination and repair 90 4.60 M Cell wall/membrane/envelope biogenesis 106 5.42 N Cell motility 12 0.61 O Posttranslational modification, protein turnover, chaperones 74 3.79 P Inorganic ion transport and metabolism 94 4.81 Q Secondary metabolites biosynthesis, transport and catabolism 21 1.07 R General function prediction only 153 7.83 S Function unknown 93 4.76 T Signal transduction mechanisms 92 4.71 U Intracellular trafficking, secretion, and vesicular transport 15 0.77 V Defense mechanisms 67 3.43 W Extracellular structures 4 0.20 X Mobilome: prophages, transposons 55 2.81 genes. Priming-GTF (p-gtf) is the enzyme that catalyzes the initial step of EPS-unit synthesis; consequently, we first found this enzyme: UDP-galactosephosphotransferase (EH245_02110) coding for proteins of 576 amino acids. Five genes were coding for the GTFs (EH245_02130, EH245_02135, EH245_02140, EH245_02145, EH245_02155). Two types of genes could be responsible for transporting of the repeat EPS-units across the cytoplasmic membrane: flippase (EH245_02150) and membrane spanning protein (EH245_02120, EH245_02180). The chain length regulation and polymerization system were also found in this EPS￾unit, which included the polymerase (EH245_02190), chain length regulator (EH245_02200) and the protein tyrosine phosphatase (EH245_02115). Another discovered feature was the presence of transposase (EH245_02125, EH245_02165, EH245_02170, EH245_02175, EH245_02185), an enzyme participated putative horizontal transfer of EPS among Frontiers in Microbiology | www.frontiersin.org 4 May 2019 | Volume 10 | Article 1044

Wang et al Anticancer Exopolysaccharide From B breve Iwo1 General function prediction only Signal transduction mechanisms Coenzyme transport and metabolism Inorganic ion transport and metabolism Carbohydrate transport and metabolism Amino acid transport and metabolism Secondary metabolites biosynthesis, transport and catabolism B breve lw01 I L Nucleotide transport and metabolism 2,313,172bp ■ Replication,rec Cell wall/membrane/envelope biogenesis Posttranslational modification, protein turnover, chaperones Cell cycle control, cell division, chromosome partitioning Translation, ribosomal structure and biogenesis Intracellular trafficking, secretion, and vesicular transport ■ Defense mechanisms Lipid transport and metabolism Chromatin structure and dynamics FIGURE 1 Circle genome map of B. breve lwo1. Genome sequences(ring 1). COG Annotated coding sequences (ring 2 and 3), KEGG enzymes(ring 4), RNA genes (ring 5), GC content (ring 6), and GC skew (ring 7) are shown. very short features were enlarged to enhance the visibility. Clustered genes, such as several rRNA genes, may appear as one-ine due to space limitations. Image was created by the software Circs. different taxa. There were also several genes coding for the bonds by transferring new sugar moieties from a donor precursor involved in the biosynthesis of EPS-unit: thiamine nucleotide sugar to the initial monosaccharide of the unit. pyrophosphate enzyme (EH245-_02160) and acyltransferase A chain length regulator and polymerization system then (EH245_02195)( Figure2A) sembled the EPS-lipophilic carrier unit and formed the finished We compared the EPS cluster information with other seven EPS-unit In the third exporting module, flippase and membrane Bifidobacteium strains(Supplementary Table S1). Interestingly, spanning protein helped this finished EPS-unit to cross the the EPS cluster region has a G+C DNA content(49.7%) cytoplasmic membrane to the extracellular face and build the final markedly lower than the B. breve strains(GC content of 58.5- EPS polymers(Figure 2B) 62.8%), which inferred that it may be acquired by horizontal DNA transfer. The comparison of amino acid sequences of p- Extraction Purification and Analysis of tf showed that it was quite similar to the strain B. longum NCC2705(with 98% identity). Partial amino acid identity of EPS P-gtf among B. breve lwOl and other seven Bifidobacterium Crude EPS extracted from 1 L of 10% skimmed milk was about stains is shown in Figure 3. The results showed that they 80 mg. Only one kind of EPS fraction was obtained from Ion process the similar sequences particularly in the carboxy exchange chromatography with 0.1 mol/L NaCl solution,which appeared at 8-18 tubes(about 50 mL). When the elution tubes exceeded 50 tubes and the concentration of NaCl was abov Hypothetical Pathway of EPs 0.3 M, no obvious polysaccharide component was eluted. After Biosynthesis in B. breve lw01 Ion exchange chromatography, we yielded about 26 mg EPS. After gel filtration chromatography and HPLC purification, 5 The hypothetical pathway of EPS biosynthesis in B. breve and 1 mg EPS were obtained, respectively. The single and the precursor synthesis. After glycose and lactose fluxed symmetric peak showed that the EPS was purified with high Iw01 was divided into three modules. First module was into the cytoplasm, activated NDP-sugar precursors modules quality(Figure 4). were formed from the intermediate molecules of glycolysis Second module was the assembling module. EPS-unit was Scanning Electron Microscopy Images of catalyzed by p-gtf enzyme(UDP-galactosephosphotransferase). EPS From B. breve lw01 with the help of p-gtf(UDP-galactosephosphotransferase), one Scanning electron microscope is a useful method to sugar-l-phosphate was transferred to the lipophilic carrier investigate the surface and three-dimensional morphology molecule and anchored to the cell membrane. Other five of biomacromolecules. The morphology of EPS observed by glycosyltransferases(GTFs)catalyzed the formation of glycosidic SEM showed that EPS was mainly composed of freely distributed FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044

fmicb-10-01044 May 8, 2019 Time: 14:36 # 5 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 FIGURE 1 | Circle genome map of B. breve lw01. Genome sequences (ring 1). COG Annotated coding sequences (ring 2 and 3), KEGG enzymes (ring 4), RNA genes (ring 5), GC content (ring 6), and GC skew (ring 7) are shown. Very short features were enlarged to enhance the visibility. Clustered genes, such as several rRNA genes, may appear as one-line due to space limitations. Image was created by the software Circos. different taxa. There were also several genes coding for the precursor involved in the biosynthesis of EPS-unit: thiamine pyrophosphate enzyme (EH245_02160) and acyltransferase (EH245_02195) (Figure 2A). We compared the EPS cluster information with other seven Bifidobacteium strains (Supplementary Table S1). Interestingly, the EPS cluster region has a G+C DNA content (49.7%) markedly lower than the B. breve strains (GC content of 58.5– 62.8%), which inferred that it may be acquired by horizontal DNA transfer. The comparison of amino acid sequences of p￾gtf showed that it was quite similar to the strain B. longum NCC2705 (with 98% identity). Partial amino acid identity of p-gtf among B. breve lw01 and other seven Bifidobacterium stains is shown in Figure 3. The results showed that they process the similar sequences particularly in the carboxy terminus regions. Hypothetical Pathway of EPS Biosynthesis in B. breve lw01 The hypothetical pathway of EPS biosynthesis in B. breve lw01 was divided into three modules. First module was the precursor synthesis. After glycose and lactose fluxed into the cytoplasm, activated NDP-sugar precursors modules were formed from the intermediate molecules of glycolysis. Second module was the assembling module. EPS-unit was catalyzed by p-gtf enzyme (UDP-galactosephosphotransferase). With the help of p-gtf (UDP-galactosephosphotransferase), one sugar-1-phosphate was transferred to the lipophilic carrier molecule and anchored to the cell membrane. Other five glycosyltransferases (GTFs) catalyzed the formation of glycosidic bonds by transferring new sugar moieties from a donor nucleotide sugar to the initial monosaccharide of the unit. A chain length regulator and polymerization system then assembled the EPS-lipophilic carrier unit and formed the finished EPS-unit. In the third exporting module, flippase and membrane spanning protein helped this finished EPS-unit to cross the cytoplasmic membrane to the extracellular face and build the final EPS polymers (Figure 2B). Extraction, Purification and Analysis of EPS Crude EPS extracted from 1 L of 10% skimmed milk was about 80 mg. Only one kind of EPS fraction was obtained from Ion exchange chromatography with 0.1 mol/L NaCl solution, which appeared at 8–18 tubes (about 50 mL). When the elution tubes exceeded 50 tubes and the concentration of NaCl was above 0.3 M, no obvious polysaccharide component was eluted. After Ion exchange chromatography, we yielded about 26 mg EPS. After gel filtration chromatography and HPLC purification, 5 and 1 mg EPS were obtained, respectively. The single and symmetric peak showed that the EPS was purified with high quality (Figure 4). Scanning Electron Microscopy Images of EPS From B. breve lw01 Scanning electron microscope is a useful method to investigate the surface and three-dimensional morphology of biomacromeolecules. The morphology of EPS observed by SEM showed that EPS was mainly composed of freely distributed Frontiers in Microbiology | www.frontiersin.org 5 May 2019 | Volume 10 | Article 1044