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 EPSencoding 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; HAPECPAD, 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 stainspecific (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 EPSproducing 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 WSUHN6 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 rRNAR: 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 EPSunit, 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 pgtf 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
Wang et al Anticancer Exopolysaccharide From B breve Iwo1 UDP-galactosephosphotransferase Protein tyrosine phosphatase Membrane spanning protein Glycosyltransferase Thiamine pyrophosphate enzyme Polymerase Acyltransferase Chain length regulator Transcription and Translation Membrane-panning protein Flippase Glycosyltransferase Polymerization Chain length regulator Glucose or Lactose Out 2888 In Lipid carrier Chain length determination 2. Assembling module Glucose or Lactose +GTF 3. Exporting module GDP-mannose UDP-arabinose UDP-glucose +UDP-galactose TDP-rhamnose mannose-6-P arabinose-1-P EMP+Glucose-6-P -Glucose-1-P+ TDP-glucose--TDP-4K-6D-mannosel 1. Precursor synthesis module FIGURE 2 Physical map of the putative EPS cluster of B breve lwo1 and a schematic diagram of EPS biosynthesis in this strain. (A) The genes were categorize ccording to their potential functions, which are indicated with the colored arrows.(B)Hypothetical EPs biosynthesis pathway in strain B breve l01 based on its mology analysis of the EPS cluster. B breve lw01 B breve UCC2003 um NCC2705 B infantis ATCC 15697 B asteroides PRL2011 B/actis DSM 10140 Q qf nv kgdms vgprp ev y I ys riV pgi t gpwq sgr B FIGURE 3 Algnment of an fragment of amino acid sequence of priming- GTF in B breve Mo1 and other Bifidobacterium strains. B at the bottom represented blocks involved in interaction with lipid carrier and c represented blocks involved in sugar-specific recognition. irregular spherical bodies, flakes and nets. EPS was relatively of branches. The use of different methods ough, and was composed of depressions and voids, which may of extraction and purification of EPS could in the different be due to the nature and structure of EPS, and the formation ultrastructure( Figure 5) FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044
fmicb-10-01044 May 8, 2019 Time: 14:36 # 6 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 FIGURE 2 | Physical map of the putative EPS cluster of B. breve lw01 and a schematic diagram of EPS biosynthesis in this strain. (A) The genes were categorized according to their potential functions, which are indicated with the colored arrows. (B) Hypothetical EPS biosynthesis pathway in strain B. breve lw01 based on its homology analysis of the EPS cluster. FIGURE 3 | Alignment of an fragment of amino acid sequence of priming-GTF in B. breve lw01 and other Bifidobacterium strains. B at the bottom represented blocks involved in interaction with lipid carrier and C represented blocks involved in sugar-specific recognition. irregular spherical bodies, flakes and nets. EPS was relatively rough, and was composed of depressions and voids, which may be due to the nature and structure of EPS, and the formation of branches. The use of different methods in the process of extraction and purification of EPS could cause different ultrastructure (Figure 5). Frontiers in Microbiology | www.frontiersin.org 6 May 2019 | Volume 10 | Article 1044
Wang et al Anticancer Exopolysaccharide From B breve Iwo1 三1.0 FIGURE 5 Scanning electron microscope images of purified EPS from B breve lo1.(A)Magnification 500x(B) In part magnified of A magnification2000×) 051015202530354045 606570758085 Tube numbers (5 mL/tube HPAEC spectra of standard monosaccharides(Figure 6). The constitution of our EPS was similar to the EPS composition produced by B animalis RH (Shang et al, 2013). Anticancer Property of B. breve lw01's EPS on HNsCC Cell Lines Anti-proliferation Effect in Aerobic and Anaerobic Environment CCK-8 assay and dAPI staining were used to examine the anti tumor effect of eps on hnscc cell lines. ccK-8 results showed that EPS could inhibit cell proliferation in a concentration dependent manner. However, different cell line showed different sensitivity to EPS. The highest effect was observed in WSU HN6, followed by SCC15 and CAL 27. On day 5, EPS inhibited 0 5 10 15 20 25 30 3 40 4s so ss (0 6s 70 75 s0 85 90 the growth of WSU-HN6 by 46% at 200 ug/mL in aerobic environment. For CAL 27 cell line. in normoxia condition 800 ug/mL EPS could inhibit CAL 27 cell line proliferation slightly, while the proliferation rate drastically decreased when culturing cells under hypoxic condition. This strengthened effect could be observed in the other cell lines as well(Figure 7) Furthermore, DAPI staining of cell nuclear with different treatment concentration presented the same tendency when cell lines were cultured in normoxia condition( Figure 8) Cell Cycle and Apoptosis-Associated Protein Expression In order to find the detailed mechanism on the anti-proliferation effect of EPS, we tested the expression of cell-cycle and apoptosis associated protein by western bolt assay in a represented cell line FIGURE 4 Purification and analysis of EPS. (A) lon exchange SCC15 in normoxia condition. Decreased expression of MCM2 chromatography.( B)gel filtration chromatography purification. (C)HPLC purification. suggested that the cell cycle arrest occurs at G1-S phase Increased expression of cell apoptosis protein caspase 3, PARP and the proportion of Cl-PARP/PARP inferred that EPS could promote the cell apoptosis( Figure 9). Chemical Composition of B. breve lwo1's EPS DISCUSSION The monomer composition of B. breve lwo1's EPS wa determined by HPAEC-PAD. The results of monomer analysis Currently, EPS obtained from probiotic bacteria Bifidobacterium indicated that EPS was a heteropolysaccharide mainly composed have been gaining increasing attention, mainly due to their of rhamnose, arabinose, galactose, glucose and mannose in role in immune system and gut microbiota modulation. In molar ratio of 0.35: 0.44: 1.38: 0.67: 1.65 compared with the the present study, we isolated a new B. breve strain FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044
fmicb-10-01044 May 8, 2019 Time: 14:36 # 7 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 FIGURE 4 | Purification and analysis of EPS. (A) Ion exchange chromatography. (B) gel filtration chromatography purification. (C) HPLC purification. Chemical Composition of B. breve lw01’s EPS The monomer composition of B. breve lw01’s EPS was determined by HPAEC-PAD. The results of monomer analysis indicated that EPS was a heteropolysaccharide mainly composed of rhamnose, arabinose, galactose, glucose and mannose in a molar ratio of 0.35:0.44:1.38:0.67:1.65 compared with the FIGURE 5 | Scanning electron microscope images of purified EPS from B. breve lw01. (A) Magnification 500×. (B) In part magnified of A (magnification 2000×). HPAEC spectra of standard monosaccharides (Figure 6). The constitution of our EPS was similar to the EPS composition produced by B. animalis RH (Shang et al., 2013). Anticancer Property of B. breve lw01’s EPS on HNSCC Cell Lines Anti-proliferation Effect in Aerobic and Anaerobic Environment CCK-8 assay and DAPI staining were used to examine the antitumor effect of EPS on HNSCC cell lines. CCK-8 results showed that EPS could inhibit cell proliferation in a concentration dependent manner. However, different cell line showed different sensitivity to EPS. The highest effect was observed in WSUHN6, followed by SCC15 and CAL 27. On day 5, EPS inhibited the growth of WSU-HN6 by 46% at 200 µg/mL in aerobic environment. For CAL 27 cell line, in normoxia condition, 800 µg/mL EPS could inhibit CAL 27 cell line proliferation slightly, while the proliferation rate drastically decreased when culturing cells under hypoxic condition. This strengthened effect could be observed in the other cell lines as well (Figure 7). Furthermore, DAPI staining of cell nuclear with different treatment concentration presented the same tendency when cell lines were cultured in normoxia condition (Figure 8). Cell Cycle and Apoptosis-Associated Protein Expression In order to find the detailed mechanism on the anti-proliferation effect of EPS, we tested the expression of cell-cycle and apoptosis associated protein by western bolt assay in a represented cell line SCC15 in normoxia condition. Decreased expression of MCM2 suggested that the cell cycle arrest occurs at G1-S phase. Increased expression of cell apoptosis protein caspase 3, PARP and the proportion of Cl-PARP/PARP inferred that EPS could promote the cell apoptosis (Figure 9). DISCUSSION Currently, EPS obtained from probiotic bacteria Bifidobacterium have been gaining increasing attention, mainly due to their role in immune system and gut microbiota modulation. In the present study, we isolated a new B. breve strain from Frontiers in Microbiology | www.frontiersin.org 7 May 2019 | Volume 10 | Article 1044
Wang et al Anticancer Exopolysaccharide From B breve Iwo1 1-Ara.2011 10 24 5Ma=11521 6420 246 224 FIGURE 6 Chromatogram of standard monosaccharide(A) and EPS monosaccharide compositions(B)on HPAEC-PAD. From left to right: 1. Rhamnose 2. Arabinose 3. Galactose 4. Glucose 5 Mannose 6. Fructose infant feces. After sequencing its genome, we predicted its 2016; Bengoa et al., 2018). In our study, slimmed milk was chosen EPS cluster and hypothetic synthesis pathway. Based on the for the initial cultivation to avoid sugar in the medium that EPS extraction and high-quality purification, we determined interferes with the EPS extraction. Crude bifido-EPS was followed its chemical composition. Finally, we focused our attention on by the further purification(Ion exchange chromatography and its anticancer activity and provided biochemical evidence to gel filtration chromatography), which yielded high purity bifido- explain it. Thus, this study provided a better understanding of EPS, thus providing good research basis for the biological activity. the function-genome cluster relation and biosynthesis in bifido- Multiple purification induced the poor yield, which was affected EPS, which could facilitate genetic and metabolic engineering by many factors such as EPS sample loading volume, eluent indispensable for production of tailor-made EPS used in food and elution rate. In the course of the research, we observed or drug industry that the low purification efficiency affected our further research Extraction and purification were necessary to examine the Therefore, future studies should focus on the elevation of the biological activity of B breve lwO1's EPS. The yield of crude EPs purification efficiency of EPS isolation. released to culture medium can be influenced by environmental Previous studies have indicated that genes responsible for the and nutritional conditions, such as the medium composition, pH, biosynthesis of EPS are presented in the form of clusters in temperature, oxygen and the growth phase (Leroy and De vuyst, Bifidobacterium genome(Leivers et al, 2011; Fanning et al., 201 FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044
fmicb-10-01044 May 8, 2019 Time: 14:36 # 8 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 FIGURE 6 | Chromatogram of standard monosaccharide (A) and EPS monosaccharide compositions (B) on HPAEC-PAD. From left to right: 1. Rhamnose 2. Arabinose 3. Galactose 4. Glucose 5. Mannose 6. Fructose. infant feces. After sequencing its genome, we predicted its EPS cluster and hypothetic synthesis pathway. Based on the EPS extraction and high-quality purification, we determined its chemical composition. Finally, we focused our attention on its anticancer activity and provided biochemical evidence to explain it. Thus, this study provided a better understanding of the function-genome cluster relation and biosynthesis in bifidoEPS, which could facilitate genetic and metabolic engineering indispensable for production of tailor-made EPS used in food or drug industry. Extraction and purification were necessary to examine the biological activity of B. breve lw01’s EPS. The yield of crude EPS released to culture medium can be influenced by environmental and nutritional conditions, such as the medium composition, pH, temperature, oxygen and the growth phase (Leroy and De Vuyst, 2016; Bengoa et al., 2018). In our study, slimmed milk was chosen for the initial cultivation to avoid sugar in the medium that interferes with the EPS extraction. Crude bifido-EPS was followed by the further purification (Ion exchange chromatography and gel filtration chromatography), which yielded high purity bifidoEPS, thus providing good research basis for the biological activity. Multiple purification induced the poor yield, which was affected by many factors such as EPS sample loading volume, eluent and elution rate. In the course of the research, we observed that the low purification efficiency affected our further research. Therefore, future studies should focus on the elevation of the purification efficiency of EPS isolation. Previous studies have indicated that genes responsible for the biosynthesis of EPS are presented in the form of clusters in Bifidobacterium genome (Leivers et al., 2011; Fanning et al., 2012; Frontiers in Microbiology | www.frontiersin.org 8 May 2019 | Volume 10 | Article 1044
Wang et al Anticancer Exopolysaccharide From B breve Iwo1 WSU-HN6 normoxia B . 80D pg/mL Time (Day) WSU-HN6 hypoxia SCC15 hy poxia 800 ugmL Time(Day) FIGURE 7 Cell proliferation inhibition of EPS from B. breve lol by CCK-8 assay(A-C)Three HNSCC cel lines were cultured in normoxia condition and treated by gradient concentration of EPs from B breve lo1(D-F)Three HNSCC cell lines were cultured in hypoxia condition. All the experiments were run in triplicate p<0.05;p<0.01 Hidalgo-Cantabrana et al, 2014). Due to the high interspecies association between the genetics and physico-chemical variability existence, and deep excavation of genome sequences characteristics (Hidalgo-Cantabrana et al., 2014). Moreover, from different database, the comparison-selected results were in this work, we did not focus on the structure of EPS used to examine the gene cluster responsible for EPS synthesis. Accordingly, future studies should focus on investigating We find complete EPs gene cluster was involved in the the genetics and physico-chemical correlation based on EPS biosynthesis of EPS and the gene cluster could be divided genome-composition-structure characters to three following groups: the gene responsible for repeated In our previous study, we already verified that the strain unit synthesis, polymerization/chain length determination and B. breve possess an anti-tumor effect on HNSCC tumor exportation. No transcriptional regulator was observed as bearing model(Wang et al, 2017). Yet, the exact mechanism in other Bifidobacterium(Ruas-Madiedo, 2014). The EPs was not fully explored. Existing studies have shown that the biosynthesis pathway was similar but not identical to that presence of bacterial molecules structure(DNA, proteins, EPS reported in B. breve UCC2003. In B. breve UCC2003, the or metabolites)could have a specific effect on the immune or EPS synthesis was bidirectional and responsible for production inflammation system(Menard et al, 2010; Hevia et al., 2014; of one or two polymers(Fanning et al., 2012). For the Hidalgo-Cantabrana et al, 2014). In the present study, we further confirmation of the exact function of each gene further examined whether the new extracted bifido-EPS may b involved in our bifdo-EPS gene cluster, mutants by deletion responsible for the anticancer bioactivity part of the EPS clusters could be used by comparing Currently, there are only few studies on the anticancer effect to their counterparts. of EPS from Bifidobacterium strains. In vitro experiments have Among the biosynthesis pathway, repeated unit synthesis shown that polysaccharide fraction extracted from B. bifidum was the key step to determine the final EPS-unit constitution BGN4 could inhibit the growth of colon cancer cell lines HT and GTF was the key enzyme involved in this part. The 29 and HCT-116, but had no effect on Caco-2 cell line. Previous amino acid identity of p-gtf among B. breve lwo1 and study has reported that the inhibition rate on HT-29 cell line other seven Bifidobacterium stains was lower. The similar was 50.5 3.6% at 80 ug/mL by Trypan blue exclusion assay sequences were observed, particularly in the carboxy terminus It is speculated that the anticancer effect may be related to the regions, due to the presence of regions involved in interaction inhibition of DNA synthesis and reduction of cell proliferation with lipid carrier (region B)and sugar specificity (region capacity(You et al, 2004). The research from Nam Joo Has group C)(Provencher et al, 2003; Yan et al, 2017). However, showed that butanol extract of B. adolescentis SPM0212 could the composition and the structure of bifido-EPS have not inhibit the growth of Caco-2, HT-29, and Sw480 cells by 70, 30, et been fully illustrated for any of the strains containing and 40%, respectively, at 200 ug/mL. The mechanism may rely on completed genomes (Hidalgo-Cantabrana et al, 2014). inducing macrophage activation and increasing the production Therefore, although the numbers and specificities of GTEs TNF-a and NO (Lee et al, 2008). In the present study, we could contribute to the different EPS-unit constitution investigated anticancer effect of bifido-EPS on HNSCC cell line, d structures, it is still not possible to fully establish the which exerted proliferation inhibition effect in dose-dependent FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044
fmicb-10-01044 May 8, 2019 Time: 14:36 # 9 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 FIGURE 7 | Cell proliferation inhibition of EPS from B. breve lw01 by CCK-8 assay. (A–C) Three HNSCC cell lines were cultured in normoxia condition and treated by gradient concentration of EPS from B. breve lw01. (D–F) Three HNSCC cell lines were cultured in hypoxia condition. All the experiments were run in triplicate. ∗p < 0.05; ∗p < 0.01. Hidalgo-Cantabrana et al., 2014). Due to the high interspecies variability existence, and deep excavation of genome sequences from different database, the comparison-selected results were used to examine the gene cluster responsible for EPS synthesis. We find complete EPS gene cluster was involved in the biosynthesis of EPS and the gene cluster could be divided into three following groups: the gene responsible for repeated unit synthesis, polymerization/chain length determination and exportation. No transcriptional regulator was observed as in other Bifidobacterium (Ruas-Madiedo, 2014). The EPS biosynthesis pathway was similar but not identical to that reported in B. breve UCC2003. In B. breve UCC2003, the EPS synthesis was bidirectional and responsible for production of one or two polymers (Fanning et al., 2012). For the further confirmation of the exact function of each gene involved in our bifdo-EPS gene cluster, mutants by deletion part of the EPS clusters could be used by comparing to their counterparts. Among the biosynthesis pathway, repeated unit synthesis was the key step to determine the final EPS-unit constitution and GTF was the key enzyme involved in this part. The amino acid identity of p-gtf among B. breve lw01 and other seven Bifidobacterium stains was lower. The similar sequences were observed, particularly in the carboxy terminus regions, due to the presence of regions involved in interaction with lipid carrier (region B) and sugar specificity (region C) (Provencher et al., 2003; Yan et al., 2017). However, the composition and the structure of bifido-EPS have not yet been fully illustrated for any of the strains containing completed genomes (Hidalgo-Cantabrana et al., 2014). Therefore, although the numbers and specificities of GTFs could contribute to the different EPS-unit constitution and structures, it is still not possible to fully establish the association between the genetics and physico-chemical characteristics (Hidalgo-Cantabrana et al., 2014). Moreover, in this work, we did not focus on the structure of EPS. Accordingly, future studies should focus on investigating the genetics and physico-chemical correlation based on EPS genome-composition-structure characters. In our previous study, we already verified that the strain B. breve possess an anti-tumor effect on HNSCC tumorbearing model (Wang et al., 2017). Yet, the exact mechanism was not fully explored. Existing studies have shown that the presence of bacterial molecules structure (DNA, proteins, EPS or metabolites) could have a specific effect on the immune or inflammation system (Menard et al., 2010; Hevia et al., 2014; Hidalgo-Cantabrana et al., 2014). In the present study, we further examined whether the new extracted bifido-EPS may be responsible for the anticancer bioactivity. Currently, there are only few studies on the anticancer effect of EPS from Bifidobacterium strains. In vitro experiments have shown that polysaccharide fraction extracted from B. bifidum BGN4 could inhibit the growth of colon cancer cell lines HT- 29 and HCT-116, but had no effect on Caco-2 cell line. Previous study has reported that the inhibition rate on HT-29 cell line was 50.5 ± 3.6% at 80 µg/mL by Trypan blue exclusion assay. It is speculated that the anticancer effect may be related to the inhibition of DNA synthesis and reduction of cell proliferation capacity (You et al., 2004). The research from Nam Joo Ha’s group showed that butanol extract of B. adolescentis SPM0212 could inhibit the growth of Caco-2, HT-29, and SW480 cells by 70, 30, and 40%, respectively, at 200 µg/mL. The mechanism may rely on inducing macrophage activation and increasing the production TNF-α and NO (Lee et al., 2008). In the present study, we investigated anticancer effect of bifido-EPS on HNSCC cell line, which exerted proliferation inhibition effect in dose-dependent Frontiers in Microbiology | www.frontiersin.org 9 May 2019 | Volume 10 | Article 1044
Wang et al Anticancer Exopolysaccharide From B breve Iwo1 0 Hg/mL 200 Hg/mL 8O pg/mL A SCC15 SCC15 normoxia (ug/mL) 了0 MCM2 ■400ygmL aspase SCC15 PARP CL-PARP 0 CAL 2 RPS18 FIGURE 8 DAPI staining images of cell nuclear after treated with gradie concentration of eps al the cells were cultured in normoxia condition FIGURE 9 I Cell cycle and apoptosis protein expression after treating SCC15 cell line with 400 ug/mL EPS. (A)Western blot assay.( B)Relative fol magnification 4x changes by calculating relative gray value using software Quantity one."p<0.05. manner. In addition, different cells presented different sensitivity to bifido-EPS et al, 2017). Besides, rhamnose has also been reported to have As for the mechanisms of the anti-cancer property of anti-tumor activity, since it can enhance the immunogenicity EPS, except for the inhibition of DNA synthesis and immune of melanoma-associated antigen A3, which stimulates antitumor modulation reported above, EPS has anti-oxidative properties immune responses(Zhang et al, 2016). The galactose is widely and can inhibit the expression of gene involved in angiogenesis used backbone of the nanocarrier in cancer therapy, beneficial for Deepak et al., 2016) Cell proliferative activity is closely related to targeted therapy of tumors (ain et al, 2018). When conjugated the cell cycle, which is regulated by the DNA replication initiation with platinum(II), the complex has well therapeutic and target factor. MCM 2, known as DNA replication licensing factor, is effect(Wu et al, 2016). In our next study, we plan to further involved in pre-replicative complex formation during Gl phase investigate the effect of our bifido-EPS in vivo d it allows for the DNA replication initiation in the subsequent To sum up, the current work isolated one new probiotic S-Phase(Gambus et al., 2011). Downregulation of MCM2 could strain B. breve lwOl and sequenced its genome, which led to increase cell death when the cells encounter replication stress discovery of a new EPS biosynthesis cluster and prediction of during S-phase(Ibarra et al., 2008). MCM2 has also been applied its biosynthesis pathway. After extraction and purification, higl as a proliferation marker in many types of cancer(Tan et al., purity of our EPS showed to possess an anticancer activity in 2001; Yousef et al, 2017; Zhou et al., 2018). In addition, its HNSCC cell lines. In addition, the composition of bifido-EPS downregulation is considered as an attractive target in tumor showed that mannose accounted for the largest proportion. The chemotherapy(Simon and Schwacha, 2014). In this study, the preliminary mechanisms could rely on its cell cycle arrest and expression of MCM2 was downregulated, which suggested that promote cell apoptosis effect. We believe that this bifido-EPS could arrest cell cycle in Gl-S phase in HNSCC cell line. could be used to facilitate genetic and metabolic engineering and PARP is activated when cells sustained dna damage and it is the to produce tailor-made EPS for further application in functional main target of caspase 3, whose activation could mediate a latent food or drug industry specific proteolytic cleavage in apoptosis(Brauns et al., 2005; Yu et al, 2006). In our study, EPS increased the expression of caspase 3 and the cleaved-PARP proportion, while inferred cell apoptosis ETHICS STATEMENT occurred in treatment group. The chemical composition of our EPS, which included The protocol was approved Biomedical Ethics rhamnose, arabinose, galactose, glucose, and mannose, could Committee of Peking University School and Hospital of explain its anti-tumor effect to some extent. It is quite interesting Stomatology. Written informed was obtained from that mannose accounted for the largest proportion of the bifido- parents of the infant. EPS, which have been reported to have a significant anti-tumor activity. Mannose could impair tumor growth by inhibiting the proliferation while promoting the apoptosis of cancer cell. AUTHOR CONTRIBUTIONS It also revealed chemo-sensitization character. Administration of doxorubicin with mannose showed an obviously enhanced GL and CG contributed the conception and design of the anti-tumor effect(Gonzalez et al., 2018). Moreover, the level study. Lw and Kt organized the database and analysis of mannose in serum is associated with the prognosis of EPs cluster information. Yw, QL, and LX carried out esophageal adenocarcinoma patients. The patients with low level the study. Yw performed the statistical of d-mannose have higher risk of recurrence and death(Gu wrote section of the manuscript. Lw wrote FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044
fmicb-10-01044 May 8, 2019 Time: 14:36 # 10 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 FIGURE 8 | DAPI staining images of cell nuclear after treated with gradient concentration of EPS. All the cells were cultured in normoxia condition. magnification 4×. manner. In addition, different cells presented different sensitivity to bifido-EPS. As for the mechanisms of the anti-cancer property of EPS, except for the inhibition of DNA synthesis and immune modulation reported above, EPS has anti-oxidative properties and can inhibit the expression of gene involved in angiogenesis (Deepak et al., 2016). Cell proliferative activity is closely related to the cell cycle, which is regulated by the DNA replication initiation factor. MCM 2, known as DNA replication licensing factor, is involved in pre-replicative complex formation during G1 phase and it allows for the DNA replication initiation in the subsequent S-phase (Gambus et al., 2011). Downregulation of MCM2 could increase cell death when the cells encounter replication stress during S-phase (Ibarra et al., 2008). MCM2 has also been applied as a proliferation marker in many types of cancer (Tan et al., 2001; Yousef et al., 2017; Zhou et al., 2018). In addition, its downregulation is considered as an attractive target in tumor chemotherapy (Simon and Schwacha, 2014). In this study, the expression of MCM2 was downregulated, which suggested that EPS could arrest cell cycle in G1-S phase in HNSCC cell line. PARP is activated when cells sustained DNA damage and it is the main target of caspase 3, whose activation could mediate a latent specific proteolytic cleavage in apoptosis (Brauns et al., 2005; Yu et al., 2006). In our study, EPS increased the expression of caspase 3 and the cleaved-PARP proportion, while inferred cell apoptosis occurred in treatment group. The chemical composition of our EPS, which included rhamnose, arabinose, galactose, glucose, and mannose, could explain its anti-tumor effect to some extent. It is quite interesting that mannose accounted for the largest proportion of the bifidoEPS, which have been reported to have a significant anti-tumor activity. Mannose could impair tumor growth by inhibiting the proliferation while promoting the apoptosis of cancer cell. It also revealed chemo-sensitization character. Administration of doxorubicin with mannose showed an obviously enhanced anti-tumor effect (Gonzalez et al., 2018). Moreover, the level of mannose in serum is associated with the prognosis of esophageal adenocarcinoma patients. The patients with low level of d-mannose have higher risk of recurrence and death (Gu FIGURE 9 | Cell cycle and apoptosis protein expression after treating SCC15 cell line with 400 µg/mL EPS. (A) Western blot assay. (B) Relative fold changes by calculating relative gray value using software Quantity One. ∗p < 0.05. et al., 2017). Besides, rhamnose has also been reported to have anti-tumor activity, since it can enhance the immunogenicity of melanoma-associated antigen A3, which stimulates antitumor immune responses (Zhang et al., 2016). The galactose is widely used backbone of the nanocarrier in cancer therapy, beneficial for targeted therapy of tumors (Jain et al., 2018). When conjugated with platinum (II), the complex has well therapeutic and target effect (Wu et al., 2016). In our next study, we plan to further investigate the effect of our bifido-EPS in vivo. To sum up, the current work isolated one new probiotic strain B. breve lw01 and sequenced its genome, which led to discovery of a new EPS biosynthesis cluster and prediction of its biosynthesis pathway. After extraction and purification, high purity of our EPS showed to possess an anticancer activity in HNSCC cell lines. In addition, the composition of bifido-EPS showed that mannose accounted for the largest proportion. The preliminary mechanisms could rely on its cell cycle arrest and promote cell apoptosis effect. We believe that this bifido-EPS could be used to facilitate genetic and metabolic engineering and to produce tailor-made EPS for further application in functional food or drug industry. ETHICS STATEMENT 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. AUTHOR CONTRIBUTIONS GL and CG contributed the conception and design of the study. LW and KT organized the database and analysis EPS cluster information. YW, QL, and LX carried out the study. YW performed the statistical analysis and wrote section of the manuscript. LW wrote the first draft Frontiers in Microbiology | www.frontiersin.org 10 May 2019 | Volume 10 | Article 1044
