Adeline Sivignon

Shin-Yi Yu

Nathalie Ballet

Pascal Vandekerckove

Nicolas Barnich

Yann Guerardel

Heteropolysaccharides from S. cerevisiae show anti-adhesive properties against E. coli associated with Crohn's disease

Keywords: mannan-glucan heteropolysaccharides, Adherent-Invasive Escherichia coli, Saccharomyces cerevisiae, anti-adhesive strategy, NMR analysis AIEC, adherent-invasive Escherichia coli, CD, Crohn's disease, CEACAM6, carcinoembryonic antigen-related cell adhesion molecule 6, YCW, yeast cell wall, Man, mannose, Glc, glucose, PPM, phospho-peptido-mannan

Introduction

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC) cause a dysfunction of the immune response to gut microbiota occurs. Among those, Crohn's disease (CD) leads to a reduction of global microbial diversity and an increase in mucosal bacteria in patients [START_REF] Gevers | The treatment-naive microbiome in new-onset Crohn's disease[END_REF][START_REF] Manichanh | Reduced diversity of faecal microbiota in Crohn's disease revealed by a metagenomic approach[END_REF][START_REF] Sommer | Microbiomarkers in inflammatory bowel diseases: caveats come with caviar[END_REF]. Into this context, ileal mucosa-associated microbiota at time of surgery was shown to be predictor of post-operative endoscopic recurrence in CD [START_REF] Sokol | Prominence of ileal mucosa-associated microbiota to predict postoperative endoscopic recurrence in Crohn's disease[END_REF]. Among this mucosa-associated community, Enterobacteriaceae, and particularly the Escherichia coli species, are over-represented at the ileal mucosa of CD patients [START_REF] Martin | Enhanced Escherichia coli adherence and invasion in Crohn's disease and colon cancer[END_REF] [START_REF] Baumgart | Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum[END_REF] [START_REF] Lopez-Siles | Mucosa-associated Faecalibacterium prausnitzii and Escherichia coli coabundance can distinguish Irritable Bowel Syndrome and Inflammatory Bowel Disease phenotypes[END_REF]. Abnormal prevalence of E. coli was observed worldwide in CD patients [START_REF] Agus | Understanding hostadherent-invasive Escherichia coli interaction in Crohn's disease: opening up new therapeutic strategies[END_REF]. These CD-associated E. coli possess adherent and invasive properties in vitro on intestinal epithelial cells, the defining characteristics of this pathobiont of E. coli that confers its name, "Adherent-Invasive E. coli" (AIEC) [START_REF] Boudeau | Invasive ability of an Escherichia coli strain isolated from the ileal mucosa of a patient with Crohn's disease[END_REF][START_REF] Darfeuille-Michaud | Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease[END_REF][START_REF] Eaves-Pyles | Escherichia coli isolated from a Crohn's disease patient adheres, invades, and induces inflammatory responses in polarized intestinal epithelial cells[END_REF]. Besides the adherent and invasive phenotypes, AIEC invade and replicate inside macrophages, which induces elevated secretions of pro-inflammatory cytokines TNF-α, and promotes granuloma in vitro [START_REF] Bringer | The Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 replicates in mature phagolysosomes within J774 macrophages[END_REF][START_REF] De La Fuente | Escherichia coli isolates from inflammatory bowel diseases patients survive in macrophages and activate NLRP3 inflammasome[END_REF][START_REF] Glasser | Adherent invasive Escherichia coli strains from patients with Crohn's disease survive and replicate within macrophages without inducing host cell death[END_REF][START_REF] Meconi | Adherentinvasive Escherichia coli isolated from Crohn's disease patients induce granulomas in vitro[END_REF]. Furthermore, AIEC bacteria induce or aggravate intestinal inflammation in various models of mice [START_REF] Carvalho | Crohn's disease-associated Escherichia coli LF82 aggravates colitis in injured mouse colon via signaling by flagellin[END_REF][START_REF] Carvalho | Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM[END_REF][START_REF] Eaves-Pyles | Escherichia coli isolated from a Crohn's disease patient adheres, invades, and induces inflammatory responses in polarized intestinal epithelial cells[END_REF][START_REF] Small | Persistent infection with Crohn's disease-associated adherent-invasive Escherichia coli leads to chronic inflammation and intestinal fibrosis[END_REF]. Therefore, it appears essential to develop therapies targeting AIEC in CD patients in order to eliminate a triggering agent of intestinal inflammation.

Anti-adhesive strategy could be of interest to the eradication of AIEC from the gut by focusing on the initial step of virulence: attachment to ileal mucosa. Bacterial adhesin FimH, located at the tip of the type 1 pili, recognizes mannose (Man) residues on host cells [START_REF] Abraham | Conservation of the D-mannose-adhesion protein among type 1 fimbriated members of the family Enterobacteriaceae[END_REF]. AIEC possess genetic polymorphisms in fimH that allow them to bind strongly to mannose residues on intestinal epithelial cells [START_REF] Dreux | Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response[END_REF]. The glycoprotein carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) was shown to be overexpressed at the ileal mucosa of CD patients [START_REF] Barnich | CEACAM6 acts as a receptor for adherent-invasive E. coli, supporting ileal mucosa colonization in Crohn disease[END_REF]. AIEC reference strain LF82 preferentially persists in the gut of transgenic mice expressing the human CEACAM6 protein (CEABAC10 mouse model) [START_REF] Carvalho | Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM[END_REF], demonstrating that oligomannosylated glycans exposed by CEACAM6 provides to AIEC increased opportunities to bind to the intestinal mucosa. From 1980's, type 1 pili-mediated interactions between enterobacteria and host cells were characterized using yeast agglutination assays [START_REF] Eshdat | Participation of pili and cell wall adhesion in the yeast agglutination activity of Escherichia coli[END_REF]. Because of the numerous mannose residues exposed on the yeast cell surface, yeasts can bind type 1 pili-expressing bacteria and thus, can act as a competitive inhibitor of the ligand system, preventing bacterial attachment to the host cells. Saccharomyces cerevisiae CNCMI-3856 was previously selected for its high ability to agglutinate AIEC bacteria and to prevent bacterial attachment to intestinal epithelial cells in vitro [START_REF] Sivignon | Saccharomyces cerevisiae CNCM I-3856 prevents colitis induced by AIEC bacteria in the transgenic mouse model mimicking Crohn's disease[END_REF]. In CEABAC10 mice, oral administration of S. cerevisiae CNCM I-3856 accelerated AIEC elimination from the gut and decreased the subsequent symptoms of colitis. Interestingly, crude extracts enriched in yeast cell wall still possess beneficial effects against AIEC infection in mice [START_REF] Sivignon | Saccharomyces cerevisiae CNCM I-3856 prevents colitis induced by AIEC bacteria in the transgenic mouse model mimicking Crohn's disease[END_REF].

In this study, the nature of glycoconjugates from S. cerevisiae that exert the anti-adhesive ability against AIEC was investigated in order to identify the active part of the yeast and to develop a rationally defined probiotic-based medicinal product targeting AIEC bacteria. Yeast mannan exerts good inhibition properties against AIEC. In comparison, a glycoconjugate fraction composed of mannan, β6-glucan, and glycogen together was shown to exhibit much superior anti-adhesive effect in vitro and in vivo. This work presents new alternatives in personalized antimicrobial therapies in CD, and more generally, to various pathologies involving type 1 pili-expressing enterobacteria.

Material and methods

Materials

All yeast products used in this study were provided by the Lesaffre Group (Marcq-en-Baroeul, France). CNCM I-3856, LV04 and LV08 are active live Saccharomyces cerevisiae yeasts commercially available in dry form. CNCM I-3856 is a patented probiotic yeast strain registered in the French National Collection of Cultures of Microorganisms (CNCM) under the number I-3856. LV04 is a specific yeast strain used in the bioethanol production process. LV08 is a specific yeast strain used in the beer high fermentation (Ale).

YCW CNCM I-3856 and YCW CNCM I-5268 are yeast cell wall fractions obtained from primary culture and then the autolysis of yeast cells from respective strain CNCM I-3856 and CNCM I-5268 [START_REF] Reed | Yeast technology[END_REF]. After cell lysis is completed, the soluble intracellular fraction (the yeast extract) is separated from the insoluble yeast cell wall fraction by centrifugation. Obtained yeast cell walls are then spray dried.

Purification of mannan fraction

50 g of yeast product (either whole yeast or yeast cell wall) was suspended in 300 mL of 0.02 M citrate buffer (pH 7), autoclaved at 121°C for 90 min. After autoclaving, the yeast extracts were cooled down, followed by centrifugation at 4300 g, at 4 °C for 30 min. The supernatant was collected, and the pellet was suspended in another 300 mL of 0.02 M citrate buffer, autoclaved at 121°C for 90 min followed by centrifugation again. Two supernatants were pooled together, called crude mannan. The pellet was kept for the extraction of glucan, which was described in section 2.4.

The conventional method to purify mannan uses Fehling solution through copper-mannan complex precipitation [START_REF] Kocourek | Method for fingerprinting yeast cell wall mannans[END_REF]. Fehling solution was prepared freshly by mixing equal volume of two prepared solutions; one solution contains 35 g of copper(II) sulfate pentahydrate, 5 mL of 2N sulfuric acid, addition of water until a final volume of 500 mL, and the other solution contains 87.5 g of potassium sodium tartrate, 38.5 g of sodium hydroxide in 500 mL of water. Fehling solution was added to the pooled supernatant (supernatant: Fehling solution, 1:1 v/v), and stirred overnight at 4°C. The mannan-copper complex was formed, and the precipitate was collected by centrifugation 4300 g at 4°C for 30 min. 100 mL of 3N HCl was added to the precipitate, and stirred at 4°C until the precipitate was dissolved to dissociate the mannan from copper. Another 300 mL of ethanol was added to precipitate mannan, and stirred overnight at 4°C. The white precipitate corresponding to mannan was collected by centrifugation at 4300 g at 4 °C for 30 min. The mannan precipitate was re-dissolved in 50 mL of water and put into dialysis membrane (MWCO 3500) against 1L of water, then stirred overnight at 4 °C. The dialyzed mannan was collected and lyophilized.

Acetolysis of mannan

Dried mannan was aliquoted into 50 mL beaker, and 10 mL of 100% acetic acid, 10 mL of acetic anhydride, 1 mL concentrated sulfuric acid were subsequently added into beaker under hood and stirred at room temperature for 24 h. Then, solution was transferred into glass tube, followed by centrifugation at 4500 g at 4 °C for 10 min. The supernatant was collected and added slowly into 40 mL of frozen ice in 250 mL beaker, on ice. Sodium bicarbonate powder was added slowly into solution over ice until pH at 6-7. The solution was transferred into separatory funnel (250 mL), and 100 mL of chloroform was added into separatory funnel, followed by agitation 5-10 times. The funnel was kept stationary after agitation until two distinct layers were formed. The peracetylated oligomannosides-containing chloroform-rich bottom layer was collected under fume hood. Another 100 mL of chloroform was added into separatory funnel for further agitation and extraction. Two bottom layers were pooled together and transferred into separatory funnel again. 20 mL of saturated sodium bicarbonate solution (in water) was added into funnel, then stirred and the bottom layer was collected. The chloroform was removed by rotary evaporator. The dried peracetylated oligomannosides was re-dissolved in 50 mL of methanol and 50 mL of 0.2 M sodium hydroxide and stirred at 4 °C for 90 min for deacetylation. The solution was concentrated until 10 mL by rotary evaporator. The concentrated solution was loaded into AG®Dowex 50W-X8 column (bed volume 50 mL, 25-50 mesh ion exchange resin, Bio-Rad, No.1421421) to remove excess sodium salts. All the eluates were collected, followed by concentration until 5 mL by rotary evaporator. For separation of oligomannosides, Bio-Gel P6 gel filtration column (80 cm long, 2 cm diameter, Bio-Rad, No.1504130) was prepared. The oligosaccharides fraction was loaded into column, and water was used as eluent. All the eluates were collected and separated to 3 fractions, called oligo F1, oligo F2, oligo F3.

Purification of soluble glucan fraction containing b6-glucan-glycogen-mannan

The methods to purify soluble glucans fractions were adapted from the methodologies developed by Manners et al. and Sugawara et al. to study b-glucans (Manners, Masson, & Patterson, 1973;[START_REF] Sugawara | Refinement of the structures of cell-wall glucans of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy[END_REF]. The pellet from yeast product treated with hot citrate buffer, as described previously in section 2.2, was solubilized in 1 L of 1N NaOH, stirred at room temperature overnight to hydrolyze the proteins, lipids and residual mannan. The pellet was collected by centrifugation at 8400 g at 4 °C for 30 min. The pellet was washed by 1 L of water followed by centrifugation at 8400 g at 4 °C for 30 min, extracted by 800 mL of 0.5 N acetic acid and stirred at 90 °C for 3 h under chemical hood. After 3 h, solution was cooled down and centrifuged at 8400 g at 4°C, for 20 min. The pellet was extracted again by 800 mL of 0.5 N acetic acid and stirred at 90 °C for 3 h and centrifuged at 8400 g at 4°C, for 20 min. The two supernatants were pooled into the same flask. The extraction of the pellet was repeated at least 5 times. The pellet was used for phosphorylation as described in section 2.5. All the supernatants were pooled together, neutralized by NaOH and concentrated by rotary evaporator down to 500 mL. This soluble fraction was put into dialysis membrane (MWCO 3500) and dialyzed against 5-10 L of water at 4 °C, overnight. This dialyzed fraction was collected and concentrated down to 5 mL by rotary evaporator.

This soluble glucan fraction was further purified by hydrophilic-lipophilic-balanced cartridge (HBL, Waters Oasis®). The soluble polysaccharides were not bound onto this reverse-phase cartridge and all the impurities containing lipids and peptides/proteins were retained on the HLB cartridge. The cartridge was equilibrated by 5mL of acetonitrile and 5 mL of 0.5 N acetic acid in a stepwise manner. This glucan fraction was loaded into HLB cartridge and eluted by 5 mL of 0.5 N acetic acid. The collected flow through was dialyzed in dialysis tube (MWCO 3500) against 2 L of water at 4 °C, overnight and lyophilized.

Phosphorylation of insoluble b3-glucan

The pellet from hot acetic acid extraction was dialyzed into dialysis membrane (MWCO 3500) against 1 L of water at 4 °C, overnight and lyophilized. This fraction contains b3-glucan, which was identified by linkage analysis and NMR analysis. To solubilize this fraction, phosphorylating b3-glucan was used [START_REF] Williams | A method for the solubilization of a (1----3)-beta-D-glucan isolated from Saccharomyces cerevisiae[END_REF]. 0.4g urea solubilized in 5 mL of dimethyl sulfoxide (DMSO) were added into the beaker containing 100 mg b3-glucan then stirred until it dissolved. 1 mL of 85% H3PO4 was added and stirred at 100°C for 6h. After cooling down, 100 mL of water was added and the product was filtered through 1.2 μm membrane. The filtered solution contains phosphorylated β3-glucan that was dialyzed against water, followed by lyophilization.

NMR Analysis

All NMR data were acquired on Avance II Bruker spectrometer equipped with BBO or TBI 5mm probe resonating at 400 MHz for 1 H, 100.6 MHz for 13 C and 162 MHz for 31 P. Mannan and b6-glucan-glycogenmannan polysaccharides were dissolved in 500 µL D2O (99.96% 2 H, EurisotopÒ) at 10 mg/mL. b3 glucan was dissolved in d6-DMSO (99.96% 2 H, EurisotopÒ) with or without D2O, at 10 mg/mL. The solubilized polysaccharides were then transferred into 5 mm Shigemi tubes (Allision Park, USA). NMR experiments were performed at 293 K for mannan, 341 K for b6-glucan-glycogen-mannan, 353 K for b3 glucan. The signals corresponding to methyl group of acetone (δ 1 H 2.225 and δ 13 C 31.55 ppm.) or signals corresponding to methyl group of DMSO at δ 1 H 2.52 and δ 13 C 40.98 ppm were used as to calibrate the spectra. The 1 H/ 1 H-COSY, 1 H/ 1 H-TOCSY, 1 H/ 13 C-HSQC experiments were performed by using the Bruker standard sequences and optimized for each experiment.

Bacterial strain and intestinal epithelial cell line

The ampicillin/erythromycin-resistant AIEC strain LF82 was isolated from an ileal biopsy specimen of a CD patient. Bacterial strain was conserved at -80 °C in glycerol stocks. Bacteria were seeded from the LF82 glycerol stock to Luria Bertani (LB, Conda, Madrid, Spain) broth and grown overnight at 37°C without shaking. The growth was spectrophotometrically evaluated at 600nm. The human intestinal epithelial T84 cell line was obtained from the American Type Culture Collection (ATCC® CCL248 TM ) and stored in liquid nitrogen. T84 cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient mixture F-12 (DMEM/F12, Gibco) supplemented with 2 mM L-Glutamine (Gibco), 0.1M HEPES buffer (Dominique Dutscher, Brumath, France), 10% fetal bovine serum (FBS, Dominique Dutscher, Brumath, France), and antibiotics-antimycotics to a final concentration of 100 U/mL penicillin G, 100 µg/mL streptomycin and 0.25µg/mL of amphotericin B (GE Healthcare Österreich, Austria). Cells were incubated at 37°C in a humidified 5% CO2 atmosphere.

Adhesion assays on AIEC LF82-infected intestinal epithelial cells in the presence of yeast glycoconjugates

T84 cells were seeded at 1.5×10 5 cells per well in 48-well plates and incubated for 48 h at 37°C. Before infection, monolayers were washed 3 times with phosphate buffer saline (PBS, Gibco) and culture medium was replaced by cell medium without antibiotics/antimycotics. LF82 bacterial suspension was prepared at a concentration of 1.2×10 7 CFU/mL in cell culture medium. Bacteria were incubated for 1 h at room temperature with increasing concentrations of yeast glycoconjugates on an orbital agitator (ratio1:1; final bacterial concentration 6×10 6 CFU/ml). Intestinal epithelial cells were infected with bacteria/yeast product mixtures for 3 h at 37°C in a humidified 5% CO2 atmosphere at a multiplicity of infection of 10 bacteria per cell. T84 cells were washed 3 times with phosphate buffer (PBS) and lysed with 1% Triton X-100 (Sigma) in deionized water. After appropriate dilutions in physiological water, adhering bacteria were plated on Luria-Bertani agar medium and were incubated overnight at 37°C to determine the number of colony-forming units (CFU). The average adhesion rates (from at least 3-4 independent experiments) were expressed as a percentage of residual adhesion, which is the ratio of the bacterial adhesion level in the presence of yeast products and the adhesion level in absence of treatment (considered as 100 %). The error bars correspond to the standard error at mean or SEM.

Animals

Mice were housed in specific-pathogen-free conditions (21-22°C, 12:12-h light-dark cycle), with access to food and water ad libitum, in the animal facility of the University Clermont Auvergne (unité de stabulation et d'expérimentation animale, Clermont-Ferrand, France). SPF FVB/N wildtype female mice purchased from Charles Rivers Laboratories (France) and FVB/N CEABAC10 transgenic male mice (heterozygous, [START_REF] Chan | Novel mouse model for carcinoembryonic antigen-based therapy[END_REF])) were coupled to obtain 50% WT mice and 50% CEABAC10 mice.

This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the University of Clermont Auvergne (Clermont-Ferrand, France). The animal protocol was approved by the Committee for Research and Ethical Issues of the Department of Auvergne (CEMEA Auvergne; Permit Number: CEMEAA, 20160111171036870).

Mice infection protocol and AIEC LF82 gut colonization assessment

Eight-week-old CEABAC10 heterozygous male mice (body weight, ∼24-26 g) were orally pretreated with 5 mg per day (in 0.2 mL of PBS, phosphate buffer saline) of the soluble glucan fraction extracted from YCW CNCM I-5268 or with PBS for non-treated mice, from day-7 to the day of the bacterial challenge (day 0). After infection, soluble glucan was administered twice a day until day 4 post-infection. Mice received 0.5 % of dextran sulfate sodium (DSS, MP Biomedicals, Illkirch, France) in drinking water from day-3 to day 4 and they were treated per os with 5 mg of streptomycin (Sigma-Aldrich) at day-1 in order to disrupt normal resident bacterial intestinal microbiota and induce a low-grade inflammation. An overnight bacterial culture of AIEC LF82 strain was harvested by centrifugation at 5 500 g for 10 min. The bacterial pellet was resuspended in PBS to reach a concentration of 2.5×10 10 bacteria/mL. Mice were challenged with 5×10 9 LF82 (in 0.2 mL) at day 0 immediately after an oral administration of 0.1 mL of sodium bicarbonate at 0.2M (Sigma-Aldrich). Body weight was monitored throughout the experiments. At days 1, 2, 3 and 4 post-infection, fresh fecal pellets (≈100 mg) were collected from individual mice and resuspended in PBS. After appropriate serial dilutions, bacteria were enumerated by plating on Luria-Bertani agar medium containing 100 µg/ml ampicillin (Sigma-Aldrich) and 20 µg/ml erythromycin (Sigma-Aldrich) to isolate AIEC LF82 and incubated at 37°C overnight. The severity of colitis was assessed by the disease activity index (DAI) score, which ranges from 0 (healthy) to 12 (high activity of colitis) [START_REF] Sivignon | Development of Heptylmannoside-Based Glycoconjugate Antiadhesive Compounds against Adherent-Invasive Escherichia coli Bacteria Associated with Crohn's Disease[END_REF]. At day 4 post-infection, they were anesthetized with isoflurane and killed by cervical dislocation. Colon specimens were collected to quantify LF82-associated to the tissue by plating onto antibiotic-selective culture medium for LF82.

Fecal lipocalin-2 assay

Fecal samples from days 2 and 3 post-infection were homogenized and centrifuged at 10,000 g for 10 min. Lipocalin-2 levels were assessed in the supernatant with the Mouse Lipocalin-2/NGAL DuoSet Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Results are expressed as ng/g of feces.

Statistical analysis

Statistical analyses were performed with GraphPad Prism 6.0 (GraphPad, La Jolla, CA, USA) software package for PC. Data are expressed as means ± standard error of the mean (SEM) or as medians. Data were compared by Student's t-test analysis (Mann-Whitney test if not parametric), or ANOVA followed by a post-Bonferroni (Kruskal-Wallis if not parametric). A p value of less than 0.05 was considered to be statistically significant. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Results

Purification of glycoconjugates from Saccharomyces cerevisiae yeasts

The yeast cell wall is a multilayered structure that provides physical protection and osmotic support to the cell. The outer layer consists of heavily glycosylated mannoproteins collectively referred as phosphopeptido-mannans (PPM) or mannan, glycogen-like a1,4-glucan, and cell wall proteins that are covalently linked or associated with the inner layer made of b1,3-glucan-chitin network and b1,6-glucans [START_REF] Klis | Dynamics of cell wall structure in Saccharomyces cerevisiae[END_REF] (Figure 1A). Initially, five yeast samples were chosen as starting material in order to compare their structures and activities, including three strains of intact dry yeasts (CNCM I-3856, LV04, LV08), and two yeast cell wall (YCW) enriched fractions generated by autolysis of yeasts (YCW CNCM I-3856 and YCW CNCM I-5268). Herein an experimental procedure aimed at purifying individual glycoconjugate fractions from the chosen whole yeasts and yeast cell walls, including PPM, b3-glucan, b6glucan, and a4-glucan, was optimized in order to subsequently assay their capacity to inhibit bacterial adhesion (Figure 1B). In brief, crude PPM fraction was solubilized from whole yeast or YCW in citrate buffer at 120°C, and enriched by Fehling method through the formation of insoluble copper-mannose complex [START_REF] Kocourek | Method for fingerprinting yeast cell wall mannans[END_REF]. Then, remaining insoluble fraction was treated with successive chemical extractions in order to purify different types of glycoconjugates [START_REF] Aklujkar | A Simplified Method for the Isolation and Estimation of Cell Wall Bound Glycogen in Saccharomyces cerevisiae[END_REF]Manners, Masson, & Patterson, 1973;[START_REF] Sugawara | Refinement of the structures of cell-wall glucans of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy[END_REF]. Herein, hot diluted acetic acid was finally utilized to separate insoluble b3-glucan from soluble b6-glucan and a4-glucan fractions. cerevisiae by autolysis. The overall fractionation pipeline was adapted from existing methodologies developped to purify mannans and b-glucans [START_REF] Kocourek | Method for fingerprinting yeast cell wall mannans[END_REF]Manners, Masson, & Patterson, 1973;[START_REF] Sugawara | Refinement of the structures of cell-wall glucans of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy[END_REF].

PPM from CNCM I-3856 yeast strain exhibits good anti-adhesive property in vitro

The outmost layer PPM consists of polymannosylated N-glycans covalently attached to proteins. In S. cerevisiae, the polymannosides are composed of a long stretch of (a-1,6)-linked D-mannopyranose units substituted in C2 positions by short side chains of (a-1,2)-linked mannose units that may be further capped by terminal Man(a-1,3) residues. So called acid-labile mannan domain is further attached to the (a-1,2)-oligomannosides through phospho-di-ester bond [START_REF] Nelson | Candida mannan: chemistry, suppression of cell-mediated immunity, and possible mechanisms of action[END_REF].

PPM fractions from different S. cerevisiae strains were extracted to identify their structures and examine their inhibitory activity effects on the AIEC LF82 adhesion to T84 intestinal epithelial cells.

The structures of PPM from the five samples were characterized by a combination of NMR experiments including 1D-1 H-NMR, 1 H-1 H COSY-NMR and 1 H-13 C HSQC-NMR analyses, based on the known structural features and NMR parameters of S. cerevisiae polymannosylated glycans [START_REF] Dulary | Investigating the function of Gdt1p in yeast Golgi glycosylation[END_REF][START_REF] Vinogradov | Structural analysis of the intact polysaccharide mannan from Saccharomyces cerevisiae yeast using 1H and 13C NMR spectroscopy at 750 MHz[END_REF]. Here, the analysis was focused on the main structural features of the polymannosylated moiety (mannan) such as the nature, the density and the terminal capping of mannosylated side chains. The anomeric region (5.5-4.8 ppm) of 1D-1 H-NMR spectra shows five broad signals annotated as I, II, III, IV, V corresponding to five different epitopes, which are internal -2)Man(a-1,2) residues (I); terminal Man(a-1,3) residues (II); -2,6)Man(a-1,6) branched residues (III); terminal Man(a-1,2) and -3)Man(a-1,2) residues (IV); unbranched -6)Man(a-1,6) residues (V), respectively (Figure 2A,2B). The identity of these signals was confirmed according to their 1 H-13 C NMR parameters as established by 1 H-13 C HSQC experiments (Supplementary Figure 1,Supplementary table 1) and to the partial spin systems of the associated monosaccharides as established by 1 H-1 H COSY experiments (data not shown). Altogether, all five PPMs expressed complete sets of I-V NMR signals, except LV08 that was totally devoid of terminal Man(a-1,3) residues, as shown by the absence of signal II. However, the relative quantification of individual signals showed that all five PPMs dispatched similar but slightly different mannose patterns (Supplementary Figure 1B). Among those, PPM extracted from whole yeast CNCM I-3856 and from YCW CNCM I-3856 showed very similar structures according to NMR data, which indicates that prior sub-fractionation of whole yeast does not modify the nature of extracted mannans. It is noteworthy that mannans from both CNCM I-3856 samples and from LV04 strain are characterized by a higher branching level than the two other strains, as shown by the very low intensity of signal V.

Intriguingly, as already mentioned, whereas the LV08 mannan does not present α3 mannose capping, LV04 mannan contains more α3 mannose capping than the four other PPM samples, accordingly to the relatively very high intensity of signal II (Figure 2A The capacity of five PPMs extracted from yeasts and yeast cell walls (Figure 3A) to inhibit bacterial adhesion was assessed in vitro. Different concentrations of PPM were pre-incubated with AIEC LF82 bacterial strain. The glycoconjugates/bacteria mixtures were then added onto T84 cells and cellassociated LF82 bacteria were quantified in order to determine the residual adhesion percentage related to the yeast treatment. Among these, PPM from CNCM I-3856 yeast exerted the highest anti-adhesive effect for LF82 adherent to T84 cells in a dose-dependent manner (Figure 3B). Indeed, at 0.25 mg/mL, PPM of CNCM I-3856 inhibited 50.06% of bacterial adhesion, whereas a 4-fold higher concentration was needed for PPM from LV04 and LV08 strains to reach the same efficiency. Intriguingly, inhibition efficiency of CNCM I-3856 is decreasing when PPM were extracted from YCW. At the maximal concentration of 1 mg/mL, PPM from YCW CNCM I-3856 led to an AIEC LF82 residual adhesion level of 54.64% compare to 22.87% for PPM from whole CNCM I-3856 (Figure 3B and3C). PPM from YCW CNCM I-3856 was less efficient to decrease AIEC LF82 attachment to cells that the one extracted from the whole yeast. Maximal inhibitory effect of PPM fraction from YCW CNCM I-5268 was slightly better than PPM from YCW CNCM I-3856 with 46.23% of residual adhesion at concentration of 1 mg/mL (Figure 3C). Altogether, combined structural and in vitro analyses showed that all PPMs exhibited anti-adhesive activities toward AIEC LF82, although with different efficiencies. The PPM activity level could not be unambiguously correlated with a specific trait such as terminal capping, which suggests that inhibitory efficiency may be dependent on other factors such as epitope presentation or density that cannot be evaluated by the used methodologies.

The production of oligomannosides from PPM does not potentiate anti-adhesive activity

In order to determine the region of mannan that plays the role of inhibition and possibly enhance the anti-adhesive potency of the purified fractions, mannans were chemically degraded in order to generate defined sets of oligomannosylated compounds. PPM was subjected to mild acetolysis that preferentially cleaves the Mana1-6Man linkage over the more stable Mana1-2Man and Mana1-3Man linkages [START_REF] Kocourek | Method for fingerprinting yeast cell wall mannans[END_REF][START_REF] Stewart | A comparison of yeast mannans and phosphomannans by acetolysis[END_REF], and then, the generated oligosaccharides were separated by gel filtration. Three major fractions F1-F3 were collected (Figure 4A). MALDI-MS profiles (Supplementary Figure 2) of these three fractions showed that fraction 1, which is the earliest eluting fraction, contains abundant phosphorylated oligosaccharides (Man)n-phosphate, n=3-10 with a prevalence for low molecular mass oligosaccharides Man3-PO3 and Man4-PO3. Early elution of phosphorylated oligosaccharides, irrespectively of their low mass range, is due to the negative charges repulsion between phosphate groups and remnant acidic groups on the polyacrylamide medium, as previously observed [START_REF] Garenaux | A single step method for purification of sulfated oligosaccharides[END_REF]. F2 contains a mixture of smaller phosphorylated oligomannoses Man3-PO3 and Man4-PO3 as well as two groups of neutral oligosaccharides with higher degree of polymerization: one group of oligomannoses Hex2 to Hex9 and one group of oligosaccharides corresponding to HexNAc2Hex4 to HexNAc2Hex6. The monosaccharide composition strongly suggests that it is composed of the trimannosylated core N-glycans (GlcNAc2Man3) further extended by a variable number of mannose residues. Finally, F3 contains smaller oligomannoses Hex2 to Hex8 dominated by Man2, Man3 and Man4.

These three fractions (F1, F2 and F3) of oligosaccharides significantly decreased LF82 adhesion to T84 cells at the concentration of 1 mg/mL, but this effect did not match the inhibition efficiency of the intact PPM at the same concentration (Figure 4B). Altogether, the complex and branched structure of the mannan from CNCM I-3856 is the best inhibitor against LF82 bacterial adhesion.

Phosphorylated b3-glucan from CNCM I-3856 has an intermediate anti-adhesive effect in vitro

β3-glucans were purified from the three intact yeast strains (CNCM I-3856, LV04 and LV08) and from YCW CNCM I-5268. According to the purification protocol, GC-MS composition analysis confirmed that these fractions were composed by more than 95% of glucose (Glc) and less than 5% of mannose. Overall, structural analysis showed that all four fractions were essentially similar. In particular, GC-MS analysis of partially methylated acetylated alditols (PMAA) showed a majority of 3-linked Glc residues and 3,6-linked Glc residues, in accordance with the presence of a b3-glucan polymer further substituted in C6 position (Supplementary Figure 3). In addition, minor signals associated to 6-linked Glc suggests the presence of either 6-linked side chains or b6-glucan.

Considering the structural similarity of all purified β3-glucans, only β3-glucan isolated from CNCM I-3856 was used to conduct in vitro assay to examine the inhibiting activity toward AIEC attachment on intestinal cells. However, because of its insolubility, β3-glucan could not be used straightly for in vitro assays. Therefore, β3-glucan from CNCM I-3856 was phosphorylated in order to solubilize it prior to assay [START_REF] Du | Preparation of Water Soluble Yeast Glucan by Four Kinds of Solubilizing Processes[END_REF][START_REF] Shi | Mechanochemical phosphorylation and solubilisation of beta-D-glucan from yeast Saccharomyces cerevisiae and its biological activities[END_REF][START_REF] Williams | A method for the solubilization of a (1----3)-beta-D-glucan isolated from Saccharomyces cerevisiae[END_REF]. Presence of phosphorylated glucan was first confirmed by observing a strong signal at 0 ppm on 1D 31 P-NMR (Supplementary Figure 4A). 1 H-31 P-HSQC NMR analysis established that the phosphate groups were in majority associated to the C6 positions of 3-linked as well as C2 and C4 position of 6-linked of b-Glc residues (Supplementary Figure 4B). Then, linkage analyses by GC-MS of phosphorylated β3-glucan (Supplementary Figure 4C) from CNCM I-3856 showed an increase in the proportions of terminal Glc and 6-linked Glc counterbalanced by a decrease in the proportion of 3-linked Glc, compared to original β3-glucan (Supplementary Figure 3). 1 H-13 C-HSQC NMR analysis of this fraction also confirmed that the phosphorylated β3-glucan was made of equivalent proportions of 3-linked and 6-linked glucans (Supplementary Figure 4D,Supplementary table 2). These results indicated that phosphorylated β3-glucan fraction is constituted by shorter chains of β3-glucan compared with the original β3-glucan. It may be caused by the high temperature phosphoric acid that partially depolymerized the β3-glucan polysaccharides. Of interest, even the small proportion of mannose in phosphorylated β3-glucan fraction (5%) compared to PPM fraction from CNCM I-3856 (92%) (known by monosaccharide analysis, data not shown), this fraction was able to inhibit 60% of bacterial adhesion at 1 mg/mL (Figure 4C).

Soluble glucan fraction exert a better anti-adhesive effect when extracted from yeast cell wall rather than intact yeast

According to the purification scheme, citrate-insoluble fraction of whole yeasts (CNCM I-3856, LV04, LV08) and of YCW (CNCM I-3856 and CNCM I-5268) treated by hot diluted acetic acid solution generates a soluble glucan fraction (Figure 1B). This soluble glucan fraction was supposedly constituted of a mixture of b6-glucan and a4-glucan [START_REF] Arvindekar | Glycogen--a covalently linked component of the cell wall in Saccharomyces cerevisiae[END_REF][START_REF] Bacon | The glucan components of the cell wall of baker's yeast (Saccharomyces cerevisiae) considered in relation to its ultrastructure[END_REF]Manners, Masson, & Patterson, 1973;[START_REF] Manners | The structure of a beta-(1--6)-D-glucan from yeast cell walls[END_REF]. In agreement to this hypothesis, 1D-1 H-NMR spectra of the glucan soluble fractions isolated from the five yeast samples showed intense signals at 4.3 ppm and at 5.37 ppm tentatively attributed to internal b6-linked glucose residues and to a4-linked glucose residues, respectively (Figure 5A). Their attribution was further confirmed by 1 H-13 C HSQC NMR experiments. These two monosaccharides were predominant in soluble glucan fractions isolated from CNCM I-3856, LV04 and LV08 as they represented from 82% (CNCM I-3856) to 96% (LV08) of the total signal intensities of anomeric protons. Unexpectedly, NMR analysis of soluble glucan fractions isolated from yeast cell walls (YCW CNCM I-3856 and YCW CNCM I-5268) showed an additional set of intense 1 H signals previously identified as a-Man residues signals I to V associated to mannan (Figure 2), which established the presence of significant quantities of mannan in these two samples (Figure 5A). The nature of these signals was further confirmed by 1 H-13 C HSQC NMR analyses (Figure 5B, Supplementary table 3). This set of signals could also be observed in the soluble fractions extracted from CNCM I-3856, LV04 and LV08, but with much lower intensity. The proportions of b-glucan, a-glucan and mannan (expressed as β-glucan/α-glucan/mannan ratios, GGM) in the soluble glucan fractions isolated from each sample were estimated by area integration of individual signals detected by 1D-1 H-NMR analyses (Supplementary Figure 5). The soluble glucan fraction isolated from CNCM I-3856, LV04 and LV08 were characterized by very high proportions of α-glucan (from 56 to 88%) and low proportions of mannan (from 2 to 18%), as depicted on Figure 5C. On the contrary, soluble glucan fractions from YCW CNCM I-3856 and YCW CNCM I-5268 contain a high content of both β-glucan and mannan but low content of α-glucan with GGM index around 40:15:45 (Supplementary Figure 5). The high mannose content of soluble glucan fraction extracted from yeast cell wall was confirmed by GC-MS monosaccharide and linkage analyses (Supplementary Figure 6). Thus, these results demonstrated that when the soluble glucan fraction is purified from whole yeasts (CNCM I-3856, LV04 and LV08), it is almost mainly constituted by α-glucan whereas the soluble glucan fraction is highly enriched in β-glucan and mannan when it is purified from cell walls. Considering the clear structural differences observed between soluble glucan fractions depending on the starting material (Figure 6A), their in vitro cellular activities toward bacterial adhesion were compared.

In intestinal epithelial cells T84, adhesion inhibition property of the soluble glucan fraction extracted from whole yeast CNCM I-3856 was shown to be moderate with a residual adhesion level of 42.3% at the concentration of 1 mg/mL (Figure 6B). At the same concentration, soluble glucan fraction from YCW CNCM I-3856 exhibits a much higher anti-adhesive effect since it decreases LF82 adhesion at 16.7% of residual adhesion (Figure 6C). Interestingly, soluble glucan fraction from YCW CNCM I-5268 exhibited the highest inhibitory effect, as 94.3% of the bacterial adhesion to T84 cells was prevented at the concentration of 1 mg/mL.

The high proportion of mannose in these fractions may be instrumental to the increased anti-adhesive activity. However, the fact that the inhibitory effect is far superior to that of the mannan fraction demonstrates that richness in mannan in not sufficient to trigger the activity. The presence of the three partners b-glucan/a-glucan/mannan in a precise ratio may be required to potentiate the anti-adhesive efficacy.

Soluble glucan fraction isolated from yeast cell wall favors the elimination of AIEC LF82 bacteria from the gut of mice

Considering the high capacity of soluble glucan fraction isolated from yeast cell wall to inhibit the bacterial adhesion to T84 cells in vitro, the in vivo activity of the most effective soluble glucan fraction isolated from YCW CNCM I-5268 was assessed in the CEABAC10 mouse model mimicking CD susceptibility infected with the AIEC LF82 bacterial strain. Bacterial infection induced a decrease of the body weight of mice for 48 hours. Yeast treatment succeeded in reversing the body weight curve from 2 days postinfection (dpi), and body weights were almost restored at the initial level at dpi 4 (Figure 7A). AIEC LF82 bacterial loads in the feces of mice were significantly decreased from dpi 2 to dpi 4 by the soluble b-glucan fraction treatment (Figure 7B). This result was confirmed by the analysis of the area under curve, which reflects the global colonization of the gut during the infection period for each mouse (2.34×10 8 cfu versus 2.95×10 7 cfu for non-treated mice and treated mice respectively, Figure 7C). Yeast treatment also reduced the number of LF82 bacteria associated to the colonic mucosa. At dpi 4, 62.5% of mice still presented AIEC associated to the mucosa, whereas percentage decreased at 42.8% in the glucan-treated group (Figure 7D). A slight improvement was observed concerning the severity of colitis as assessed by the disease activity index (DAI) score (Figure 7E) and the concentration of lipocalin-2, a fecal marker of intestinal inflammation, was significantly decreased at dpi 2 (Figure 7F). To conclude, soluble glucan fraction extracted from YCW from CNCM I-5268 was highly effective to prevent AIEC LF82 attachment to intestinal epithelial cells, but also exerts beneficial properties in vivo in decreasing LF82 gut colonization and in reducing a fecal biomarker of intestinal inflammation, lipocalin-2.

The tri-hetero partners present in soluble glucan fraction is optimal to decrease efficiently the AIEC gut colonization.

Discussion

The potential of probiotics in CD management still remains unclear as standard probiotic strategies have hitherto failed to demonstrate any clear benefits [START_REF] Oka | Microbial-Based and Microbial-Targeted Therapies for Inflammatory Bowel Diseases[END_REF]. Even so, Saccharomyces yeast, especially Var. boulardii CNCM I-745, has been shown to decrease risks of relapse or improve intestinal permeability in inactive CD [START_REF] Vilela | Influence of Saccharomyces boulardii on the intestinal permeability of patients with Crohn's disease in remission[END_REF][START_REF] Guslandi | Saccharomyces boulardii in maintenance treatment of Crohn's disease[END_REF], but these clinical data are rare and rely on relatively small groups of patients (32 and 25 patients for these two studies). Specific pathobiont-targeted strategies, and more precisely AIEC-targeted strategy, should be of interest to the disease management, with a requisite stratification of AIEC-carrier patients. The present work stems from the seminal observation that selected Saccharomyces cerevisiae CNCM I-3856 strain exhibited significant ability to prevent adhesion of AIEC to intestinal epithelial cells in culture and to human ileal biopsies isolated from CD patients abnormally expressing the mannosylated protein CEACAM6 [START_REF] Sivignon | Saccharomyces cerevisiae CNCM I-3856 prevents colitis induced by AIEC bacteria in the transgenic mouse model mimicking Crohn's disease[END_REF]. Using a preclinical model, it is shown that CNCM I-3856 significantly decreased AIEC colonization and colitis symptoms of infected transgenic mice expressing CEACAM6 (CEABAC10 model) and restored impaired barrier function of intestinal mucosa. Both the intact yeast and the S. cerevisiae cell wall derivatives administered to mice showed protective properties toward AIEC, strongly suggesting that the beneficial effect was associated with a surface component. As AIEC are known to colonize the ileal mucosa of CD patients via the interaction of the mannose-specific adhesin FimH with CEACAM6 mannosylated protein expressed on epithelial cell surface, we hypothesized that yeast polymannosylated glycoconjugates were involved in the anti-adhesive properties by competing with natural ligand of FimH. In order to identify molecular motifs responsible for the anti-adhesive properties of the yeast, the main glycoconjugates associated with yeast cell wall were sub-fractionated to individually assay their biological properties. Both intact yeast cells and yeast cell wall (YCW) preparations were used in order to uncover potential differences in the activity of non-surface expressed molecules and to potentially improve the originally observed activity. In parallel, the impact of strain-specific structural differences was evaluated with the objective of providing insights into the structure-to-function activity of the selected molecules. Based on FimH recognition specificity, we originally expected the highly mannosylated surface glycoconjugates would present the highest anti-adhesive activity. As expected, PPM isolated from all samples exhibited good in vitro anti-adhesive properties toward T84 cells. On average, PPM isolated from intact yeast showed better activities (about 20 to 40% residual adhesion) at high concentration (1mg/mL) than those isolated from YCW (about 45 to 55% residual adhesion). One may speculate that pre-purification of the cell wall removes some surface components that potentiate the activity of mannan or sub-fractionates mannans with different activities. Indeed, comparative analysis showed that polymannosylated moieties of PPM from different origins exhibited variable structural features such as the extend of Mana1-3 terminal capping (Figure 2, peak II) that varies from 0 in LV08 to 20% in LV04, and the degree of Mana1-2 branching of the -6Mana1-chain (Figure 2, peak III). However, none of these structural features could be directly correlated with the anti-adhesive activity, strongly suggesting that the linkage pattern of mannan has no influence on the overall activity of PPM. Similarly, depolymerization of the mannan chain into more homogenous smaller oligosaccharides did not improve the anti-adhesive activity. On the contrary, the maximum activity was decreased for all purified fractions, which strongly suggests that polymerization increasing avidity is important to reach optimal activity, as previously observed for numerous bacterial lectins, including FimH from uropathogenic E. coli and LecA from Pseudomonas aeruginosa [START_REF] Palmioli | Targeting Bacterial Biofilm: A New LecA Multivalent Ligand with Inhibitory Activity[END_REF][START_REF] Sauer | Binding of the Bacterial Adhesin FimH to Its Natural, Multivalent High-Mannose Type Glycan Targets[END_REF]. Thus, highly purified PPMs, irrespective of their origins, did not exhibit higher anti-adhesive potential than the intact yeasts [START_REF] Sivignon | Saccharomyces cerevisiae CNCM I-3856 prevents colitis induced by AIEC bacteria in the transgenic mouse model mimicking Crohn's disease[END_REF] as more than 80% of inhibition in the used experimental conditions could not be reached.

Unexpectedly, the highest inhibition levels were observed with soluble glucan fractions, especially those extracted from yeast cell wall (YCW) preparations. Structural analyses revealed that soluble glucan fractions, exclusively from YCW, were enriched in mannan. This mannan was covalently attached to αand β-glucans according to previous works [START_REF] Arvindekar | Glycogen--a covalently linked component of the cell wall in Saccharomyces cerevisiae[END_REF][START_REF] Kollar | Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3glucan, and chitin[END_REF][START_REF] Kwiatkowski | A Study of Saccharomyces cerevisiae Cell Wall Glucans[END_REF]. Thus, presence of the three partners b-glucan, a-glucan and mannan in this soluble glucan fraction with a ratio 45:15:40 potentializes anti-adhesive effect, as AIEC LF82 adhesion to T84 cells was nearly abrogated. As the maximal efficiency was obtained with soluble glucans extracted from YCW CNCM I-5268, this fraction was tested in the CEABAC10 mouse model. CNCM I-5268 soluble glucans were able to accelerate AIEC elimination from mice gut, to prevent LF82 attachment to intestinal mucosa and to decrease intestinal inflammation, as demonstrates by decreased fecal lipocalin-2 level.

These data demonstrate that upstream yeast processing has a direct effect on the activity of the soluble glucan fraction but the precise role of the "strain-effect" in the anti-adhesive effects should be established. Indeed, despite a similar β-glucan/a-glucan/mannan ratio, soluble glucans from YCW CNCM I-5268 has a superior biological activity compared to those from YCW CNCM I-3856. A hypothesis should be the ligand presentation for FimH adhesin, that is different between the two yeast strains.

Conclusions

For better inflammatory diseases management, it seems essential to develop therapies rationally designed to manipulate dysbiotic microbiota. Here, a bioactive fraction in yeast wall responsible for competitive blocking of AIEC bacteria to intestinal epithelium through FimH was identified. This soluble glucan fraction represents a promising pathobiont-targeted tool to detach AIEC from the intestinal mucosa. To demonstrate the efficacity of such therapy in CD patients, AIEC-carriage should be considered as an essential criterion of inclusion. Use of a concomitant companion diagnostic test to rapidly guide patients towards an AIEC-targeted treatment is necessary and heptyl-mannose-grafted cellulose represents a promising method to easily detect AIEC in biological fluids [START_REF] Cauwel | Heptylmannose-functionalized cellulose for the binding and specific detection of pathogenic E. coli[END_REF]. It should also be interesting to propose this soluble glucan as a post-biotic strategy in patients susceptible to be colonized by AIEC, for example after surgery to limit AIEC implantation at the ileal mucosa, in order to decrease postoperative recurrence and thus to extend periods of remission. Finally, as FimH is broadly expressed by enterobacteria, this soluble glucan fraction containing b-glucan/a-glucan/mannan with a ratio can be applied more generally in prophylactic or therapeutic treatments against FimH-expressing bacteria responsible for gastro-intestinal acute or others chronic disorders. Administration of this bioactive yeast fraction in combination with a live probiotic yeast could be considered in order to cumulate anti-adhesive properties against FimH-producers with another beneficial effect like antiinflammatory response.

Declaration of Interest

Nathalie Ballet and Pascal Vandekerckove are full-time employed by the company Lesaffre International, Lesaffre Group, Marcq-en-Baroeul, France. All other authors declare no competing interests.

conducive to achieving this work. We thank Sebastian Ku from Lesaffre Singapore for English language proofreading. This study was supported by Lesaffre international, the Ministère de la Recherche et de la GC-MS (TRACE GC Ultra, Thermo Fisher Scientific). The initial oven temperature was held at 120°C, increased to 230°C at 2°C/min, and then, 270°C for 10 min.

Mass Spectrometry Analyses

The oligosaccharides were aliquoted for permethylation. In brief, the dried oligosaccharides in glass tube were permethylated with NaOH/DMSO and methyl iodide according to Ciucanu and Kerek method (Ciucanu & Kerek, 1984) (https://doi.org/10.1016/0008-6215(84)85242-8) extracted by chloroform/water partitioning. Repeated water-chloroform partitions were used, and the sample was dried under a stream of nitrogen. The permethylated oligosaccharides were dissolved in 100% acetonitrile and pre-mixed with 2,5-dihydroxybenzoic acid matrix solution (10 mg/ml in 50% acetonitrile/water, v/v, Sigma), then spotted on target. MS spectra were acquired by using a MALDI-TOF/TOF 4800 mass spectrometer (Applied Biosystems) equipped with Nd:YAG laser, operated in the reflectron mode. Data acquisition was normally comprised of 40 sub-spectra of 50 laser shots, and the laser energy is set 5000-5300.

-

Figure 1

 1 Figure 1. (A) Schematic representation of major glycoconjugates on the cell wall of S. cerevisiae. (B)

  , Supplementary Fig 1B). Altogether, the five PPMs could be categorized in four different groups according to the combination of structural features of their mannan moieties, as depicted in Figure 2C.

Figure 2 .

 2 Figure 2. Structural characterizations of mannan fractions extracted from CNCM I-3856, LV04, LV08

Figure 3 .

 3 Figure 3. Mannan fractions extracted from CNCM I-3856, LV04, LV08 yeasts and CNCM I-3856, CNCM I-

Figure 4

 4 Figure 4 Activity of mannan-derived oligomannosides and phosphorylated b3-glucan from CNCM I-3856

Figure 5 .

 5 Figure 5. Structural characterizations of soluble glucan fractions extracted from CNCM I-3856, LV04,

Figure 6 .

 6 Figure 6. Soluble glucan fractions extracted from CNCM I-3856 and YCW from CNCM I-3856, CNCM I-

Figure 7 .

 7 Figure 7. Soluble glucan fraction extracted from YCW CNCM I-5268 accelerates elimination of AIEC

  Technologie (Université Clermont Auvergne), Inserm (UMR 1071), INRAE (USC-2018) and by the French government IDEX-ISITE initiative 16-IDEX-0001 (CAP 20-25), I-SITE project (CAP 2025) of the University of Clermont Auvergne.

table 3 .

 3 Chemical shifts (ppm) of 1 H and 13 C of the glycosyl residues of soluble glucan (GGM) fraction, dissolved in D2O and measured at 431K. These values were deduced from the interpretation of the 1D-1 H NMR, 2D-1 H-1 H COSY, TOCSY, and 1 H-13 C HSQC spectra

	3)Glc(b1,3)	H	4.53	3.31	3.48	3.27	3.27	3.71	3.49
		C	102.9	72.6	86.1	68.4	76.2	60.82	60.82
	Glc(b 1,3)	H	4.39	3.12	3.26	-	-	-	-
		C	103.6	76.57	-	-	-	-	-
	3,6)Glc(b 1,3)	H	4.36	3.27	3.41	-	-	-	-
		C	102.77	-	87.11	-	-	-	-
	-6)Glc(b1,6)	H	4.28	3.04	3.21	3.14	3.33	4.02	3.62
		C	102.87	73.32	76.48	70.0	75.4	68.3	68.3
		C	100.77	71.59	-	-	-	-	-
	"-" undetectable/undetermined value

Acknowledgements

We thank Abdelkrim Alloui for animal care (unité de stabulation et d'expérimentation animale, Université Clermont Auvergne, Clermont-Ferrand, France). We are grateful to Emeline Calichon for her technical assistance and to the PAGes core facility (http://plateforme-pages.univ-lille1.fr) and UMS 2014 -US 41 -Plateformes Lilloises en Biologie & Santé for providing the scientific and technical environment

Supplementary Data

Heteropolysaccharides from S. cerevisiae show anti-adhesive properties against E. coli associated with Crohn's disease

Adeline Sivignon a,1 , Shin-Yi Yu b,1 , Nathalie Ballet c , Pascal Vandekerckove c , Nicolas Barnich a,2 , Yann Guerardel b,d,2, * a Université Clermont Auvergne/Inserm U1071; USC-INRAE 2018, Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte (M2iSH), Clermont-Ferrand, France.

Supplementary methods

Monosaccharide Analysis and Linkage Analysis

For monosaccharide analysis of mannan and glucan, 100 µg of inositol and 100 µg of target sugars were taken and put in the same tube. 500 µL of 4 N trifluroacetic acid (TFA) were added and incubated at 100 °C, for 4 h to hydrolysis polysaccharides into monosaccharides, followed by drying the sample by under stream of nitrogen at room temperature. Reduction monosaccharides by 500 µL of 20 mg/mL NaBH4 in 2N NH4OH, room temperature, overnight. The reaction was quenched by adding 500 µL of 10% acetic acid in methanol, and dry under stream of nitrogen, again. Repeat 4-5 times. Then, reduced monosaccharides were peracetylated by adding 500 µL of acetic anhydride, 100°C, for 4 h, and derivatized monosaccharides were extracted by 1 mL of chloroform. 1 mL of water was added to do water-chloroform partition to remove excess salts. Repeat 5 times extraction and dry under stream of nitrogen. The derivatized monosaccharides were re-dissolved in 500 µL chloroform. 1 µL of sample was injected into GC-FID (TRACE GC Ultra, Thermo Fisher Scientific). The capillary column is SOLGEL (30m x 0.25 mm, 0.25 µm film thickness, SGE Analytical Science). The initial oven temperature was held at 120°C, increased to 230°C at 3°C /min, and then, 270°C for 10 min.

For linkage analysis of mannan and glucan, 200 µg of target polysaccharides were dissolved in 100 µL of DMSO. Polysaccharides were derivatized by modified (Hakomori, 1964). 33 mg of NaI was taken in one glass tube and washed by pentane several times to remove glycerol. NaI was dried under nitrogen and the dissolved polysaccharides were added into NaI-contained glass tube, followed by addition of 100 µL of CH3I. Reaction was agitated for 10 minutes at room temperature. The reaction was quenched by adding 1 mL of water on ice and permethylated polysaccharides were extracted by 2 mL chloroform/water partition. The chloroform phase was dried under nitrogen. Samples were hydrolyzed by 4 N TFA at 100°C, for 4hr and reduced by 1 mL NaBD4 (20 mg/mL) in 2 N NH4. The reaction was quenched by 1 mL of 10% acetic acid in methanol. Then, sugars were peracetylated and chloroform/water extracted as described above. The derivatized monosaccharides were dissolved in 1 mL of chloroform and 1 µL were injected into

Supplementary figures

Supplementary Figure 1. Supplementary table 1. Chemical shifts (ppm) of 1 H and 13 C of the glycosyl residues of mannan, dissolved in D2O and measured at 300K. These values were deduced from the interpretation of the 1D-1 H NMR, 2D-1 H-1 H COSY, TOCSY, and 1 H-


Petit Sabine
email: sabine.petit@univ-poitiers.fr

BARON FABIEN Decarreau Alain

Sabine Petit

Controls on tetrahedral Fe(III) abundance in 2:1 phyllosilicates --Discussion

Keywords: 2:1 phyllosilicates, tetrahedral Fe, partition coefficient, smectite, nontronite

Cuadros et al. (2019) used a wide range of data from dioctahedral and trioctahedral Fe 3+ -bearing 2:1 phyllosilicates, to propose a model describing how tetrahedral occupancy by Fe 3+ takes place in both dioctahedral and trioctahedral 2:1 phyllosilicates. The partition coefficient approach (Decarreau and Petit, 2014) while focusing on distribution of Al 3+ and Fe 3+ between octahedral and tetrahedral sites of dioctahedral smectites has been disregarded in the Cuadros et al. (2019)'s study. This approach was applied here on the Cuadros et al. (2019)'s set of data. The partition coefficient value linked to the distribution of Al 3+ and Fe 3+ between octahedral and tetrahedral sites determined from natural and synthetic dioctahedral smectites applies well to trioctahedral phyllosilicates too. Data from synthetic iron-rich 2:1 smectites fit also well with both Cuadros et al. (2019) and Decarreau and Petit (2014) models.

 [START_REF] Cuadros | Controls on tetrahedral Fe (III) abundance in 2:1phyllosilicates[END_REF]used a wide range of data (70 samples) concerning dioctahedral and trioctahedral Fe 3+ -bearing 2:1 phyllosilicates, to propose a model describing how tetrahedral occupancy by Fe 3+ takes place in both dioctahedral and trioctahedral 2:1 phyllosilicates. The data came from the investigation of 2:1 phyllosilicates of submarine hydrothermal origin (29) and from literature (41). [START_REF] Cuadros | Controls on tetrahedral Fe (III) abundance in 2:1phyllosilicates[END_REF] wrote: "With respect to cation competition for specific sites in phyllosilicates, it appears that the radius and charge of Si 4+ , Al 3+ , Fe 3+ , Fe 2+ , and Mg 2+ only allow Al 3+ and Fe 3+ to occupy both tetrahedral and octahedral sites. The relative stability of these two cations in the two sites should be a control for Fe(III) distribution between both sites". On the basis of this largely accepted assumption, and using the formalism for intra-crystalline homovalent ions exchange between two nonequivalent sites, [START_REF] Decarreau | Fe 3+/ Al 3+ partitioning between tetrahedral and octahedral sites in dioctahedral smectites[END_REF] proposed a preexisting model based on a partition coefficient approach contradicting Cuadros et al.

(2019)'s claim to report for the first time a model of Fe 3+ distribution in 2:1 phyllosilicates. [START_REF] Decarreau | Fe 3+/ Al 3+ partitioning between tetrahedral and octahedral sites in dioctahedral smectites[END_REF] showed that the distribution of Al 3+ and Fe 3+ for a large variety of both di-and tri-octahedral 2:1 phyllosilicates. A single Kd (4/6) value suits to most samples irrespective of their di-or tri-octahedral character and of their amount of M 2+ (Fig. 1).

It is possible to evaluate the amount of tetrahedral Fe 3+ from the total amount of Fe 3+ and the Kd (4/6) value determined by [START_REF] Decarreau | Fe 3+/ Al 3+ partitioning between tetrahedral and octahedral sites in dioctahedral smectites[END_REF], fixing the amount of octahedral M 2+ cations and the layer charge. The Cuadros et al ( 2019)'s data of figure 1 were reported on figure 2. Most of di-and tri-octahedral samples fit well using a Kd (4/6) value of 0.006, a tetrahedral charge of 1, and an amount of M 2+ cations from 0.2 to 0.8 (for O 20 (OH) 4 ) (Fig. 2). The trioctahedral samples that are out of the range are micas and Fe-rich saponites with none or verylow total amount of Al. Nevertheless, the data are consistent with a low Kd (4/6) value (Fig. 2).

For the selection of literature data, [START_REF] Cuadros | Controls on tetrahedral Fe (III) abundance in 2:1phyllosilicates[END_REF] disregarded some relevant data concerning micas [START_REF] Semenova | The structural chracteristics of micas with tetrahedral iron content regarding isomorphous substitutions[END_REF] and excluded some data from synthetic nontronites [START_REF] Petit | Hydrothermal synthesis of dioctahedral smectites: The Al-Fe 3+ chemical series. Part II: Crystal-chemistry[END_REF][START_REF] Baron | Influence of aqueous Si and Fe speciation on tetrahedral Fe (III) substitutions in nontronites: a clay synthesis approach[END_REF] because tetrahedral Fe 3+ was mainly obtained using IR spectroscopy. Due to their high total Fe 3+ and high Fe 3+ 4 amounts compared to natural samples, the series of well characterized synthetic nontronites are interesting samples to study iron status in clay minerals [START_REF] Petit | Synthesis of nontronite and other Fe-rich smectites: a critical review[END_REF]. Several studies gave strong evidence that the wavenumber of several IR bands, and notably the main Si-O band at about 1000 cm -1 can be used efficiently to quantify the octahedral and tetrahedral Fe 3+ amounts [START_REF] Petit | Hydrothermal synthesis of dioctahedral smectites: The Al-Fe 3+ chemical series. Part II: Crystal-chemistry[END_REF][START_REF] Baron | Influence of aqueous Si and Fe speciation on tetrahedral Fe (III) substitutions in nontronites: a clay synthesis approach[END_REF]. These samples of [START_REF] Baron | Influence of aqueous Si and Fe speciation on tetrahedral Fe (III) substitutions in nontronites: a clay synthesis approach[END_REF] were also studied by chemical analysis using scanning electron microscope equipped with an energy dispersive spectrometer, Mössbauer spectroscopy (Baron et al., 2017), and by X-ray diffraction that supported well the IR results. [START_REF] Cuadros | Controls on tetrahedral Fe (III) abundance in 2:1phyllosilicates[END_REF] alleged that these data from synthetic nontronites fit their regression. Why thus exclude these data for their study?

Figure 2

 2 Figure2

between octahedral and tetrahedral sites of dioctahedral smectites was controlled by a partition coefficient Kd

  This is the peer-reviewed, final accepted version for American Mineralogist, published by the Mineralogical Society of America. The published version is subject to change. Cite as Authors (Year) Title. American Mineralogist, in press. DOI: https://doi.org/10.2138/am-2021-7865. http://www.minsocam.org/ and Petit, 2014). All data of Cuadros et al. (2019) (supplementary file S1) were plotted on a

	(4/6) = 6 ) to evaluate a Kd value (Fig. 1). Most of the data 4 = Fe 3+ 4 ], (Fe 3+ 6 * Al 3+ 6 ]/ [Fe 3+ 4 * Al 3+ [Fe 3+ 4 vs Fe 3+ are consistent with Kd (4/6) values ranging from 0.006 to 0.02 except two trioctahedral samples (ferriphlogopite with no Al 3+ 6 and talc/smectite with almost no Fe 3+ 6 and Al 3+ 6 ) and three nontronites (HQ and two NG1 with different structural formulae). The partition coefficient / (Fe 3+ + Al classical geochemical diagram (Fe 3+ approach of Decarreau and Petit (2014), established for dioctahedral smectites, appears efficient

3+

) molar ratio in tetrahedra, 4 and 6 referring to tetrahedral and octahedral sites; similar relations for Al 3+ ), and a Kd (4/6) value of 0.006 was obtained from the fit of data from natural smectites formed at low temperature. This very low Kd (4/6) value was consistent with the physical model of

[START_REF] Brice | Some thermodynamic aspects of the growth of strained crystals[END_REF]

, widely used for the partitioning of elements in geochemistry. The model of

[START_REF] Brice | Some thermodynamic aspects of the growth of strained crystals[END_REF]

also predicts an increase in Kd (4/6) with the increase of temperature of mineral formation. Accordingly, a Kd (4/6) value of 0.02 was measured from synthesis experiments of dioctahedral smectites at 200°C

(Decarreau

  This is the peer-reviewed, final accepted version for American Mineralogist, published by the Mineralogical Society of America.The published version is subject to change. Cite as Authors (Year) Title. American Mineralogist, in press. DOI: https://doi.org/10.2138/am-2021-7865. http://www.minsocam.org/

Figure captions

This is the peer-reviewed, final accepted version for American Mineralogist, published by the Mineralogical Society of America.

The published version is subject to change. Cite as Authors (Year) Title. American Mineralogist, in press. DOI: https://doi.org/10.2138/am-2021-7865. http://www.minsocam.org/ Always consult and cite the final, published document. See http:/www.minsocam.org or GeoscienceWorld


Johan Quispe

Stéphane Régnier

Magnetic miniature swimmers with multiple rigid flagella

In this paper, we introduce a novel miniature swimmer with multiple rigid tails. The tails' geometry is based on spherical helices that benefit the swimmers for transporting objects with their flagellar bundle. When the swimmer is rotated, their tails provide a considerable propulsive force to generate a net displacement. Thus, achieving propulsion speeds up to 6 mm/s at 3.5 Hz (small rotation frequencies) for a 6-mm in size prototypes. We study the efficiency of different bundle distribution for a 2-flagella swimmer by varying the phase angle between tails. Moreover, it is demonstrated that these swimmers experience a great sensibility when changing their tail height. Besides, the swimmers demonstrate to be effective for cargo carrying tasks since they can displace objects up to 3.5 times their weight. Finally, the confinement effect is studied with multi-tailed swimmer robots considering 2 containers with 20 and 50-mm in width. Results showed speeds' increments up to 59% when swimmers are actuated in the smaller container.

I. INTRODUCTION

Over the years, the microworld has drawn much attention of various research areas because of its fascinating and nonintuitive way of working. At small scales, inertia and gravity forces are less significant. Conversely, surfaces forces play an important role for microorganism propulsion. These species must perform nonreciprocal patterns in order to propel [START_REF] Purcell | Life at low Reynolds number[END_REF]. Multifarious of such species are provided of single or multiple flagella through an evolutionary process where optimization was performed through survival and reducing energy consumption [START_REF] Liu | Propulsion by a helical flagellum in a capillary tube[END_REF]. Moreover, microorganisms developed different propulsion mechanisms, namely, a bundle rotating in a corkscrew fashion or a beating flagellum propelling by planar undulations. The former is inspired by prokaryotic cells such as E. coli bacteria, R. sphaeroides, Spiroplasma, and so on [START_REF] Lauga | The hydrodynamics of swimming microorganisms[END_REF]. During last decades, single helical flagellum swimming have been well reviewed in the literature. Great efforts have been made to find an optimal shape for the single flagellum. The influence on the swimming efficiency of parameters such as the pitch [START_REF] Xu | Influence of geometry on swimming performance of helical swimmers using doe[END_REF], pitch angle [START_REF] Yang | Kinematics of the swimming of spiroplasma[END_REF], [START_REF] Wada | Model for self-propulsive helical filaments: kink-pair propagation[END_REF], number of turns [START_REF] Xu | Influence of geometry on swimming performance of helical swimmers using doe[END_REF], total length [START_REF] Hakan O Caldag | Experimental characterization of helical swimming trajectories in circular channels[END_REF], magnetization [START_REF] Henry C Fu | Magnetization directions and geometries of helical microswimmers for linear velocity-frequency response[END_REF], flagellum thickness [START_REF] Xu | Influence of geometry on swimming performance of helical swimmers using doe[END_REF], and so on have been widely studied through experiments and simulations [START_REF] Fujita | Optimum shape of a flagellated microorganism[END_REF], [START_REF] Bet | Efficient shapes for microswimming: From three-body swimmers to helical flagella[END_REF], [START_REF] Liu | Propulsion by a helical flagellum in a capillary tube[END_REF], [START_REF] Shum | Microswimmer propulsion by two steadily rotating helical flagella[END_REF]. This trend of studying the single-flagellum propulsion born because real microorganisms present multiple flagella that frequently generate a single rotating bundle to self-propel. Nonetheless, organisms such as magnetotactic bacteria include the strain MO-1 that generates two bundles which rotate independently from well-defined locations [START_REF] Christopher T Lefèvre | Isolation and characterization of a magnetotactic bacterial culture from the mediterranean sea[END_REF]. Each *This work was supported by the French National Research Agency through ANR MultiFlag (grant number ANR-16-CE33-0019).

Johan Quispe and Stéphane Régnier are with the Institut des Systèmes Intelligents et Robotique (ISIR), Sorbonne Université, 75005 Paris, France. quispe@isir.upmc.fr bundle is composed of 7 flagellar filaments and numerous fibrils enveloped in a sheath [START_REF] Ruan | Architecture of a flagellar apparatus in the fastswimming magnetotactic bacterium mo-1[END_REF]. The main advantage is that this structure can propel the bacterium at speeds of up to 300 µm/s, an order of magnitude faster than many other flagellated bacteria. In [START_REF] Shum | Microswimmer propulsion by two steadily rotating helical flagella[END_REF] is demonstrated that this structure with two bundles achieves a better propulsion speed when bundles are far apart. In addition, this 2-bundle prototype is proven to escape with ease from surfaces, conversely to their single-bundle counterparts. Other microorganisms such as Paramecium have independent beating cilia around their plasma membranes that allow them to self-propel [START_REF] Lauga | The hydrodynamics of swimming microorganisms[END_REF]. Previous studies have demonstrated propulsion enhancement by increasing the number of flexible flagella [START_REF] Zhou Ye | Rotating magnetic miniature swimming robots with multiple flexible flagella[END_REF], [START_REF] Singleton | Micro-scale propulsion using multiple flexible artificial flagella[END_REF]. In those works, straight-flexible flagella were manufactured and placed on a magnetized head with an offset distance and an anchoring angle with respect to the robot's axis of rotation. When the robot's body rotates, their tails experienced bending angles achieving a chiral structure, and thus provoking a net axial force to propel in the viscous medium. The authors in [START_REF] Wright Carlsen | Magnetic steering control of multi-cellular bio-hybrid microswimmers[END_REF] studied bio-hybrid microswimmers propelled by multiple bacterial cells. Such swimmers consist of Serratia marcescens bacteria attached to a 6 µm-diameter superparamgnetic bead. Authors demonstrated that using remote magnetic control reduces the stochasticity of motion, enabling steering control. Besides, a direct proportion between the number of attached bacteria and the reached speed is proven as well. Notwithstanding these results, the bundle shapes was studied in [START_REF] Danis | Thrust and hydrodynamic efficiency of the bundled flagella[END_REF] considering rigid millimetric prototypes using CFD simulations and PIV techniques. The authors observed that the most efficient configuration is for a single flagellum. Furthermore, they showed that 2-flagella configurations with 90 and 180 degrees in phase (angular tail separation) are ineffective, conversely to previous results considering flexible tails. In [START_REF] Bet | Efficient shapes for microswimming: From three-body swimmers to helical flagella[END_REF], using bead-shell formulation the authors found different optimal structures considering the same volume and varying the flagellum helical radius and pitch angle. Some of these optimal structures are not found in nature, bringing new criteria for fabricating artificial microrobots. In this article, miniature robot designs with multiple flagella are introduced. Such structures consider some of the previous discussed points, namely, a multi-flagellated structure, a chiral shape inspired by spherical helices, and a large helical radius, in such a manner to create a shape not seen before in nature but with effective propulsion. Although their geometries are quite different from real micro-species, their propelling mechanism remains the same. The experiments with these millirobots were performed in a low Reynolds regime using pure glycerol. However, even if gravity force at this regime is smaller than viscous forces, the gravity term will act as a perturbation term for the swimmer dynamics. We study the bundle distribution of a 2-flagella swimmer finding an optimum distribution when tails are separated a phase angle of 180 0 . Moreover, the in-phase distribution (phase angle 0 0 ) is ineffective since the swimmer does not achieve to propel, confronting the results in [START_REF] Danis | Thrust and hydrodynamic efficiency of the bundled flagella[END_REF] with the typical helical geometries. Furthermore, the proposed 2-flagella swimmer with 3-mm in tail height demonstrates to be susceptible to tail height variations. Results demonstrate that just an small increment of 1-mm of the height can duplicate the speed while the decrease of 1-mm can cause a null propulsion speed. In addition, this swimmer could be helpful for cargo carrying applications. Its great helical radius allow it to carry objects with even 3.5 times its weight. Finally, the last contribution relies on the analysis of the confinement effect for different symmetric bundle distributions of 2, 3 and 4 tails in 2 containers with 20 and 50-mm in width. The swimmers in all cases achieve increments on propulsion speed, reaching an increment up to 59% for a 3-flagella robot. In this study as well, it is demonstrated that the 2-flagella distribution is the most effective for this class of robots. The manuscript is organised as follows: in section II, the conception and actuation of the robot are presented. Moreover, the theoretical background for modeling the proposed robot is treated with finite element method (FEM) formulation through a commercial software, COMSOL Multiphysics. In Section III, we present simulation and experimental results accompanied by a brief discussion. Results are related to geometric optimization, maximum cargo, and confinement effect experimented by the proposed robot. Finally, in section IV, we synthesize discussion preceding of conclusions, and perspectives for future work.

II. CONCEPTION, MODELING AND SIMULATION

A. Conception and actuation

The millimetric prototypes are composed of spherical helical tails conceived by 3D printing with Visijet M3 black as a UV curable plastic material. The magnetization is given through a disc-shaped neodymium magnet (φ = 1.0mm in diameter, and δ d = 0.5mm in thickness) attached to a cylindrical head (cf. figure 1.b). The tail geometry follows the next parameterization:

x = R 2 -t 2 cos(t/s) (1a) y = R 2 -t 2 sin(t/s) (1b) z = t (1c)
This parameterization stands for a helical flagellum drawn on a spherical surface with radius R. Parameters t and s determine the number of turns and helix curvature. In most of experiments we keep R = 3, except for the experiments concerning the tail height. Variable t is strong correlated with R, and in order to not obtain a complex number in the parameterization, t might range from 0 to R. Finally, by convenience we kept the parameter s equal to R in all experiments, thus maintaining helix shape for the tail height experiments. The experiments were carried out in pure glycerol with the finality of reproducing a low Reynolds environment. The robots are guided harnessing magnetic fields generated by a 3D nested Helmholtz coil system (cf. figure 1.a). The maximum magnetic field achieved by the system is 12mT. However, the robots were just actuated by 8mT rotating magnetic field to avoid high temperatures in the workspace.

B. Modeling and simulation -finite element formulation

The finite element method is an alternative discretization method for solving partial differential equations (PDEs). This method approximates the solutions of these PDEs by subdividing a large system into smaller, simpler parts that are called finite elements. A priori, the problem that we face involves fluid-structure interaction (FSI) formulation since the swimming robot rotates around its axis generating a net displacements in a viscous fluid. Nonetheless, this interaction is just one-way coupled. In other words, since the robot has a rigid structure, fluid will not be able to deform it. Hence, only the fluid deformation caused by the rotation of the rigid structure is considered. This simplification tremendously reduced the computation time. The one-way coupled FSI problem groups Navier-Stokes (N-S) equations with the mesh treatment. N-S equations (Ω f fluid domain) solves the fluid dynamics, the rotating structure dynamic (Ω s structure domain) is solved by mesh equations that deal with mesh translation and deformation. The boundary condition (∂ Ω f s fluid-structure boundary) on the robot-fluid interface relates mesh translation and fluid dynamics through all time evolution (τ time interval). N-S equations are composed of the momentum conservation and the mass conservation equations. They are expressed as follows:

∂ u ∂t + ρ(u.∇)u = ∇.Γ + F in Ω f × τ (2) ∂ ρ ∂t + ∇(ρ.u) = 0 in Ω f × τ (3)
Γ = -pI + µ(∇u + (∇u) T ) (4)
With u as the fluid velocity, p pressure, ρ density, µ dynamic viscosity, Γ depicting the stress on an infinitesimal fluid volume (∇.Γ surface forces) and F the volume forces.

Concerning the rigid robot structure, a rotation movement is along the robot helix axis. That can be translated in a prescribed mesh motion (dX) since there is no structure deformation.

dX = dX(r, ω,t) in Ω m × τ ( 5
)
Where r is the helix axis, ω is the angular frequency and t is time and that stands for the coupling between N-S equations and the structure movement is given by:

Ω m ⊂ Ω s . The boundary condition in ∂ Ω f s
u = u s in ∂ Ω f s × τ (6)
u s = ∂ X ∂t in ∂ Ω f s × τ (7)
Where u s is the solid structure velocity in one point situated on its surface. Another boundary condition in the limits of the container (∂ Ω f ) is:

u = 0 in ∂ Ω f × τ (8)
Null fluid speed in the fluid-container boundary. Finally, force components are computed by integrating the total stress (Γ) over all the robot's surface.

F i = ∂ Ω s Γn f .e i ds in ∂ Ω f s × τ (9)
Being i = x, y, and z for all different components, n f the normal vector to the surface of each finite element on the robot surface, ds the surface element, and finally, e i representing the unit vectors for x, y, and z of the global frame.

For the robot dynamics we compute the net displacement by using the Newton's second law.

m dv z dt = F z + gV (ρ f luid -ρ robot ) (10)
dx z dt = v z (11)
Where dx z is the displacement in the forward direction, v z is the robot speed. The term gV (ρ f luid -ρ robot ) (perturbation term) is the buoyancy force, where g is the gravity and V is the robot volume. Forces in x and y are not considered Finally, the total force is decomposed in its x, y and z-components (green, red and blue respectively in fig. 4.c). While the z-component is null through the time evolution, the components in x and y have an harmonic behaviour because of the symmetry disposition of the robot's tails. When solving a multiphysics model, there are two approaches that can be taken to solving the (usually nonlinear) system of equations that describe the solution. the fully coupled approach forms a single large system of equations that solve for all the unknowns (the fields) and includes all of the couplings between the unknowns (the multiphysics effects) at once, within a single iteration. On the other hand, the "segregated" approach will not solve for all of the unknowns at one time. Instead, it subdivides the problem up into two or more "segregated steps". These individual segregated steps are smaller than the full system if equations that are formed with the fully coupled approach.

The segregated steps are solved sequentially within a single iteration, and thus less memory is required. I gives an extra-information of mesh study concerning the number of elements for each mesh size and the time taken for each simulation in a 28 GB RAM computer with 7 cores. Finally, as an additional study, we considered the case when the robot is propelling in an inverted fashion (with the head up and down). Results shown a decrease in the forward speed that can be caused by the change of the aerodynamic profile when the robot is inverted while going forward.

III. EXPERIMENTAL AND SIMULATION RESULTS

A. Optimal angular position for a second tail

Through experimentation, the optimal angular position for a second tail is studied. Considering the proposed tail geometry, we fabricated different 2-flagella robots with different angular separations (cf. fig. 6.b). These robots were actuated under 8mT rotating magnetic field at 4 Hz. The forward Fig. 7. Analysis of height influence on propulsion speed. Solid lines and dashed lines stand for simulation and experimental results, respectively. Blue and red lines depict results for the 3-mm and 4-mm in tail length robots, respectively. Simulation and experimental results show an increase of speed when tail height is increased. The error bars in experiments are calculated as the standard deviation of 3 trials for each frequency and robot.

propulsion speed was measured for each configuration 3 times (negligible error) and plotted in polar coordinates in fig. 6.a. The radial axis depicts the forward speed in (mm/s) while the angular separation is represented by the angles in the graph. Figure 6.b depicts a robot with an angular separation θ between tails. The tail on the left is kept while the other is rotated forming different angles θ . In the experiments were considered θ = 0, 30, 60, 90, 120, 150, 180 degrees because of the robot symmetry. The in-phase configuration (θ = 0) demonstrates, in this case, to generate insufficient propulsion force to get off the ground. Different from the result obtained in [START_REF] Danis | Thrust and hydrodynamic efficiency of the bundled flagella[END_REF]. Finally, in figure 6.c stands for the optimal angular distribution of tails, being 180 degrees the most performant. It means that both tails have to be as far as possible distributed in order to avoid hydrodynamic interactions between each other and thus achieving high performance. A similar result was obtained recently in [START_REF] Shum | Microswimmer propulsion by two steadily rotating helical flagella[END_REF] for a 2-bundled robot with each bundle rotating independently.

B. Height influence

This study is devoted to analyze the effect on forward speed of the length increment and decrease. The experiments were carried out through finite element simulations and corroborated experimentally. Initially, we started considering 3 robots with tails height of: 2, 3 and 4 mm or more precise with parameter R = 2, 3, and 4. However, the 2-mm robot tails were not able to provide the enough net force to displace the robot. hence, figure 7 only include results for the robots of 3-mm, and 4-mm in tail heights. In both cases, simulations and experiments, the tail length increased the forward speed. The robot in 4-mm tail height experienced an increment of practically 100% in forward speed with respect to its smaller counterpart just by increasing 1-mm the tail height. On the other hand, if we reduce in 1-mm the tail height of the robot in 3-mm tail height, it will not be able to compensate the robot apparent weight and propulsion becomes non-achievable.

C. Cargo influence

As part of the study, the feasibility for cargo transporting was assessed considering spheres with different weights attached inside flagella of a 2-tailed robot with 3-mm in tail height. Simulations and experiments were performed in order to analyze the correlation between the cargo weight and the propulsion speed. The tested weights for this experiment were 25, 34, and 56 mg as can be seen in figure 8. Results confirm the intuition that the heavier cargo, the less propulsion speed. The interesting part of this is the robot capacity to transport even objects 3.5 times heavier than its weight (2-flagella robot's weight is 16mg).

D. Confinement effect

Future applications in microrobotics involve medicament delivery in tiny, constrained, and difficult-to-reach environments; where confinement effect will change drastically the robot dynamics. Several studies have stated that confinement effect benefit robot propulsion since speed increases [START_REF] Howard | Chemotaxis of bacteria in glass capillary arrays. escherichia coli, motility, microchannel plate, and light scattering[END_REF], [START_REF] Biondi | Random motility of swimming bacteria in restricted geometries[END_REF]. In spite of this increment, the viscous drag increases as well as the viscous fluidic torque producing a decrease of cutoff frequency in case we set the magnetic torque constant. In other words, in order to avoid a cut-off frequency, we would need to increase the magnetic field value in order to increase as well the magnetic torque and overcome the fluidic torque. The experiment consists of analysing the confinement effect of 3 different multi-flagella robots with 2, 3, and 4 tails arranged symmetrically (cf. figure 2). Each robot is placed in two containers with 20 and 50 mm in diameter respectively, then, actuated in 8 mT rotating magnetic field at frequencies ranging from 0 to 3.5 Hz. The obtained results for containers with 50-mm and 20-mm in diameter are depicted in figure 9.a and 9.b respectively. The results are divided in FEM simulations (solid lines) and experimental (dashed lines). In both cases, comparing frequency by frequency for each robot in each container (before the respective cut-off frequency), speed increases considerably. In the inset of figure 9.b, a bar graph is depicting the increment ratio of the speed of each robot at cut-off frequencies when placed in the 20-mm diameter container. This bar graph shows that the maximum a speed increment is for a 3-tailed robot, achieving a 59% with respect to the speed when swimming in the 50-mm container. However, as aforementioned, the problem of the cut-off frequencies can be overcome if we increases the magnetic field. Finally, comparing the effectiveness of the multi-tailed robots, we could see that for the 50-mm container the speeds of the 2 and 3-tailed were close. However, when analysing the results for a 20-mm container, the 2-tailed robot resulted the most effective.

IV. CONCLUSIONS AND PERSPECTIVES

Through these studies, we analysed the optimal position for a second tail in a miniature robot, the tail height effect on propulsion, the influence of cargo on propulsion, and the boundary effect with rigid multi-flagella prototypes (2, 3 and 4 tails). These studies were performed in most of cases using two approaches: finite element simulations and experimentation. Thus, corroborating the simulation data. We found that the optimal angular position for a second tail in a 2-flagella configuration is 180 o , as far as possible from each other. Furthermore, the influence of the tail height was demonstrated to be strongly correlated to the forward speed of the robot. The more tail height the larger speed value. On the other hand, the influence of the cargo is inversely proportional to the forward speed. The proposed prototype demonstrates to carry large amounts of cargo. However, the forward speed reduces drastically. Besides, the boundary effect was investigated using three multiflagella prototypes with a symmetric tail distribution. For that experiment, two containers with 20 and 50 mm in width were used. Simulation and experiments demonstrates an increment on propulsion speed at same frequencies when robots are immersed in the container of smaller width. Experimentally we achieved a relative increment up to 59%. Nonetheless, the cut-off frequencies is reduced since the fluidic torque increases in the small container. Finally, the effectiveness of the multi-tailed robots was discussed showing that the 2tailed robot was the best one from our experiments. The next step of this work is to scale down our flagella prototypes in order to see their viability for microscale applications.

Fig. 1 .

 1 Fig. 1. Experimental set-up and swimmer prototype. (a) The system is mainly composed of a side camera with an endoscope, a top camera, and a 3D Helmholtz coil system for the magnetic actuation. (b) The swimmer prototype is composed of multiple 3D printed tails with a disc magnet placed inside the cylindrical head.

Fig. 2 .

 2 Fig. 2. Robot designs with multiples tails 2,3 and 4. The magnet is found inside the cylindrical head with 1.6mm in diameter and 2mm in height. The real printed prototypes are flanked respectively the CAD's. For the case study with finite elements we use the 2-tailed robot, the rest is used for the confinement effect experiments.

Fig. 3 .

 3 Fig. 3. Kinematics of the 2-flagella robot. (a) Depicts the robot displacement and the fluid speed around for 4 frames.(b) shows a graph representing the position and speed reached during 0.9 seconds. (c) Draw the isocurves of speed for the top and side views.

Fig. 4 .

 4 Fig. 4. Dynamics of the 2-flagella robot. (a) separates the total force in z (green) in its pressure (blue), viscous (red) components. (b) Depicts how the viscous force (red arrows on the robot's surface) is opposite to fluid velocity. (c) shows the total force decomposed in its 3 components in x, y and z. While the total force in z-component converges rapidly to zero, the y and x-components shows an harmonic behaviour because of the tail disposition and symmetry.

Fig. 5 .

 5 Fig. 5. Convergence study. (a) Segregated solver error. (b) Time-dependent solver, reciprocal time-step. (c) Mesh effect on speed accuracy. (d) effect on speed of having the robot with head up or head down.

Fig. 6 .

 6 Fig. 6. Looking for the optimal position for a second tail to enhance propulsion. (a) Polar plot depicting the forward speed (mm/s) in the radial axis vs. angular separation between tails θ ( o ). The measurements for this experiment were done 3 times and the error bars were negligible. (b) A sample of a robot with an angular separation of θ . (c) The angle that maximizes propulsion speed is 180 0 .

  Figure 5.a depicts the convergence error of the segregated steps per each iteration. In fig. 5.b is shown the reciprocal of step size vs. the time step. Besides, a mesh susceptibility study was performed considering the case study with different mesh sizes: coarse, normal, fine, finer. We analysed the speed evolution during 0.1 s of the velocity when setting different meshes sizes. These results are shown in fig. 5.c. Moreover, table

Fig. 8 .

 8 Fig. 8. Cargo influence on propulsion speed. Solid and dashed lines represent simulation and experimental data, respectively. Blue, red and green color represents the cargo weight of 25, 34 and 56 mg respectively. Simulation and experimental results have the same trend, the larger cargo amount, the less propulsion speed. The inset on the bottom right shows in the same frame different positions of the robot with the 25mg cargo. The experimental error bars are calculated as the standard deviation of three trials for each frequency and robot.

Fig. 9 .

 9 Fig. 9. Confinement effect, (a) Robots with 2 (blue), 3 (red) and 4 (green) tails are assessed in a 50-mm in width container. The 2 and 3-tailed robots have close speed values. (b) The robots are tested in a 20-mm in width container experiencing cut-off frequencies because of the increase of the fluid torque associated to the degree of confinement. The inset on the bottom left show the percentage of speed increment at cut-off frequencies of each multi-flagellated robot. Being the 3-tailed robot the one who experiences a percentage up to 59%.The experimental error bars are calculated as the standard deviation of three trials for each frequency and robot.