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Bioengineered 2′-fucosyllactose and 3-fucosyllactose inhibitthe adhesion of Pseudomonas aeruginosa and enteric pathogensto human intestinal and respiratory cell lines

Stefan Weichert a,⁎, Stefan Jenneweinb, Eric Hüfner b, Christel Weiss c, Julia Borkowskia,Johannes Putzea, 1, Horst Schrotena, 1

a Pediatric Infectious Diseases, University Children's Hospital Mannheim of Heidelberg University, Germanyb Jennewein Biotechnology, Rheinbreitbach, Germanyc Institute of Medical Statistics and Biomathematics, Medical Faculty Mannheim of Heidelberg University, Germany

A R T I C L E I N F O

Abbreviations: CFU, colony forming units;human milk oligosaccharide; HPAEC-PAD, hiLNFP, lacto-N-fucopentaose; MOI, multiplicit⁎ Corresponding author. Pediatric Infectious

Kutzer-Ufer 1-3, 68167 Mannheim, GermanyE-mail address: Stefan.Weichert@medma

1 These authors contributed equally to this

0271-5317/$ – see front matter © 2013 Elsevihttp://dx.doi.org/10.1016/j.nutres.2013.07.009

A B S T R A C T

Article history:Received 8 May 2013Revised 6 July 2013Accepted 8 July 2013

Human milk oligosaccharides help to prevent infectious diseases in breastfed infants.Larger scale testing, particularly in animalmodels and human clinical studies, is still limiteddue to shortened availability of more complex oligosaccharides. The purpose of this studywas to evaluate 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) synthesized by whole-cell biocatalysis for their biological activity in vitro. Therefore, we have tested theseoligosaccharides for their inhibitory potential of pathogen adhesion in two different humanepithelial cell lines. 2′-FL could inhibit adhesion of Campylobacter jejuni, enteropathogenicEscherichia coli, Salmonella enterica serovar fyris, and Pseudomonas aeruginosa to the intestinalhuman cell line Caco-2 (reduction of 26%, 18%, 12%, and 17%, respectively), as could beshown for 3-FL (enteropathogenic E coli 29%, P aeruginosa 26%). Furthermore, adherence of Paeruginosa to the human respiratory epithelial cell line A549was significantly inhibited by 2′-FL and 3-FL (reduction of 24% and 23%, respectively). These results confirm the biologicaland functional activity of biotechnologically synthesized human milk oligosaccharides.Mass-tailored human milk oligosaccharides could be used in the future to supplementinfant formula ingredients or as preventatives to reduce the impact of infectious diseases.

© 2013 Elsevier Inc. All rights reserved.

Keywords:Human milk2′-fucosyllactose3′-fucosyllactoseBacterial adhesionCell culture techniques

1. Introduction

Human breast milk is a complex mixture of carbohydrates,fats, proteins, and other molecules that provide the primarysource of nutrients for newborns and infants, as well as anumber of additional protective and health-promoting factors

EPEC, enteropathogenic Egh pH anion exchange chy of infection.Diseases, University Ch

. Tel.: +49 621 383 1299; fa.uni-heidelberg.de (S. Wework.

er Inc. All rights reserved

that cannot be replicated in artificial formulas. The exactcomposition of breast milk varies during the neonatal andpostnatal period in accordance with the nutritional require-ments of the child. It also varies during feeding, and accordingto the diet and health of the mother [1–3]. A better under-standing of the composition and function of human breast

scherichia coli; 2′-FL, 2′-fucosyllactose; 3-FL, 3-fucosyllactose; HMO,romatography with pulsed amperometric detection; Fuc, L-fucose;

ildren's Hospital Mannheim of Heidelberg University, Theodor-x: +49 621 383 3818.ichert).

.

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milk has revealed its diverse interactions with microbes andthe immune and digestive systems of nursing infants. In thiscontext, recent research has focused on the health-promotingrole of human milk oligosaccharides (HMOs).

HMOs represent the third largest solid componentofhumanbreast milk after lactose and lipids, reaching concentrationsexceeding 20 g/L in colostrumand5–10 g/L inmaturemilk [4,5].They are highly diverse unconjugated lactose-based carbohy-drates,whichare clarified into 13 core series andmore than 200different HMO structures are composed by the attachment of L-fucose (Fuc) and N-acetylneuraminic acid to the core oligosac-charides [5–7]. Human breast milk is unusual compared to themilk of other mammals in the complexity and high concen-tration of fucosylated oligosaccharides, promoting studies todetermine the biological roles of these molecules [5,8–10].

HMOshelp to prevent infectious diseases by acting as decoyreceptors and inhibiting the adhesionof pathogenic bacteria tospecific receptors on epithelial cell surfaces, one of the firststeps towards colonization [11,12]. This has been demonstrat-ed by using in vitro cell culture models in the presence ofdifferent fractions or specific purified HMOs from donatedhuman breast milk. For example, Coppa and colleagues [13]showed that different HMO fractions could prevent theadhesion of Salmonella enterica serovar fyris, enteropathogenicEscherichia coli (EPEC) serotype O119 and Vibrio cholerae to ahuman intestinal epithelial cell line, and Ruiz-Palacios et al[14] showed that fractionated neutral and fucosylated HMOsreduced the adhesion of different Campylobacter jejuni strainsin cell culture and reduced colonization rates in a mousemodel. HMOs also provide protection against respiratorypathogens such as Pseudomonas aeruginosa by inhibiting theirbinding to human respiratory epithelial cells [15]. Bindingstudies confirmed that the fucose-specific P aeruginosa lectinPA-IIL was blocked by certain fucosylated HMOs [16].

The studies discussed above primarily involved the use of invitromodels because the fractionation of donated human breastmilk yields only minute quantities of neutral and acidic HMOsandevensmaller amountsof trisaccharides. Larger-scale testing,particularly in animal models and human clinical studies, willrequire amore reliable source of HMOs and preferably the abilityto test specific HMO structures individually.

In this study we evaluated 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) synthesized by whole-cell biocatalysisfor their biological activity. Therefore, we have tested theseoligosaccharides for their inhibitory potential of pathogenadhesion in the human intestinal cell line Caco-2 and thehuman respiratory cell line A459. Typically enteric pathogens(C jejuni, enteropathogenic E coli, and S fyris) as well as themainly respiratory pathogen P aeruginosa were tested in thesein vitro systems.

2. Methods and materials

2.1. Synthesis of HMOs

We synthesized 2′-FL and 3-FL by whole-cell biocatalysis aspreviously described [17,18] using E coli strains expressing theBacteroides fragilis FKP gene encoding the bifunctional enzymeL-fucokinase/GDP-fucose pyrophosphorylase (which converts

L-fucose to fucose-1-phosphate and then to GDP-fucose) aswell as either 3-fucosyltransferase or 2′-fucosyltransferase(facilitating the synthesis of 3-FL and 2′-FL, respectively) and asugar efflux exporter, enabling the recovery of the synthesizedoligosaccharides from the medium. The E coli cells werecultivated by fed-batch fermentation in M9 minimal mediumsupplemented with L-fucose and lactose. The exportedoligosaccharides were separated from the biomass by filtra-tion, desalted, purified by gel filtration, and freeze-dried. Theidentities of the purified oligosaccharides were confirmed bynuclear magnetic resonance spectroscopy and a purity of>95% was determined by high-performance liquid chroma-tography. The other carbohydrates present were identified aslactose and L-fucose. For the biological assays, the HMOs weredissolved in water at a final concentration of 10 mg/mL andfilter sterilized. D-(+)-mannose (Sigma Aldrich, St. Louis, MO,USA) and D-(+)-lactose-monohydrate (Applichem, Darmstadt,Germany) were used as controls.

2.2. Human breast milk

Written informed consent was obtained from all mothers whodonated human breast milk. The samples were separated intoaqueous and lipid phases by centrifugation (4.600 rounds perminute for 15 minutes) at 4°C. The aqueous fraction was filtersterilized anddiluted 1:10 into the cell culturemediumtouse asexperimental controls. For the preparation of the analysis ofhuman milk oligosaccharides milk was centrifuged (3.500rounds per minute for 1 h) at 4°C and then processed asdescribed elsewhere [19]. Briefly, the lipid layer was removedand the aqueous phase decanted and filtered (0.2 μmpore size).By adding an equal volume of pre-cooled acetone, proteinswere precipitated. Lactose was removed by repeated crystalli-zation. Carbohydrate-containing fractions were identified bythe anthrone and Ehrlich methods. Fractions with residualpeptides (ninhydrin-positive fractions) were excluded from theanalysis. Then, salt-free but carbohydrate-positive fractionswere pooled and lyophilized. The milk oligosaccharide compo-sition was determined by high pH anion exchange chromatog-raphy with pulsed amperometric detection (HPAEC-PAD).Briefly, HPAEC-PAD on a Carbo Pac PA-1 column(250 × 4.6 mm ID) equipped with a guard column and a ModelPAD 2 detector (Dionex, Sunnyvale, CA, USA) were used forcharacterization of neutral and acidic HMOs as describedpreviously [20]. The separating conditions were as follows:eluent A, 100 mmol/L sodium hydroxide; eluent B, 100 mmol/Lsodiumhydroxide and250 mmol/L sodiumacetate. The elutionprogram started with 3 mL of buffer A followed by a gradient ofup to 100% buffer B in 30 min and a re-equilibration volume of5 mL buffer A. The flow-rate of 1.0 mL/min was used and 25 μlof a 1 mg/mL solutions were injected. The analysis has beenperformed in the laboratory of Clemens Kunz (Institute ofNutritional Sciences, University of Giessen, Germany).

2.3. Bacterial strains and culture conditions

Four different bacterial strains were tested: S enterica serovarfyris (serotype 4,12:1, v,4,2), an uncommon group B Salmonellaserotype isolated from infants during a small outbreak [13];EPEC strain O119 (both pathogens were kindly provided by G.

833N U T R I T I O N R E S E A R C H 3 3 ( 2 0 1 3 ) 8 3 1 – 8 3 8

Coppa, Ancona, Italy, on behalf of The Italian Study Group onGastrointestinal Infections) [13,21]; P aeruginosa PAO strainDSM1707 (purchased from the DSMZ); and C jejuni strain 81-176 (generous gift of M Heimesaat, Institute for Microbiologyand Hygiene, Charite, Berlin, Germany). We used E coli K-12strain HB101 as a control [22] (kindly provided by U. Dobrindt,Institute for Hygiene, Münster, Germany). S enterica serovarfyris, EPECO119 and P aeruginosaDSM1707were grownonLuria-Bertani agar whereas C jejuni 81-176 was grown on Columbiablood agar. For the infection assays, 3 of the bacterial strainswere inoculated into liquid growth medium (Luria-Bertanibroth for S enterica serovar fyris and P aeruginosa, and brainheart infusion broth for EPEC O119). C jejuni strain 81-176 wastaken directly from the Columbia blood agar plates. Liquidcultureswere incubated at 37°Cwith agitation, whereas C jejuniwas cultured under microaerophilic conditions (CampyGen,Oxoid) at 42°C. For the infection of eukaryotic cells, bacterialsuspensions were adjusted to the correct inoculum afterspectrophotometric analysis, and the number of colony form-ing units (CFU) was determined by plating serial dilutions.

2.4. Cell lines

The human colorectal adenocarcinoma cell line Caco-2 [23]forms confluent andpolarizedmonolayerswith tight junctions,but post-confluent cultivation induces differentiation and thedevelopment ofmicrovilli resembling those of enterocytes [24].Caco-2 cells (AmericanTypeCultureCollection)were cultivatedin 75 cm2 flasks in Dulbecco's modified Eagle's medium(Invitrogen, Darmstadt, Germany) containing 4.5 g/L glucose,4 mmol/L L-glutamine, non-essential amino acids, 10% fetalcalf serum at 37°C and 5% CO2. Cells were subcultured once ortwice weekly. After reaching approximately 90% confluence,the cells were trypsinized (Trypsin, Invitrogen) and seeded into24-well plates at a density of 4 × 105 cells/well. Once the cellsdeveloped a confluent monolayer, they were cultivated to apost-confluent state for 14 days changing the medium everysecond day.

The lung carcinoma cell line A549 is derived from type 2alveolar epithelial cells. A549 cells (American Type CultureCollection) were cultivated as above for Caco-2 cells althoughin 1:1 Dulbecco's modified Eagle's medium/Ham's F12 medi-um (Invitrogen) containing 2 mmol/L L-glutamine, 10% fetalcalf serum and were subcultured 2 to 3 times weekly. Afterreaching approximately 90% confluence, the cells weretrypsinized and seeded into 24-well plates at a density of2.5 × 105 cells/well for 24 hours.

2.5. Infection experiments

Confluent monolayers of A549 cells and post-confluent mono-layers of Caco-2 cellswere infectedwith bacteria at amultiplicityof infection (MOI) of 10 for 2 h at 37 °C and 5%CO2, except for theinfection of A549 cells with P aeruginosa for which the infectiontime was shortened to 1 h to limit potential cytotoxic effects.Infections were carried out in the presence of different oligosac-charides, including D-(+)-lactose as a non-inhibiting control andD-(+)-mannose as a potential inhibitor of E coli and S entericaserovar fyris type 1 fimbriae. The aqueous phase of humanmilkwas included as an inhibition control. Cells were pre-incubated

for 1 h with all the sugars (10 mg/mL) and the aqueous phase ofhumanmilk (1:10 v/v), respectively. Following infection, the cellswere washed 5 times with pre-warmed Hanks' balanced saltsolution (Invitrogen) to remove non-adherent bacteria, and thenlysed by adding Triton X-100 (Sigma Aldrich; final concentration0.025%) to release adherent bacteria without affecting theirviability. Serial dilutions of the recovered bacteria were plated,the number of total bacteria after infectionwas determined, andthe percentage of cell-associated bacteria was calculated usingthe equation (number of recovered bacteria/number of totalbacteria) × 100. All experiments were performed five times(except C jejuni; four times), with triplicates measured for eachsingle condition. Results are shownasmeans ± standard error ofthe mean (means ± SEM).

2.6. Measurement of cell viability

The viability of the cells after infection with different bacterialstrains was determined using a Live/Dead assay (Invitrogen)according to the manufacturer’s instructions. The resultswere photo-documented by fluorescence microscopy. Thecytotoxic effects of HMOs and control sugars were determinedwith alamarBlue cell viability reagent (Invitrogen) according tothe manufacturer's instructions.

2.7. Immunoglobulin A depletion and quantification inhuman breast milk

IgA was depleted from the fractionated donated humanbreast milk by affinity chromatography, as described else-where [25]. Briefly, IgA antibody (Sigma Aldrich) was incu-bated with Affigel 10 (Bio-Rad) overnight at 4 °C in 0.1 mol/L3N-morpholinopropanesulfonic acid at pH 7.5. After blockingfree binding sites with ethanolamine HCl, the gel wastransferred to a column. Free reagents were eluted withcoupling buffer, the column was washed repeatedly withelution buffer (1 mol/L acetic acid, 0.5 mol/L NaCl, 0.5 mol/Lglycine, pH 3.2) and finally equilibrated using starting buffer(0.01 mol/L phosphate-buffered saline, pH 7.4). IgA concen-trations before and after depletion were determined by ELISA(ImmunDiagnostik).

2.8. Statistical analyses

All experiments were carried out five times independently intriplicates (C jejuni; four times). All data are presented asmeans ± SEM. Significant differences were determined byanalysis of variance followed by Dunnett test. P < .05 wasconsidered statistically significant; <.01, very significant; and<.001, highly significant. All statistical calculations wereperformed with the SAS system, release 9.2 (SAS InstituteInc, Cary, NC, USA).

3. Results

3.1. Bacterial adhesion in the absence of HMOs

The percentage of cell-associated bacteria (as a proportionof the growth control) was determined in the presence of

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different HMOs and control sugars by counting the number ofCFUs per mL of serially diluted detergent washes frominfected cell plates. In the absence of HMOs, 21.39 ± 1.51% ofP aeruginosa remained associated with A549 lung epithelialcells, and 3.53 ± 0.23% remained associated with the differen-tiated Caco-2 epithelial cells, whereas 15.86 ± 0.84% of EPEC,1.96 ± 0.16% of S fyris, and 0.40 ± 0.04% of C jejuni remainedassociated with the differentiated Caco-2 epithelial cells (allvalues are means with SEMs). Percentage of adhesion wascalculated relative to an internal growth control, taking intoaccount ongoing bacterial growth throughout the experi-ments. For control, E coli K-12 strain HB101 did not show anyrelevant adhesion to the tested epithelial cell lines (results notshown). None of the HMOs we tested had any impact on hostcell or bacterial growth per se at a concentration of 10 mg/mLas determined by growth curves and cytotoxicity assays(Supplementary Fig. S1, Fig. S2). Donated human breast milkwas prepared as described above and diluted 1:10 into the cellculture medium. The HMO profile of the donated humanbreast milk was analyzed by HPAEC-PAD (Fig. 1). The highestpeak observed in the HMO pool was 2′-FL, whereas morecomplex penta- or hexasaccharides were less prominent. Dueto simultaneous elution of 3-FL with other HMOs (similarretention times), 3-FL could not specifically been quantified.Given the high level of fucosylated oligosaccharides (espe-cially 2′-FL), donated human breast milk was henceforth usedas a positive control for oligosaccharide-mediated inhibitionof adhesion throughout the experiments.

3.2. Bacterial adhesion to gastrointestinal and respiratoryepithelium in the presence of HMOs

Although none of the HMOs directly inhibited bacterialgrowth, the adhesion of S enterica serovar fyris to post-

Fig. 1 – Compositional analysis of donated human breast milk: Hamperometric detection (HPAEC-PAD) of HMOs. The chromatograneutral HMOs. The resulting peaks represent the following oligosLNFP III, LNFP II, lactodifucotetraose (LDFT), lactose, 2′-FL, LNFP I,6lactose (6′-SL), Sialylα2-3lactose (3′-SL), disialyl-lacto-N-tetraosretention times of other oligosaccharides.

confluent Caco-2 cells was inhibited 11.67% ± 6.94% by 2′-FL(P = 0.08, Student t test; adjusted n.s.), 13.37% ± 10.7% by 3-FL(n.s.) and 69.20 ± 2.63% by mannose (P < 0.001) (Fig. 2). Breastmilk had no inhibitory effect when diluted 1:10 to a HMOequivalent volume (results not shown) but showed a tendencytoward inhibition of adhesion at a higher dose with a dilutionof 1:5 (inhibition of 16.98% ± 8.79%, n.s.). The adhesion of EPECstrain O119 to post-confluent Caco-2 cells was significantlyinhibited by 2′-FL (inhibition of 18% ± 5.59%, P = 0.025) andmannose (13.84% ± 3.58%, P = .02) and was even more prom-inent in the presence of 3-FL (28.92% ± 3.38%, P < .0001) or 1:10human breast milk (43.62% ± 4.34%, P < .0001) (Fig. 2). Theadhesion of C jejuni to post-confluent Caco-2 cells wasinhibited 25.77% ± 8.87% (P = .0023) by its known bindingpartner 2′-FL and 23.21% ± 7.08% (P = .0059) by 1:10 breastmilk (Fig. 3). We did not test 3-FL in this assay.

The adhesion of P aeruginosa to post-confluent Caco-2 cellswas inhibited 16.91% ± 5.45% by 2′-FL (P = .0113) and 26.18% ±3.84% by 3-FL (P < .0001). Interestingly, adhesion of P aerugi-nosa was inhibited 52.06% ± 5.72% by 1:10 human breast milk(P < .0001) and 33.76% ± 7.3% by IgA-depleted human breastmilk (P = .0042). The adhesion of P aeruginosa to A549 cells wasinhibited 23.64% ± 4.7% by 2′-FL (P < .0001), 22.64% ± 4.95% by3-FL (P < .0001) and 20.78% ± 6.89% by 1:10 breast milk (P =.0367) (Fig. 4).

4. Discussion

The adhesion of pathogens to human epithelial cells is usuallythe first step towards successful colonization and subsequentsystemic infection. Human milk oligosaccharides mimic cellsurface receptor structures and are therefore thought to act assoluble decoys to prevent the colonization of epithelial surfaces

igh pH anion-exchange chromatography with pulsedm shows one experiment of the separation of acidic aswell asaccharides (left to right): lacto-N-difucohexaose II (LNDFH II),lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), Sialylα2-e (DS-LNT). 3-FL could not be quantified due to similar

Fig. 2 – Adhesion of S fyris (□) and EPEC ( ) to differentiatedCaco-2 cells. Effects on pathogen adhesion of the saccharides2′-FL, 3-FL and mannose (Man), and of human breast milk(BM) are shown in % (relative adhesion) compared to lactose(Lac) (n 5, triplicates for each condition, values are means,with standard error of themeans (means ± SEM) representedby vertical bars, *P < .05, **P < .01, ***P < .0001, #usedrespectively). Sugars were used at a concentration of 10 mg/mL. Human breast milk was used in an equivalent volumecompared to the sugars (1:10, EPEC assay), and in a higherconcentration in the S fyris adhesion assay (1:5).

ig. 4 – Adhesion of P aeruginosa to A549 (□) and toifferentiated Caco-2 ( ) cells. Effect of the saccharides 2′-FL,-FL and human breast milk (BM) (relative adhesion (%)ompared to lactose (Lac); n 5, triplicates for each singleondition, values are means, with standard error of theeans (means ± SEM) represented by vertical bars); *P < .05,

*P < .01, ***P < .0001). Sugarswere used in a concentration of0 mg/mL, andhumanbreastmilkwas used in an equivalentolume compared to the sugars (1:10 dilution). In thedhesion assays with Caco-2 cells BM as well as breast milkhich had been depleted of secretory immunoglobulin AMΔIgA) was used. After depletion sIgA levels in BMΔIgAere 0.1 mg/mL.

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and reduce the incidence of gastrointestinal and respiratoryinfectious diseases [26,27]. In our study, we demonstratedsuccessfully that the synthesized oligosaccharides 2′-FL and 3-FL can inhibit the adhesion of enteric and respiratorypathogensto the human epithelial cell lines Caco-2 and A549.

The addition of 3-FL inhibited the adhesion of EPEC by 29%butwas less effective for S fyris (13%, n.s.). Similar patterns couldbe observed for 1:10 diluted human breastmilk, which inhibitedthe adhesion of EPEC by 39%, but did not affect the adhesion of Sfyris, although a higher concentration (1:5) was successful. The1:10 dilution was chosen to achieve equivalent volumes of

Fig. 3 – Adhesion of C jejuni to differentiated Caco-2 cells.Effects of the oligosaccharide 2′-FL and of human breastmilk (BM ) on pathogen adhesion are shown in % (relativeadhesion) compared to lactose (Lac□) (n 4, triplicates for eachcondition, values are means, with standard error of themeans (means ± SEM) represented by vertical bars; *P < .05,**P < .01, ***P < .0001). Sugarswere used at a concentration of10 mg/mL, and breast milk was used in an equivalentvolume compared to the sugars (1:10).

Fd3ccm*1vaw(Bw

trisaccharides in both assays, but was not adjusted for equalconcentrations of HMOs in the donated human breast milk andthe synthesized HMOs. Also, the lower inhibitory impact of thebreast milk toward S fyris probably reflects the variableconcentration and composition of HMOs among lactatingwomen [28,29]. Our results are consistent with those of Coppaet al [13], who demonstrated that pooled and purified HMOfractions from donated human breast milk could inhibit theadhesion of EPEC and S fyris to Caco-2 cells. The most potentinhibitors of E coli adhesionwere neutral high-molecular-weightfucosylated oligosaccharides (up to 42% inhibition), whereasneutral low-molecular-weight oligosaccharides were moreeffective for S fyris (up to 25% inhibition). Notably, 3-FL inhibitedthe adhesion of E coli by up to 30% and of S fyris by up to 16%,whereas 2′-FL had no effect on EPEC adhesion. In contrast, wefound that2′-FL inhibitedEPECadhesionby18%, buthada lesserimpact on S fyris adhesion (inhibition by 12%). The observeddifferences could reflect thehigherdoses of trisaccharides inourassays and the different incubation and infection-times. D-(+)-mannose is known to inhibitmannose-specific adhesins of typeI fimbriated E coli and S fyris and accordingly we observed theinhibitionof bothpathogens (14%and69%, respectively). It isnotsurprising, that D-(+)-mannose as a specific binding partner forSalmonella inhibited pathogen adhesion more efficient com-pared to the neutral oligosaccharides 2′-FL and 3-FL. Also, thiseffectwas observed less prominent inE coli (EPEC), which exhibittype 1 fimbriae, but lack the high mannose binding affinity asseen in Salmonella. These results not only validate our assay, butalso demonstrate the importance ofmultiple bacterial adhesinsfor initial pathogen-host cell interaction and binding [30,31].

Carvioto et al. [32] showed that lacto-difucotetraose(Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]Glc) and other fucosylated tetra-saccharides and pentasaccharides from human breast milk

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strongly inhibited the adhesion of different EPEC strains toHEp-2 cells (up to 92% inhibition, at 3 mg/mL), whereassmaller disaccharides and trisaccharides such as lactose, 2′-FL and 3-FL displayed no inhibitory activity at 10 mg/mL andlarger or non-fucosylated oligosaccharides displayed limitedinhibitory activity. We demonstrated that 3-FL was able toinhibit EPEC adhesion by 29%, which may reflect the uniqueproperties of the Caco-2 cell line or the EPEC strain we used, orthe counting method (CFUs rather than fixing and stainingcells). Nevertheless, the strong inhibitory activity of fucosy-lated tetrasaccharides and pentaoligosaccharides indicatesthat fucosylated HMOs have the potential to inhibit pathogenattachment and adhesion even at lower concentrations thanin our assays. This shows a possible limitation of our study,where we used trisaccharides only, whichmight not reach theanti-adhesive properties of more complex HMOs. Difficultiesin manufacturing complex HMOs means that so far simpleoligosaccharides based on galactose or fructose have beeneconomically attractive and therefore have been furtherinvestigated for their anti-adhesive and mainly for theirpotential prebiotic properties. Commercially available fruc-tose-based oligosaccharides have demonstrated only margin-al inhibition (<5%). However galacto-oligosaccharides (GOS)were shown to inhibit the adhesion of EPEC strain E2348/69 toHEp-2 and Caco-2 cells by 65% and 70%, respectively [33].Inhibition could only be achieved with relatively high doses ofGOS (16 mg/mL) compared to fractionized oligosaccharidesused in other studies and the trisaccharides used in our study[13]. Therefore, the inhibitory activity of GOS may reflectdosing rather than the specificity of pathogen binding tofucosylated and/or sialylated residues. This dose-dependenteffect may also apply to the trisaccharides we used, because adose of 10 mg/mL is within the expected average range for allcombined oligosaccharides naturally found in human breastmilk [5] but exceeds the amount of each single oligosaccha-ride. Nevertheless, fucosylated oligosaccharides represent thelargest fraction in the HMO pool [6,34].

The diarrhea-causing bacterial pathogen C jejuni binds tospecific α(1,2)-fucosylated host cell epitopes (H2 antigen). Thisspecific inhibition of C jejuni adhesion and colonization hasbeen already demonstrated in vitro, ex vivo and in a mousemodel by applying analogs of these carbohydrate receptors[14]. To validate our adhesion assays, we investigated theability of ournovel synthesized2′-FL to inhibitC jejuniadhesionin vitro. We found that adhesion to differentiated Caco-2 cellswas inhibited 26% by 2′-FL and 23% by human breast milk. Inadhesion assays with alternative C jejuni strains, fucosylatedfractions of HMOs applied at 3 mg/mL inhibited C jejuniadhesion to HEp-2 cells by up to 50% [14]. The differencesobserved when compared to our results could reflect differentC jejuni strains used and in vitro systems used, but are alsolikely to reflect the complex nature of α(1,2)-fucosylatedoligosaccharides.. Interestingly, the above investigation dem-onstrated that 2′-FL could inhibit the binding of pathogenic Cjejuni strains to the H2 antigen at concentrations as low as10 μg/mL. In a mouse model, 3 mg/mL 2′-FL inhibited C jejuniadhesion and colonization by 69% but strikinglymore complexfucosylated oligosaccharide fractions inhibited the bacteria by93%. C jejuni binding to 2′-FL has also been characterized bysurface plasmon resonance spectroscopy, and preincubation

with unlabeled 2′-FL led to a 60% reduction in signal response,probably reflecting theoccupationofC jejuni binding sites by 2′-FL [35]. Our results confirm the inhibitory activity of thismolecule and the lower levels of inhibition may reflectdifferences in the experimental setup including the C jejunistrains. Interestingly, we found that 2′-FL showed similarlevels of inhibitory activity as donated human breast milkcontaining immunoglobulin fractions as well as the wholeoligosaccharide spectrum, including α(1,2)-fucosylated oligo-saccharides. The extent to which these data can be extrapo-lated into in vivo models remains to be seen, and furtherresearch isneeded todefine theoptimalmixtureand/ ordosingof synthesized HMOs for the inhibition of specific pathogens.

P aeruginosa is an opportunistic pathogen that predominant-ly causes respiratory diseases in susceptible individuals, but itcan also colonize the gastrointestinal tract and cause dissem-inated disease in immunocompromized individuals [36,37]. A Paeruginosa fucose-binding lectin (PA-IIL) can be blocked specif-ically by the HMOs Lea (lacto-N-fucopentaose II) and 3-FL [13].Therefore, we investigated the ability of our synthesizedoligosaccharides to inhibit P aeruginosa adhesion using bothintestinal and respiratory epithelial cell lines. We showed forthe first time that the adhesion of P aeruginosa to differentiatedCaco-2 cells is inhibited by 2′-FL (17%) and 3-FL (26%), as well asby 1:10 human breast milk (52%) and its IgA-depleted variant(34%). It has previously been shown that N-acetyl-D-galactos-amine can inhibit the adhesion of P aeruginosa to Caco-2 cellsand attenuate the loss of transepithelial electrical resistance bybinding to PA-IL, but PA-IIL ligands such as fucose do not havethe same effect [38]. Differences observed in our study mayagain reflect the use of different bacterial strains or Caco-2 cells(C2 cells, a clone which already exhibits characteristics ofdifferentiated cells) [39]. It seems plausible that the simplemonosaccharide fucose could lack the ability of more complextrisaccharides to interfere with bacterial binding sites. Our datasuggest that the 26% inhibition achieved using 3-FL (comparedto >30% inhibition by IgA-depleted breast milk) could also beincreased byusingmore complex and specific oligosachcarides.Also, other anti-adhesive factors like glycoproteins mightcontribute to the observed differences. Interestingly, P aerugi-nosa adhesion to Caco-2 cells can be inhibited by approximately50% by pre-incubating the cells with the probiotic bacteriumBifidobacterium longum [40]. Prebiotic HMOs could thereforerestore epithelial barrier functions and prevent pathogenattachment directly, but also indirectly by promoting thegrowth of specific probiotic bacteria. Further studies are neededto address the potential prebiotic, growth-promoting propertiesof our synthesized HMOs.

We found that the adhesion of P aeruginosa to A549respiratory epithelial cells was inhibited 24% by 2′-FL, 23%by 3-FL, and 21% by 1:10 breast milk. In contrast, Thomas andBrooks [15] found that the same HMOs achieved only 9% and0.6% inhibition, respectively, whereas the sialylated oligosac-charides 3-sialyllactose and 6-sialyllactose achieved up to 61%inhibition of P aeruginosa adhesion. Of note, fucosylatedoligosaccharides were applied in a concentration of 0.07 mg/mL and sialylated oligosaccharides in an even lower concen-tration (0.02 mg/mL). Interestingly, 2′-FL and 3-FL were able toinhibit the adhesion of Burkholderia cenocepacia to A549 cells by57% and 53%, respectively. These differencesmay reflect the use

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ofdiversePaeruginosa strains ineachstudy, andalso themethodsused (eg, pre-incubation of bacteria and oligosaccharides in a 1:1ratio versus pre-incubation of cells and oligosaccharides).Furthermore, the absolute adhesion of P aeruginosa to A549 cellswas 1.3% according to Thomas and Brooks [15] but 21% in ourinvestigation, whichmight be explained by the use of different Paeruginosa strains, inoculum sizes and assaymethods. Data froma more recent study using both an in vitro cell culture model(A549 cells) and an in vivomousemodel showed that the lectinsPA-IL and PA-IIL contribute to the pathogenicity of Pseudomonas-mediated lung injury [41].Mutant strainsmissing the lectingeneswere 50% less adherentwhen comparedwith thewild type strainon A549 cells, and specific lectin inhibitors such as N-acetyl-D-galactosamine, α-methyl-D-galactoside for PA-IL, and α-methyl-L-fucoside for PA-IIL, also reduced lung injury, disseminateddisease, and mortality (PA-IL inhibitors only). These resultsconfirm that lectins are crucial for pathogen-cell interactionand adhesion, and fucosylated oligosaccharides (α-methyl-L-fucoside) could prevent infections by interacting with specificlectins (PA-IIL). Further studies would be required to investigatethe potential anti-cytotoxic effects and lectin-specific bindingcharacteristics of novel synthesized oligosaccharides. The dem-onstration of such a specific interaction between the fucosylatedHMO lacto-N-fucopentaose II (LNFP) II and P aeruginosa lectin PA-IIL [16] led to an in vivo investigation that revealed an inversecorrelation between the amount of LNFP II in breast milk andinfant stools, and the occurrence of respiratory problemsat 6 and12 weeks postpartum [42]. This, together with evidence showingthat HMOs inhibit the adhesion of various respiratory pathogenssuch as Streptococcus pneumoniae, Haemophilus influenza andinfluenza virus [43–45] suggests that the HMOs used in thisinvestigation should be further analyzed to determine theirimpact on the adhesion of other respiratory pathogens.

We have demonstrated that engineered HMOs produced bywhole-cell biocatalysis are biologically active and can inhibitthe adhesion of different pathogens in two human cell culturemodels (A549 and Caco-2 cells). Our data are consistent withprevious studies using purified HMOs from donated humanbreast milk. We see limitations of our study, mainly due to therelative simple structure of the novel trisaccharides, whencompared to more complex natural occurring HMOs. Never-theless, our results suggest that mass-tailored HMOs could beused in the future to supplement nutritional ingredients or aspreventatives to reduce the impact of infectious diseases.However, it is important to replicate these in vitro data usinganimal challenge models and ultimately human clinicalstudies to determine the protective efficacy of mass-tailoredHMOs through the inhibition of pathogen adhesion, prebioticeffects and immunomodulation. It is tempting to speculatethat synthesized HMOs, which are structurally similar tonaturally-occurring HMOs, could be developedwith additionalfunctions beyond their anti-adhesive and prebiotic effects.

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.nutres.2013.07.009.

Acknowledgments

We are grateful for the support of Clemens Kunz (Institute ofNutritional Sciences, University of Giessen, Germany), who

analyzed the breast milk samples. We further thank CarolinStump and Natascha Quednau for their technical assistance.Bacterial strains of S fyris and EPEC O119 were kindly providedby “The Italian Study Group on Gastrointestinal Infections”(Giovanni Coppa, Università Politecnica delle Marche, Ancona,Italy) and C jejuniwas kindly provided byMarkusM. Heimesaat(Department of Microbiology and Hygiene, Charité-UniversityMedicine Berlin, Berlin, Germany). This project was partlysupported by the German Federal Ministry of Economics andTechnology (“ZIM Kooperations-Projekt KF2876401”). The au-thors have no conflict of interest to declare.

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