Inhibition of Type 1 Fimbriae-mediated Escherichia Coli Adhesion and Biofilm Formation by Trimeric Cluster Thiomannosides Conjugated to Diamond Nanoparticles

HAL Id: pasteur-01378734 https://hal-pasteur.archives-ouvertes.fr/pasteur-01378734 Submitted on 17 Nov 2016 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Distributed under a Creative Commons Attribution - NonCommercial - ShareAlike| 4.0 International License Inhibition of type 1 fimbriae-mediated Escherichia coli adhesion and biofilm formation by trimeric cluster thiomannosides conjugated to diamond nanoparticles. Manakamana Khanal, Fanny Larsonneur, Victoriia Raks, Alexandre Barras, Jean-Sebastien Baumann, Fernando Ariel Martin, Rabah Boukherroub, Jean-Marc Ghigo, Carmen Ortiz Mellet, Vladimir Zaitsev, et al. To cite this version: Manakamana Khanal, Fanny Larsonneur, Victoriia Raks, Alexandre Barras, Jean-Sebastien Baumann, et al.. Inhibition of type 1 fimbriae-mediated Escherichia coli adhesion and biofilm formation by trimeric cluster thiomannosides conjugated to diamond nanoparticles.. Nanoscale, Royal Society of Chemistry, 2015, 7 (6), pp.2325–2335. �10.1039/c4nr05906a�. �pasteur-01378734� ! 1! TOC image Inhibition(of biofilm formation Inhibition(of( bacterial(binding(to(cells ! 2! Inhibition of type 1 fimbriae-mediated Escherichia coli adhesion and biofilm formation by trimeric cluster thiomannosides conjugated to diamond nanoparticles Manakamana Khanal,a, # Fanny Larsonneur,b,c,# Victoriia Raks,d,e Alexandre Barras,a Jean- Sébastien Baumann,f Fernando Ariel Martin,b Rabah Boukherroub,a Jean-Marc Ghigo,b Carmen Ortiz Mellet,e Vladimir Zaitsev,e,g Jose M. Garcia Fernandez,h Christophe Beloin,b,* Aloysius Siriwardena,f* Sabine Szunerits,a,* a Institut de Recherche Interdisciplinaire (IRI, USR CNRS 3078), Université Lille 1, Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d'Ascq, France. b Institut Pasteur, Unité de Génétique des Biofilms, 28 rue du Dr. Roux, 75724 Paris cedex 15, France. c Université Paris Diderot, Ecole Doctorale Bio Sorbonne Paris Cité (BioSPC), Cellule Pasteur, rue du Dr. Roux, 75724 Paris cedex 15, France. d Taras Shevchenko University, 60 Vladimirskaya str., Kiev, Ukraine e Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, c/ Profesor García González, 41012 Sevilla, Spain. f Laboratoire de Glycochimie des Antimicrobiens et des Agroressources (FRE 3517 CNRS), Université de Picardie Jules Vernes, 33 Rue St Leu, 80039 Amiens, France. g Chemistry Department, Pontifical Catholic University of Rio de Janeiro, Rua Marques de Sao Vicente, 225-Gavea, Rio de Janeiro, 22451-900, Brazil h Investigaciones Químicas (IIQ), CSIC – Universidad de Sevilla, Avda. Américo Vespucio 49, 41092 Sevilla, Spain Keywords: diamond nanoparticles, glycoclusters, anti-adhesives, E. coli, FimH, biofilm !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! # these authors contributed equally to the work * to whom correspondence should be sent: [email protected]; [email protected]; [email protected]; ! ! 3! Abstract: Recent advances in nanotechnology have seen the development of a number of microbiocidal and/or anti-adhesive nanoparticles displaying activity against biofilms. In this work, trimeric thiomannoside clusters conjugated to nanodiamond particles (ND) were targeted for investigation. NDs have attracted attention as a biocompatible nanomaterial and we were curious to see whether the high mannose glycotope density obtained upon grouping monosaccharide units in triads might lead to the corresponding ND-conjugates behaving as effective inhibitors of E. coli type 1 fimbriae-mediated adhesion as well as of biofilm formation. The required trimeric thiosugar clusters were obtained through a convenient thiolene “click” strategy and were subsequently conjugated to alkynyl-functionalized NDs using a Cu(I)-catalysed “click” reaction. We demonstrated that the tri-thiomannoside clusterconjugated NDs (ND-Man3) show potent inhibition of type 1 fimbriae-mediated E. coli adhesion to yeast and T24 bladder cells as well as of biofilm formation. The biofilm disrupting effects demonstrated here have only rarely been reported in the past for analogues featuring such simple glycosidic motifs. Moreover, the finding that the tri-thiomannoside cluster (Man3N3) is itself a relatively efficient inhibitor, even when not conjugated to any ND edifice, suggests that alternative mono- or multivalent sugar-derived analogues might also be usefully explored for E. coli-mediated biofilm disrupting properties. ! 4! 1. INTRODUCTION Bacterial infectious diseases pose a major threat to human health. Several share clinical characteristics such as chronic inflammation and tissue damage, and are greatly exacerbated when microorganisms grow as biofilms on mucosal surfaces or medical devices.1, 2 Biofilms enable the bacteria residing within them to counter and resist the action of the human immune system and to enhance their tolerance towards antibiotics, leading to infections that are very difficult to eradicate.3-5 The threat of biofilm-related infections has been greatly aggravated with the emergence of multidrug resistant bacteria, a phenomenon that has been compounded in the past decades with the overuse and misuse of antibiotics. These and other considerations have generated an increased interest in the development of non-biocidal anti-infective strategies as alternatives to antibiotics, as these would be expected to show reduced tendency to provoke the appearance of resistant strains.6-13 One such approach is the development of a number of microbiocidal and/or anti-adhesive nanoparticles displaying activity against biofilms.14-19 Among the targets that have been identified for anti-adhesive nanoparticles are type 1 fimbriae, which constitute major virulence factors produced by Escherichia coli (E. coli).20 Type 1 fimbriae are filamentous tubular structures each of 0.2-2.0 µm in length and 5- 7 nm in diameter that are distributed over the entire surface of the bacterium.21 In various E. coli strains the lectin located at the extremity of type 1 fimbriae, FimH, contributes to tissue colonization through its specific recognition of the terminal α-D-mannopyranosyl units present on cell-surface glycoproteins. FimH-mediated adhesion to such mannosyl moieties has been demonstrated to be crucial for the interaction of E. coli with uroplakins and consequently for bladder colonization.22 Disruption of this interaction has been proposed as a promising strategy for the development of an anti-adhesive therapy.23, 24 While a number of multivalent as well as monovalent sugar-based ligands have been reported to show promise as effective inhibitors of E. coli adhesion to eukaryotic cells,9, 20, 25-31 multivalent presentation of carbohydrate ligands on appropriate scaffolds has been demonstrated, in several instances, to lead to significantly increased affinities for their appropriate lectin target compared to a monovalent references.32-37 These avidities can be dramatically superior to those arising from a simple additive effect. The types of multivalent structures targeting FimH thus far reported are very varied and range from small- to mediumsized scaffolds presenting carbohydrate-derived ligands, to larger entities such as sugardecorated polymers and nanoparticles, and a multitude of creatively designed compounds in between.20, 38, 39 ! 5! We and others have been interested in exploring whether the reported characteristic properties of nanodiamonds (NDs) might be taken advantage of in the development of useful inhibitors of type 1 fimbriae-mediated E. coli adhesion.40-42 ND particles are completely inert, optically transparent, biocompatible and moreover, easily functionalizable via a variety of strategies depending on their intended application.43-50 Although their in vivo toxicity depends in particular on their surface characteristics (as well as the nature of the ligands they carry on their surface),51 ND particles have thus far been reported not to induce significant cytotoxicity in a variety of cell types.51-54 The demonstration that our 1st-generation sugarconjugated NDs do show marked anti-adhesive activity in cell-based assays without displaying toxicity against eukaryotic cells conforted us in our choice of particle and convinced us that sugar-NDs should indeed be further pursued as biomaterials. Particularly striking was the unexpected observation that these ND-mannose conjugates are able to inhibit E. coli-induced biofilm formation. Such a feature has indeed only been observed rarely for ligands of FimH but would be expected to constitute a very desirable additional attribute in any potential anti-adhesive molecule.9, 40, 55 Moreover, anti-biofilm disrupting activity had not apparently been described previously for alternative glyco-nanoparticles (glyco-NPs) such as glycofullerenes, gold-based glyco-NPs or for other multivalent mannose-derived molecules.33, 36, 41 The coupling strategy used for the fabrication of our 1st-generation glyco-NDs was selected with the expectation that it would ensure not only a convenient means of conjugating carbohydrate moieties to the ND core, but also provide a linker that would itself constitute an extended ligand for FimH. In that approach propargyl sugar derivatives were ligated using a Cu(I)-catalysed Huisgen cycloaddition reaction (“click” reaction) to NDs decorated with surface azidophenyl functions. To further scrutinize the origin of the bacterial adhesion and biofilm growth inhibition activities observed for our 1st-generation glyco-NDs, we embarked on the investigation of a second, structurally complimentary, family of sugar-conjugated NDs. It was decided that the 2nd-generation ND-sugar conjugates were to be obtained through an alternative sugar-conjugation strategy and, in addition, would feature a trimeric thiomannoside cluster motif as a contrasting mode of surface-sugar presentation. Indeed various O-glycoside-derived trimeric clusters have been shown to be strong ligands for FimH compared with their corresponding monovalent analogues.20, 28 Moreover, it has been demonstrated that trimeric mannoside and thiomannoside clusters, related to those proposed, often give relatively large multivalent effects towards mannose-specific lectins.56-60 The sugar-linker of our 2nd-generation ND-sugar conjugates is quite different from the one ! 6! featured in the 1st-generation NDs, (synthesized through the “clicking” of propargyl glycosides to ND-grafted azido functions) and would thus very probably make different secondary interactions with the sugar-binding pocket in FimH. Furthermore, the trimeric thiosugar cluster backbone would be expected to be relatively flexible and, in addition, its peripheral thiomannosyl moieties held much further away from the ND surface than the mannosyl units featured in the 1st-generation NDs. Taken together, we suspected that all these factors would serve to render the sugar moieties present in the targeted 2nd-generation ND- conjugates more accessible to FimH receptors on the bacterial surface than those featured in our 1st-generation conjugates and thus give contrasting behavior in the projected biological assays. An additional feature of the second family of glyco-NDs proposed in this work is the installation of thioglycoside linkages which would render the anomeric tethering function much more robust to acidic or enzymatic hydrolysis than the O-glycosidic functions featured in our initial ND-sugar conjugates. This strategy has been a design feature of a multitude of thioglycoside-based ligands61 and we were surprised to discover that such a functional group motif had rarely been integrated into potential inhibitors of FimH and FimH-mediated E. coli adhesion events. Yet another difference between the 1st- and targeted 2nd-generation sugar- NDs is that the concentration of surface triazole functions relative to that of conjugated mannosyl moieties in the later family would be much lower than in the original ND sugarconjugates. We were curious to ascertain if inhibition of type 1 fimbriae-mediated adhesion might in some way be connected to: (i) the presence and accessibility of surface triazole functions; (ii) to the presence of multiple surface-conjugated mannosyl units; (iii) to the inherent physico-chemical characteristics of the ND core itself, or even to some combination of the three. We show in this paper the successful integration of trimeric thiomannosyl clusters onto alkynyl-terminated NDs, to give the targeted 2nd-generation sugar-conjugated NDs (ND- Man3) (Figure 1). Thiolactoside trimer-ND conjugates (ND-Lac3) and ND-OH particles have also been prepared as negative controls. These compounds have all been tested as inhibitors of E. coli adhesion to yeast and also to T24 bladder cells. The thiomannosyl trimer-NDs (ND- Man3), but not the negative controls, have been found to be strong inhibitors of both E. coli adhesions in both assays. In addition, these ND-Man3 particles are shown also to inhibit E. coli-driven biofilm growth significantly. 2. EXPERIMENTAL ! 7! 2.1. Materials Reagents and solvents were purchased from commercial sources and used without further purification. Azo-bis(isobutyronitrile), dichloromethane, trifluoromethanesulfonic anhydride, pyridine, and N,N-dimethylformamide are indicated by the acronyms AIBN, DCM, Tf2O, Py, and DMF, respectively. Thin-layer chromatography (TLC) was carried out on aluminum sheets coated with Kieselgel 60 F254, with visualization by UV light and by charring with 10% H2SO4 or 0.2% ninhydrin. Column chromatography was carried out on silica gel 60 (230−400 mesh). 2.2. Synthesis of tri-thiomannoside (Man3N3) and tri-thiolactoside (Lac3N3) cluster ligands for ND conjugation The synthesis of the new trivalent clusters (Man3N3) and (Lac3N3) (Figure 2) was achieved following a four-step reaction sequence involving: (i) radical addition of the corresponding per-O-acetyl-1-thiosugar B or C to tri-O-allylpentaerythritol A, using either UV (250 nm) light in DCM (for B; room temperature, 1 h) or AIBN in dioxane (for C; 75 ºC, 3 h) as radical initiator; (ii) activation of the focal hydroxyl group in the resulting adducts by triflation with Tf2O-Py in DCM (-25 ºC, 40 min); (iii) nucleophlic displacement of triflate by azide ion by reaction of the crude triflic esters with NaN3 in DMF (room temperature, 3 h; 73 and 50% yield over three steps for the previously reported per-O-acetylated azide-armed trimannoside D62 and trilactoside E,63 respectively); and (iv) final catalytic deacetylation with sodium methylate in dry methanol as detailed below. The precursor triallylated pentaerythritol derivative A (Figure 2) required for the synthesis of Man3N3 and Lac3N3 was prepared following the reported procedure.64 2,3,4,6-tetra-O-acetyl-1-thio-α-D- mannopyranose (B) and 2,3,6,2′,3′,4′,6′-hepta-O-acetyl-1-thio-β-lactose (C) were prepared from the corresponding sugar per-O-acetates in three steps: transformation into the corresponding glycosyl halides, treatment with thiourea, and subsequent hydrolysis of the resulting isothiouronium salt with potassium metabisulfite (K2S2O5) (Figure 2).65, 66 Full spectral data are reported in supporting material (Figures S2-S5). 2,2,2-Tris[5-(α-D-mannopyranosylthio)-2-oxapentyl]ethyl azide (Man3N3). To a solution of 2,2,2-tris[5-(2,3,4,6-O-tetra-acetyl-α-D-mannopyranosylthio)-2-oxapentyl]ethyl azide (D) (294 mg, 0.214 mmol) in dry MeOH (20 mL) was added methanolic MeONa (1 M, 0.1 equiv per mol of acetate). The reaction mixture was stirred at room temperature for 30 min, then ! 8! neutralized with Amberlite IRA-120 (H+) ion-exchange resin, concentrated, and the resulting residue was freeze-dried to afford Man3N3 (189 mg, quant.) as a white solid. [α]D +154.3 (c 0.56, H2O). Rf 0.19 (10:20:1 CH3CN-H2O-NH4OH). 1H NMR (500 MHz, CD3OD) δ 5.23 (bs, 3 H, H-1Man), 3.92 (bs, 3 H, H-2Man), 3.89 (ddd, 3 H, J4,5 = 11.9 Hz, J5,6b = 5.6 Hz, J5,6a = 2.4 Hz, H-5Man), 3.81 (dd, 3 H, J6a,6b = 11.9 Hz, H-6aMan), 3.(dd, 3 H, H-6bMan), 3.66 (m, 6 H, H-3Man, H-4Man), 3.51 (t, 6 H, 3JH,H = 6.0 Hz, H-3Pent), 3.33 (t, 2 H, 3JH,H = 7.0 Hz, CH2N3), 3.33 (m, 6 H, H-1Pent), 2.72 (m, 6 H, H-5Pent), 1.88 (m, 6 H, 3JH,H = 6.6 Hz, H-4Pent). 13C NMR (125.7 MHz, CD3OD) δ 86.5 (C-1Man), 74.9 (C-5Man), 73.7 (C-2Man), 73.2 (C-3Man), 70.8 (C- 3Pent), 70.6 (C-1Pent), 68.9 (C-4Man), 62.7 (C-6Man), 53.1 (CH2N3), 44.7 (Cq) 30.8 (C-4Pent), 28.8 (C-5Pent). ESIMS: m/z 892.4 [M + Na]+. Anal. Calcd for C32H59N3O18S3: C, 44.18; H, 6.84; N, 4.83; S, 11.06. Found: C, 43.6; H, 6.66; N, 4.51; S, 10.79. 2,2,2-Tris[5-(β-lactopyranosylthio)-2-oxapentyl]ethyl azide (Lac3N3). To a solution of 2,2,2-tris[5-(2,3,6,2’,3’,4’,6’-hepta-O-acetyl-β-lactopyranosylthio)-2-oxapentyl]ethyl azide (E) (294 mg, 0.131 mmol) in dry MeOH (20 mL) was added methanolic MeONa (1 M, 0.1 equiv per mol of acetate). The reaction mixture was stirred at 40º C for 45 min, then neutralized with Amberlite IRA-120 (H+) ion-exchange resin, concentrated, and the resulting residue was freeze-dried to afford Lac3N3. (180 mg, quant.) as a white solid. [α]D -7.4 (c 0.60, H2O). Rf 0.17 (6:3:1 CH3CN-H2O-NH4OH). 1H NMR (500 MHz, D2O): δ 4.42 (m, 6 H, H-1Lact, H-1’Lact), 4.00-3.30 (m, 54 H, H-2Lact to H-6a,bLact, H-2’Lact to H-6’a,bLact, H-1Pent, H- 3Pent and CH2N3), 2.81 (2 dt, 6 H, J4’,5’ = 7.0 Hz, J5a’,5b’ = 14.0 Hz, H-5Pent), 1.91 (m, 6 H, H- 4Pent); 13C NMR (125.7 MHz, CD3OD) δ 105.1 (C-1′Lact), 87.2 (C-1Lact), 80.7 (C-5Lact), 80.5 (C-4Lact), 77.9 (C-3Lact), 77.1 (C-5′Lact), 74.9 (C-3′Lact), 74.1 (C-2Lact), 72.6 (C- 2′Lact), 70.9 (C-3Pent), 70.6 (C-1Pent), 70.4 (C-4′Lact), 62.5 (C-6′Lact), 62.3 (C-6Lact), 53.2 (CH2N3), 46.8 (Cq), 31.3 (C-4Pent), 28.0 (C-5Pent). ESIMS: m/z 1378.4 [M + Na]+. Anal. Calcd for C50H89N3O33S3: C, 44.27; H, 6.61; N, 3.10; S, 7.09. Found: C, 44.12; H, 6.56; N, 2.87; S, 6.73. 2.3. Tri-thiomannosyl and tri-thiolactosyl cluster conjugation to NDs (respectively, ND- Man3 and ND-Lac3) 4-Pentynoic acid (0.20 mmol), DCC (0.22 mmol) and DMAP (0.066 mmol) were dissolved in 5 mL anhydrous DMF. A suspension of ND-OH particles in anhydrous DMF (10 mg in 5 mL) was added and the mixture stirred at room temperature for 24 h under nitrogen. The ! 15! abiotic surfaces can be inhibited by α-mannopyranosyl containing O-glycosides and O- glycans, implicating the integral role of the FimH lectin in this process.69 The biofilm disrupting ability of the various sugar ligands and conjugated-nanostructures fabricated in this work was evaluated, as previously described, using an assay that measures their ability to inhibit E. coli MG1655_ λATT::amp_GFP_kmPcL_fimAICDFGH biofilm formation on polyvinyl chloride (PVC) surfaces (Figure 4).40 Whereas neither the ND-OH nor ND-Lac3 controls proved active (data not shown), both the unconjugated tri-thiomannoside cluster Man3N3 and ND-Man3 displayed a strong disrupting effect on biofilm formation as compared to α-mmp. The biofilm inhibitory potency of the ND-Man3 described herein is significantly greater than that observed for our 1st generation sugar-NDs (ca. 10 fold).40 However, the relatively small increase in the biofilm inhibition potency of ND-Man3N3 relative to that of the Man3N3 (a factor of 2) is in sharp contrast to the large increases in adhesion inhibition observed upon conjugation of Man3N3 to NDs in the corresponding yeast agglutination and T24 bladder cells binding assays and perhaps deserves comment. Adhesion of bacteria to bladder cells and yeast agglutination are exclusively dependent on type 1 fimbriae, whereas biofilm formation of E. coli cells is known to be mediated not only by type 1 fimbriae but also through the interplay of number of additional cell surface appendages. Additionally biofilms are constituted of a complex matrix of high molecular weight constituents including polysaccharides and this would be expected to impede diffusion of large molecules such as NDs conjugates relative to that of smaller entities. CONCLUSIONS In this work we demonstrate that sugar-conjugated nanodiamonds have marked detrimental effects on E. coli-mediated biofilm formation and that this phenomenon is related to their ability to interfere with FimH-mediated bacterial adhesion. The conjugation strategy developed for these 2nd-generation sugar conjugated NDs, using alkynyl-functionalized NDs, proves as efficient as the one described previously which was based on azido-functionalized NDs.40 Having in hand this pair of complementary strategies for surface modification of ND particles, makes possible the application of the Huisgen Cu(I) “click” methodology to a wide range of propargyl- or azido-armed ligand counterparts thus greatly broadening its scope. The demonstration that the tri-thiomannoside cluster-NDs (ND-Man3) fabricated here are able to effectively impede type 1 fimbriae-mediated bacterial adhesion in two independent assay ! 16! formats is consistent with our earlier findings that mannose-conjugated NDs have a marked E.coli anti-adhesive activity. The ability of the new glycocluster-NDs to significantly inhibit E. coli-mediated biofilm formation is remarkable. The fact that both the 1st-(glycoside) and 2nd-(thioglycoside) generations of glyco-NDs both manifest this property is also notable.40 Moreover, the finding that the unconjugated trimeric thiomannoside cluster Man3N3 shows a non-negligible activity as a biofilm inhibitor, despite its low relative molecular weight was unexpected. Indeed, rarely have sugar-based inhibitors of E. coli-generated biofilms been reported although a number have for biofilms mediated by Pseudomonas aeruginosa.70, 71 The fact that Man3N3 does not feature any triazole segment in the vicinity of the sugar moiety strongly suggests that the presence of the heterocycle as an integral feature of the 1st-generation NDs is not critical for their ability to inhibit biofilm formation. In addition, neither the ND-OH nor ND- Lac3 controls are seen to show any anti-adhesive activity, underlining that the activities observed for the thiomannosyl conjugates are sugar-specific. Taken together, the data supports that it is the presence of mannosyl residues in the thiosugar clusters that constitute the primary ingredient driving the biofilm-inhibitory activity observed for the ND- conjugates: neither the presence of triazole functions or the interplay of some intrinsic physico-chemical property of the nanodiamond core itself have an obvious influence on this process. Although it would be premature to advance a detailed explanation for this observation at this point, such biofilm inhibition effects would constitute a useful additional feature of any antiadhesive lead and has rarely been reported in the past for the alternative mono- or multivalentmannose derivatives. We suspect that the activities brought to light in this work might not be exclusive to nanodiamond-based sugar conjugates. Moreover, the finding that the tri-thiomannosyl cluster Man3N3 itself is a relatively efficient inhibitor, even when not conjugated to any ND scaffold, suggests that alternative mono- and medium- to low-valency mannosyl conjugates might also demonstrate significant E. coli-mediated biofilm disrupting properties, a hypothesis that deserves to be further investigated ACKNOWLEDGEMENTS A.B, M.K., R.B and S.S. gratefully acknowledge financial support from the Centre National de Recherche Scientifique (CNRS), the Université Lille 1, the Nord Pas de Calais region and the Institut Universitaire de France (IUF). A.S. gratefully acknowledges financial support ! 17! from the CNRS and the IFCPAR (Indo-French Centre for the Promotion of Advanced Research) (Project 3905-1) and the Region Picardie for a doctoral fellowship to J.-S. Baumann. F. L. is supported by a MENESR (Ministère Français de l’Éducation Nationale, de l’Enseignement Supérieur et de la Recherche) fellowship. C.B. and J-M.G. are supported by the Institut Pasteur, the French Government's Investissement d'Avenir program Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (grant n°ANR-10-LABX-62-IBEID) and from Fondation pour la Recherche Médicale grant "Equipe FRM DEQ20140329508". C.O.M. and J.M.G.F. are greatful to the Spanish Ministerio de Economía y Competitividad (contract numbers SAF2013-44021-R and CTQ2010-15848), the Junta de Andalucía (contract number FQM2012 1467) and the European Regional Development Funds (FEDER and FSE) for financial support. We also acknowledge support from the European Union through the FP7-PEOPLE-2010-IRSES action “Photorelease” (grant number 269099) and the COST action CM1102 “MultiGlycoNano”. ! 18! REFERENCES 1 N. Hoiby, O. Ciofu, H.K. Johansen, Z.J. Song, C. Moser, P.O. Jensen, S. Molin, M. Givskov, T. Tolker-Nielsen, T. Bjarnsholt, Int J Oral Sci, 2011, 3 55-65. 2 D. Lebeaux, A. Chauhan, O. Rendueles, C. Beloin, Pathogens, 2013, 2 288-356. 3 G. O'Toole, H.B. Kaplan, R. Kolter, Annual review of microbiology, 2000, 54 49-79. 4 N. Hoiby, T. Bjarnsholt, M. Givskov, S. Molin, O. Ciofu, Int J Antimicrob Agents, 2010, 35 322-332. 5 D. Lebeaux, J.M. Ghigo, C. Beloin, Microbiol Mol Biol Rev, 2014, 78 510-543. 6 A. Chauhan, A. Bernardin, W. Mussard, I. Kriegel, M. Esteve, J.M. Ghigo, C. Beloin, V. Semetey, J Infect Dis, 2014, 210 1347-56. 7 O. Rendueles, J.B. Kaplan, J.M. Ghigo, Environ Microbiol, 2013, 15 334-346. 8 L.R. Rodrigues, Adv Exp Med Biol, 2011, 715 351-367. 9 C.K. Cusumano, J.S. Pinkner, Z. Han, S.E. Greene, B.A. Ford, J.R. Crowley, J.P. Henderson, J.W. Janetka, S.J. Hultgren, Sci Transl Med, 2011, 3 109ra115. 10 A.M. Krachler, K. Orth, Virulence, 2013, 4 284-294. 11 D. Romero, E. Sanabria-Valentin, H. Vlamakis, R. Kolter, Chem Biol, 2013, 20 102-110. 12 N. Sharon, Biochim Biophys Acta, 2006, 1760 527-537. 13 M. Totsika, M. Kostakioti, T.J. Hannan, M. Upton, S.A. Beatson, J.W. Janetka, S.J. Hultgren, M.A. Schembri, J Infect Dis, 2013, 208 921-928. 14 R.P. Allaker, K. Memarzadeh, Int J Antimicrob Agents, 2014, 43 95-104. 15 S. Chernousova, M. Epple, Angew Chem Int Ed Engl, 2013, 52 1636-1653. 16 M.R. Das, R.K. Sarma, S. Borah, R. Kumari, R. Saikia, A.B. Deshmukh, M.V. Shelke, P. Sengupta, S. Szunerits, R. Boukherroub, Colloids Surf B Biointerfaces, 2013, 105 128-136. 17 I. Francolini, G. Donelli, FEMS Immunol Med Microbiol, 2010, 59 227-238. 18 A. Herman, A.P. Herman, J Nanosci Nanotechnol, 2014, 14 946-957. 19 E. Taylor, T.J. Webster, Int J Nanomedicine, 2011, 6 1463-1473. 20 M. Hartmann, T.K. Lindhorst, Eur J Org Chem, 2011, 3583-3609. 21 P. Klemm, M. Schembri, Type 1 Fimbriae, Curli, and Antigen 43: Adhesion, Colonization, and Biofilm Formation, in: A. Böck, R. Curtiss III, J.B. Kaper, F.C. Neidhardt, T. Nyström, K.E. Rudd, C.L. Squires (Eds.) EcoSal- Escherichia coli and Salmonella: cellular and molecular biology, ASM Press, Washington, D.C., 2004. 22 X.R. Wu, T.T. Sun, J.J. Medina, Proc Natl Acad Sci U S A, 1996, 93 9630-9635. 23 N. Jayaraman, Chem Soc Rev, 2009, 38 3463-3483. 24 I. Ofek, D.L. Hasty, N. Sharon, FEMS Immunol Med Microbiol, 2003, 38 181-191. 25 S. Brument, A. Sivignon, T.I. Dumych, N. Moreau, G. Roos, Y. Guerardel, T. Chalopin, D. Deniaud, R.O. Bilyy, A. Darfeuille-Michaud, J. Bouckaert, S.G. Gouin, J Med Chem, 2013, 56 5395-5406. 26 Z. Han, J.S. Pinkner, B. Ford, E. Chorell, J.M. Crowley, C.K. Cusumano, S. Campbell, J.P. Henderson, S.J. Hultgren, J.W. Janetka, J Med Chem, 2012, 55 3945-3959. 27 X. Jiang, D. Abgottspon, S. Kleeb, S. Rabbani, M. Scharenberg, M. Wittwer, M. Haug, O. Schwardt, B. Ernst, J Med Chem, 2012, 55 4700-4713. 28 N. Nagahori, R.T. Lee, S. Nishimura, D. Page, R. Roy, Y.C. Lee, Chembiochem, 2002, 3 836-844. 29 A. Patel, T.K. Lindhorst, Carbohydr Res, 2006, 341 1657-1668. 30 R.J. Pieters, Org Biomol Chem, 2009, 7 2013-2025. 31 M. Touaibia, A. Wellens, T.C. Shiao, Q. Wang, S. Sirois, J. Bouckaert, R. Roy, ChemMedChem, 2007, 2 1190-1201. 32 L.L. Kiessling, J.E. Gestwicki, L.E. Strong, Curr Opin Chem Biol, 2000, 4 696-703. 33 C.C. Lin, Y.C. Yeh, C.Y. Yang, C.L. Chen, G.F. Chen, C.C. Chen, Y.C. Wu, J Am Chem Soc, 2002, 124 3508-3509. ! 19! 34 J.J. Lundquist, E.J. Toone, Chem Rev, 2002, 102 555-578. 35 M. Mammen, S.-K. Choi, G.M. Whitesides, Angew. Chem. Int. Ed., 1998, 37 2754-2899. 36 M. Durka, K. Buffet, J. Iehl, M. Holler, J.F. Nierengarten, J. Taganna, J. Bouckaert, S.P. Vincent, Chem Commun (Camb), 2011, 47 1321-1323. 37 Y.C. Lee, R.T. Lee, Acc. Chem. Res., 1995, 28 321-327. 38 A. Bernardi, J. Jimenez-Barbero, A. Casnati, C. De Castro, T. Darbre, F. Fieschi, J. Finne, H. Funken, K.E. Jaeger, M. Lahmann, T.K. Lindhorst, M. Marradi, P. Messner, A. Molinaro, P.V. Murphy, C. Nativi, S. Oscarson, S. Penades, F. Peri, R.J. Pieters, O. Renaudet, J.L. Reymond, B. Richichi, J. Rojo, F. Sansone, C. Schaffer, W.B. Turnbull, T. Velasco-Torrijos, S. Vidal, S. Vincent, T. Wennekes, H. Zuilhof, A. Imberty, Chem Soc Rev, 2013, 42 4709- 4727. 39 M.A. Mintzer, E.L. Dane, G.A. O'Toole, M.W. Grinstaff, Mol Pharm, 2012, 9 342-354. 40 A. Barras, F.A. Martin, O. Bande, J.S. Baumann, J.M. Ghigo, R. Boukherroub, C. Beloin, A. Siriwardena, S. Szunerits, Nanoscale, 2013, 5 2307-2316. 41 M. Hartmann, P. Betz, Y. Sun, S.N. Gorb, T.K. Lindhorst, A. Krueger, Chemistry, 2012, 18 6485-6492. 42 A. Siriwardena, A. Barras, F.A. Martin, O. Bande, J.S. Baumann, J.M. Ghigo, C. Beloin, R. Boukherroub, S. Szunerits, Glycoconj. J., 2011, 28 216. 43 A. Barras, J. Lyskawa, S. Szunerits, P. Woisel, R. Boukherroub, Langmuir, 2011, 27 12451-12457. 44 A. Barras, S. Szunerits, L. Marcon, N. Monfilliette-Dupont, R. Boukherroub, Langmuir, 2010, 26 13168-13172. 45 Y.R. Chang, H.Y. Lee, K. Chen, C.C. Chang, D.S. Tsai, C.C. Fu, T.S. Lim, Y.K. Tzeng, C.Y. Fang, C.C. Han, H.C. Chang, W. Fann, Nat Nanotechnol, 2008, 3 284-288. 46 S.A. Dahoumane, M.N. Nguyen, A. Thorel, J.P. Boudou, M.M. Chehimi, C. Mangeney, Langmuir, 2009, 25 9633-9638. 47 A. Krüger, Angew.Chem. Int. Ed., 2006, 45 6426-6427. 48 A. Krüger, Chemistry, 2008, 14 1382-1390. 49 Y. Liang, M. Ozawa, A. Krueger, ACS Nano, 2009, 3 2288-2296. 50 V.N. Mochalin, O. Shenderova, D. Ho, Y. Gogotsi, Nat Nanotechnol, 2012, 7 11-23. 51 L. Marcon, F. Riquet, D. Vicogne, S. Szunerits, J.-F. Bodart, R. Boukherroub, J. Mater. Chem., 2010, 20 8064-8069. 52 K.-K. Liu, C.-L. Cheng, C.C. Chang, J.-I. Chao, Nanotech., 2007, 18 325102. 53 A.M. Schrand, H. Huang, C. Carlson, J.J. Schlager, E. Omacr Sawa, S.M. Hussain, L. Dai, J Phys Chem B, 2007, 111 2-7. 54 S.J. Yu, M.W. Kang, H.C. Chang, K.M. Chen, Y.C. Yu, J Am Chem Soc, 2005, 127 17604-17605. 55 A. Wellens, C. Garofalo, H. Nguyen, N. Van Gerven, R. Slattegard, J.P. Hernalsteens, L. Wyns, S. Oscarson, H. De Greve, S. Hultgren, J. Bouckaert, PLoS One, 2008, 3 e2040. 56 J.M. Benito, M. Gomez-Garcia, C. Ortiz Mellet, I. Baussanne, J. Defaye, J.M. Garcia Fernandez, J Am Chem Soc, 2004, 126 10355-10363. 57 J.L. Jimenez Blanco, C. Ortiz Mellet, J.M. Garcia Fernandez, Chem Soc Rev, 2013, 42 4518-4531. 58 A. Martinez, C. Ortiz Mellet, J.M. Garcia Fernandez, Chem Soc Rev, 2013, 42 4746- 4773. 59 J. Rodriguez-Lavado, M. de la Mata, J.L. Jimenez-Blanco, M.I. Garcia-Moreno, J.M. Benito, A. Diaz-Quintana, J.A. Sanchez-Alcazar, K. Higaki, E. Nanba, K. Ohno, Y. Suzuki, C. Ortiz Mellet, J.M. Garcia Fernandez, Org Biomol Chem, 2014, 12 2289-2301. 60 K.H. Schlick, J.R. Morgan, J.J. Weiel, M.S. Kelsey, M.J. Cloninger, Bioorg Med Chem Lett, 2011, 21 5078-5083. ! 20! 61 M. Gingras, Y.M. Chabre, M. Roy, R. Roy, Chem Soc Rev, 2013, 42 4823-4841. 62 M. Gomez-Garcia, J.M. Benito, R. Gutierrez-Gallego, A. Maestre, C.O. Mellet, J.M. Fernandez, J.L. Blanco, Org Biomol Chem, 2010, 8 1849-1860. 63 M. Gomez-Garcia, J.M. Benito, A.P. Butera, C. Ortiz Mellet, J.M. Garcia Fernandez, J.L. Jimenez Blanco, J Org Chem, 2012, 77 1273-1288. 64 A. Lubineau, A. Malleron, C. Le Narvor, Tetrahedron Lett., 2000, 41 8887-8891. 65 D.A. Fulton, J.F. Stoddart, J Org Chem, 2001, 66 8309-8319. 66 K.L. Matta, R.N. Girotra, J.J. Barlow, Carbohydr Res, 1975, 43 101-109. 67 C.G. Korea, R. Badouraly, M.C. Prevost, J.M. Ghigo, C. Beloin, Environ Microbiol, 2010, 12 1957-1977. 68 A. Roux, C. Beloin, J.M. Ghigo, J Bacteriol, 2005, 187 1001-1013. 69 L.A. Pratt, R. Kolter, Mol Microbiol, 1998, 30 285-293. 70 J.L. Reymond, M. Bergmann, T. Darbre, Chem Soc Rev, 2013, 42 4814-4822. 71 E.L. Dane, A.E. Ballok, G.A. O'Toole, M.W. Grinstaff, Chem Sci, 2014, 5 doi: 10.1039/C3SC52777H. ! 21! Table 1: Selected physical properties of the sugar-conjugated NDs Diameter (nm) PIa Zeta potential (mV) Sugar loading (µg mg-1 ND) N 1s at. % S 2p at. % ND-OH 89 ± 13 0.246 ±0.002 35.3 ± 1.6 - 1.5 - ND-alkynyl 126 ± 3 0.168 ±0.021 34.2± 1.4 - 1.5 - ND-Man3 125 ± 9 0.345 ±0.003 27.2 ± 0.5 168 ± 12 5.3 3.9 ND-Lac3 138 ± 8 0.258 ±0.062 31.2 ± 0.4 135 ± 18 5.2 3.6 a Polydispersity Index; mean ± SD, n = 3 1! ! 22! Table 2: Inhibition of type 1 fimbriae-mediated yeast agglutination Compound IT (µg mL-1) a RIT (µM) b RIP50 (RIC50 α-mmp/ RIC50 of the compound) RIP50 (RIC50 (Man3N3)/RIC50 of the compound) α-mmp - 7000 1 Man3N3 63.4 218.8 32 ND-Man3 3.14 2.4 2970 91 ND-Lac3 >100 c - ND-OH >100 c - ND-mannosed 19.4 6.98 1003 a IT= inhibition titre, bRIT= relative inhibition titre= IT × 3.45 µmol mannose/mg for Man3N3 or 0.75 µmol mannose/mg for ND-Man3 or 0.49 µmol lactose/mg for ND-Lac3, RIP50= relative inhibition potency of either α-mmp or Man3N3/RIC50 of the corresponding ND- conjugate. All relative inhibition parameters are expressed as micromolar concentration of carbohydrate. c Values not determined. Sigmoïdal fitting of data not possible. d These parameters correspond to those reported for 1st-generation mannose-NDs.40 Table 3: Inhibition of type 1 fimbriae mediated adhesion to T24 bladder cells Compound IC50 (µg mL-1) RIC50 (µM) a RIP50 (RIC50 α-mmp/ RIC50 of the compound) RIP50 (RIC50 (Man3N3)/RIC50 of the compound) α-mmp - 22511 1 Man3N3 28.5 98.2 229 ND-Man3 0.98 0.738 30502 133 ND- Lac3 >100 b ND-OH >100 b ND-mannose c 7.6 2.7 9259 a RIC50= relative IC50 = IC50 x 3.45 µmol mannose/mg for Man3N3 or 0.75 µmol mannose/mg for ND-Man3 or 0.49 µmol lactose/mg for ND-Lac3, RIP50= relative inhibition potency of αmmp or Man3N3/RIC50 of the compound. All relative inhibition parameters are expressed as micromolar concentration of carbohydrate. b Values not determined. Sigmoïdal fitting of data not possible. c These parameters correspond to those reported for 1st-generation mannose-NDs.40 ! 23! Figure 1. Schematic illustration of the stepwise chemical functionalization of diamond nanoparticles (ND) to give the target ND-conjugated trimeric thiosugar clusters (2nd- generation ND). For comparison, the structure of the 1st-generation ND (ND-mannose)40 is presented. ! 24! Figure 2. Synthetic routes to tri-thiomannoside Man3N3 and tri-thiolactoside Lac3N3 clusters. O! R! O! R! O! R! O! R! O! S! O! O! R! O! R! O! R! O! R! O! S! O! R! O! R! O! R ! O! O! R! S! N! 3! O! O! O! R! O! O! R! O! R! S! O! O! R! O! R! O! O! R! S! O! O! R! R! O! O! R! S! O! O! O! R! O! O! R! O! R! O! O! R! O! R! O! R! O! R! O! O! R! O! O! R! O! R! O! R! O! O! R! O! N! 3! O! O! H! O! O! O! A! c! O! A! c! O! A! c! O! O! A! c! S! H! O! O! A! c! A! c! O! O! A! c! S! H! O! A! c! O! A! c! O! A! c! O! O! A! c! O! A ! B! C ! 1! )! U! V! (! λ! 2! 5! 4! )! ,! M! e! O! H! ,! r! t! ,! 1! h! 2! )! T! f! 2! O! ,! P! y! ,! D! C! M! ,! -! 2! 5! º! C! ,! 4! 0! m! i! n! 7! 3! %! o! v! e! r! a! l! l! 1! )! A! I! B! N! ,! d! i! o! x! a! n! e! ,! 7! 5! º! C! ,! 3! h! 3! )! D! M! F! ,! N! a! N! 3! ,! r! t! ,! 3! h! 5! 0! %! o! v! e! r! a! l! l! D! ,! R! =! A! c! Man3N3! ! R = OH ! ! N! a! O! M ! e! /! M! e! O! H! r! t! ,! 3! 0! m! i! n! ,! 1 ! 0! 0! %! E! ,! R ! =! A! c! ! ,! R ! =! O! H! N! a! O! M! e! /! M! e! O! H! r! t! ,! 3! 0! m! i! n! ,! 1! 0! 0! %! Lac3N3!