Force Nanoscopy as a Versatile Platform for ... - ACS Publications

Jan 26, 2016 - Here, we establish atomic force microscopy (AFM)-based nanoscopy as a platform for assessing the antiadhesion activity of multivalent m...
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Force Nanoscopy as a Versatile Platform for Quantifying the Activity of Antiadhesion Compounds Targeting Bacterial Pathogens Audrey Beaussart,†,⊥ Marta Abellán-Flos,‡ Sofiane El-Kirat-Chatel,†,∥,# Stéphane P. Vincent,*,‡ and Yves F. Dufrêne*,†,§ †

Université catholique de Louvain, Institute of Life Sciences, Croix du Sud, 4-5, bte L7.07.06., B-1348 Louvain-la-Neuve, Belgium University of Namur, Department of Chemistry, Rue de Bruxelles 61, 5000 Namur, Belgium § Walloon Excellence in Life sciences and Biotechnology (WELBIO) 1300 Wavre, Belgium ‡

S Supporting Information *

ABSTRACT: The development of bacterial strains that are resistant to multiple antibiotics has urged the need for new antibacterial therapies. An exciting approach to fight bacterial diseases is the use of antiadhesive agents capable to block the adhesion of the pathogens to host tissues, the first step of infection. We report the use of a novel atomic force microscopy (AFM) platform for quantifying the activity of antiadhesion compounds directly on living bacteria, thus without labeling or purification. Novel fullerene-based mannoconjugates bearing 10 carbohydrate ligands and a thiol bond were efficiently prepared. The thiol functionality could be exploited as a convenient handle to graft the multimeric species onto AFM tips. Using a combination of single-molecule and single-cell AFM assays, we demonstrate that, unlike mannosidic monomers, multivalent glycofullerenes strongly block the adhesion of uropathogenic Escherichia coli bacteria to their carbohydrate receptors. We expect that the nanoscopy technique developed here will help designing new antiadhesion drugs to treat microbial infections, including those caused by multidrug resistant organisms.

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globular multivalent presentation of mannose residues capable to interfere with adhesion.11 The development of novel ultrasensitive adhesion and antiadhesion assays would clearly contribute to increase our understanding of the antiadhesion activity of such glycoconjugates. Here, we establish atomic force microscopy (AFM)-based nanoscopy as a platform for assessing the antiadhesion activity of multivalent mannofullerenes directed against E. coli FimH. We show that fullerenes bearing multiple peripheral mannose residues show greatly enhanced adhesion and antiadhesion activity toward FimH, thus indicating that the multivalent presentation of sugars on nanoparticles is a valuable strategy for antiadhesion therapy. Unlike existing bioassays, the method enables us to quantify the antiadhesion efficiency of glycoconjugates directly on living bacteria, without the need for labeling or purification. Results and Discussion. Nanoscale Imaging of E. coli. We studied the interaction of glycoconjugates with E. coli FimH, taken as a prototype of mannose-binding adhesin. As FimH is expressed at the tip of type 1 pili, we initially imaged the surface morphology of cells from the UTI89 clinical isolate that were engineered for continuous type-1 fimbriation (hereafter, “FimH E. coli cells”). Bacteria were decorated with

he advent of multiresistant bacteria has urged the need for alternative drugs capable to fight bacterial infections. A promising approach is the use of antiadhesion compounds which target the adhesion of the pathogens to host tissues, the first stage of infection.1−5 Antiadhesive agents do not kill the pathogens, meaning the development of resistant strains is less likely than with antibiotics. The development of efficient antiadhesion therapies to treat bacterial infections requires the identification of new antiadhesive compounds and the availability of innovative techniques for the reliable assessment of antiadhesion activity. As many bacterial pathogens bind to their host cells via carbohydrate−protein interactions, the ability of sugars to protect against bacterial infections has already been recognized 35 years ago.2,6 The affinity of carbohydrates for bacterial lectins is low, meaning they must be used at high concentration. However, presentation of multiple copies of carbohydrates leads to high-affinity multivalent binding.7,8 FimH from uropathogenic Escherichia coli bacteria is a widely investigated prototype of sugar-specific bacterial adhesion protein. This adhesin, which is expressed at the tip of type 1 bacterial pili, binds to terminal mannose moieties on host epithelial cells. While soluble mannose is capable to inhibit bacterial adhesion by blocking FimH proteins, the multivalent presentation of carbohydrates on synthetic scaffolds, like dendrimers, is a key strategy for achieving high affinity interactions with FimH, thus to efficiently block cell adhesion.9,10 Recently, fullerenes were also used to achieve a © 2016 American Chemical Society

Received: November 17, 2015 Revised: January 5, 2016 Published: January 26, 2016 1299

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Figure 1. Studying the interaction between glycofullerenes and E. coli FimH. (a, b) AFM deflection images of FimH (a) and WT (b) E. coli UTI89 bacteria recorded in air. (c, d) Structure of disulfide-containing bis-mannofullerene 1 (c) and thiolated heptylmannoside 2 (d) used in this study.

numerous fibers displaying an average length of 544 ± 213 nm and an average diameter of 4.3 ± 1.1 nm (Figure 1a), consistent with the structure of type 1 pili.12 As expected, wild-type E. coli UTI89 displayed a smooth morphology devoid of any fibrillar structures (Figure 1b). Synthesis of Glycoconjugates. To probe glycofullerenebacterial adhesive interactions, we synthesized a disulfidecontaining bis-mannofullerene 1 displaying 20 peripheral mannose residues (Figure 1c) from a [5:1] heterovalent fullerene scaffold (the whole synthesis is depicted in Figure 2). Heterovalent and clickable [5:1] fullerene derivatives were first prepared by Nierengarten and co-workers for 10 + 2 grafting of diverse functional ligands such as aromatic residues13 or ferrocenes.14 Recently, we, along with the groups of Martin, Rojo and Nierengarten, reported the 10 + 1 fullerene scaffold 3 employed herein, which displays ten azides and one TMSalkyne, for further carbohydrate-derivatization.15 As reported, scaffold 3 was synthesized through two sequential Bingel− Hirsch cyclopropanations from C60 and the malonates of interest. Next, derivatization of 3 was carried out by two consecutive CuAAC reactions, first with propargylated

heptylmannoside 416 and then with bis-azide pegylated linker 5 possessing a central disulfide bond,17,18 which provided the desired 20-valent mannocluster 1 in excellent yield (see Figure 2). This bis-fullerene was characterized by 1H NMR, 13C NMR and IR. In particular, the 13C NMR spectrum analysis was of paramount importance by demonstrating the high symmetry of the molecule as shown earlier in our previous studies.11,19−22 The choice of disulfide linker 5 was strategically made in order to avoid the presence of the strong Cu-complexing thiol group during the click reactions. Moreover, generating directly a disulfide instead of a thiol also allows to avoid the partial oxidation of the final thiol during its purification or its storage, and thus to facilitate the characterization of the final molecule(s). An alternative strategy would have consisted in using a protective group on the sulfur group for the last click reaction, followed by a deprotection. However, thiols being prone to rapid (air) oxidation, they are extremely difficult to store without their corresponding disulfides. To overcome this problem, we privileged here the direct synthesis of a disulfide 1, and showed that the presence of the disulfide in the linker 5 does not poison the CuAAC copper catalyst. To functionalize 1300

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Figure 2. Synthesis of disulfide bonded glycofullerene 1 and in situ reduction to 6 for AFM tip functionalization. Reagents and conditions: 4, CuSO4/NaAsc, THF/DMSO/H2O (3:1:1), 16 h, room temperature and 2 h, 80 °C; 5, CuSO4/NaAsc, TBAF, DMSO:H2O (9:1), 16 h, room temperature (85%); (iii) TCEP.

interaction between heptylmannoside 2 and FimH E. coli cells are shown in Figure 3a,b (n = 2048 curves from two independent tips and two independent cells). Many force profiles showed single or multiple adhesion events of ∼200− 2500 pN magnitude that were preceded by constant force plateaus, and showed extended rupture lengths of ∼500−5000 nm. As described in Figure 3c, the adhesion force histogram was built by considering the maximum force exerted on the cantilever (measured relative to the baseline), whereas the rupture length histogram was generated using the last rupture peak. Adhesive events were never seen on cells lacking FimH (Figure 3e,f), and were abolished upon injection of soluble heptylmannoside 2 (Figure 3a, inset), leading us to believe they represent specific interactions between the mannose-derivatized tip and FimH. Consistent with this, the long ruptures indicate that elongated structures like pili were stretched. All heptylmannoside-FimH interactions displayed single or multiple constant force plateaus, with the mean magnitude of single plateaus being 226 ± 29 pN (mean ± s.d.; n = 926 plateaus from two independent experiments) (Figure 3c,d). In agreement with earlier studies on type 1 pili,30,31 this behavior reflects the force-induced unfolding of the pilus helical quaternary structure. That Gram-negative pili elongate under force is in line with the notion that they are formed by noncovalent interactions. Pilus elongation also explains why rupture distances up to 5 μm, thus much longer than the pili

gold-coated AFM tips with glycofullerenes, the thiol− glycofullerene 6 could be quantitatively generated in situ using TCEP (Figure 2), as a reducing reagent. So the in situ generated thiol functionality could be exploited as a convenient handle to graft the multimeric species onto the tips. Such a strategy had been already successfully exploited by Wong et al. for the grafting of complex thiolated biomolecules, generated in situ from their corresponding disulfides, onto gold microarrays.23,24 Interestingly, a complex thiolated molecule such as 6, bearing 10 biologically relevant ligands could be exploited for the functionalization of any gold-based surfaces or materials (e.g., microarrays, nanoparticles). For comparative analysis, we also used the thiol-terminated heptyl α-D-mannoside 2 (Figure 1d) prepared in a few steps from D-mannose.25 Heptylmannoside was chosen on a rational basis owing to the work of Bouckaert et al., who showed that heptyl mannose is among the most potent monovalent ligands of FimH due to strongly attractive interaction of the alkyl chain with the lectin’s tyrosine gate.26 Mannofullerenes Strongly Bind to FimH. We first asked whether mannofullerenes are capable of forming strong bonds with FimH. Single-molecule force spectroscopy (SMFS) with functionalized tips27−29 was used to measure the adhesion forces between heptylmannoside sugars or mannofullerenes and living E. coli bacteria (Figures 3 and 4). Adhesion forces, rupture lengths and representative force curves obtained for the 1301

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Figure 3. Single-molecule force spectroscopy of the FimH-mannose adhesion. (a) Adhesion force histogram and (b) rupture length histogram with representative force curves obtained by recording force curves in TBS buffer between mannose-terminated tips and FimH E. coli bacteria. Data from two different cells from independent cultures are pooled (n = 2048 curves). (c) Typical adhesive force curve showing how we defined the key parameters, i.e. adhesion force, rupture length, plateau force and baseline. (d) Histogram showing the distribution of plateau forces measured from 926 plateaus from 2 different cells. (e) Adhesion force histogram and (f) rupture length histogram obtained in the same conditions for pili-less WT E. coli bacteria (n = 2048 curves from two cells). Similar results were obtained for 4 other FimH cells and another WT cell from independent cultures. Mannoconjugates, AFM tips, cells, and pili in the cartoons are not drawn to scale.

baseline was not well-defined, see dashed line in the lower force curve) with multiple plateaus (up to 7) and rupture events (Figure 4a, b). Assuming that each plateau may represent a bundle of several pili (see above), the 3−7 plateaus observed in many of the curves suggest that up to 20 pili may bind the mannofullerene. This is in line with isothermal titration calorimetry (ITC) data showing that fullerene glycoconjugates can trigger the aggregation of FimH.11 The density (or concentration) of mannose residues in the mannofullerene-tip should not be higher than that of the heptylmannose-tip as the latter was functionalized at high density using the Au-thiol chemistry. Therefore, we believe that the strongly enhanced adhesion observed with mannofullerenes results from the multivalent presentation of sugars, enabling to achieve highaffinity interactions with multiple pili.

length, were often observed. We note that our 226 pN minimal plateau force is larger than that reported earlier (60−80 pN),30,31 suggesting strongly that multiple pili were stretched together in our assay, possibly as bundles made of 3 or 4 fibers. Plateau forces of 400−500 pN were also observed (Figure 3d), which could reflect the elongation of larger bundles (6−8 pili). SMFS data obtained for thiol-bound mannofullerene 6 (Figure 4a,b) strongly differed from the monovalent heptylmannoside 2 results. Many curves showed complex shapes without a clear baseline. Although the physical meaning of these profiles is unclear, they are likely to result from strongly adhesive interactions. Supporting this view, we also observed curves showing large adhesion forces, 1952 ± 733 pN (mean ± s.d. from n = 53 curves from several independent experiments; note that this value is a lower estimate as the 1302

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Figure 4. Mannofullerenes exhibit greatly enhanced adhesion toward FimH. (a) Adhesion force histogram with representative force curves, and (b) average number of force plateaus observed in the force profiles, obtained by SMFS in TBS buffer between mannofullerene-terminated tips and FimH E. coli bacteria. The dashed line in part a shows the estimated baseline. The inset in part b shows the plateau numbers observed using mannoseterminated tips. For each condition, data from two different cells from independent cultures are pooled (n = 53 curves). The cartoon is not to scale.

Figure 5. Single-cell force spectroscopy quantifies adhesion forces between E. coli bacteria and mannose-coated substrates. (a, b) Adhesion force (a) and rupture length (b) histograms obtained by recording multiple force−distance curves in TBS buffer between FimH cells and mannose substrates (n = 100 curves from 2 cells; similar results were obtained in four other cells from independent cultures). The inset in part b is an optical microscope image of a living bacterial cell attached to the colloidal cantilever probe (green color). (c) Variation of the maximum adhesion force observed upon recording 500 consecutive force curves between a FimH cell and a mannose substrate or a glass substrate. The same trends were observed with four different cells from independent cultures. (d) Representative force profiles observed on mannose substrates. The cartoon in part a is not to scale.

Mannofullerenes Show Greatly Enhanced Antiadhesion Activity. We then addressed the issue as to whether mannofullerenes show enhanced antiadhesion properties against FimH as compared to monovalent heptylmannose. Therefore, we used single-cell force spectroscopy (SCFS)32,33 to measure the adhesion forces between whole E. coli cells and mannose-coated substrates, prior (Figure 5) and after (Figure 6) addition of mannofullerene 1 or soluble heptylmannose 2.

Cells were attached onto colloidal cantilevers coated with the wet adhesive polydopamine (Figure 5a, inset). Labeling of the cell probe showed that the method is nondestructive (Figure 5b, inset, green color). Figure 5a,b shows the adhesion forces and rupture lengths obtained between single FimH E. coli cells and mannose-coated substrates (data from 2 different cells are pooled). All force profiles showed adhesion events of ∼500− 1000 pN magnitude and ∼2000−5000 nm rupture length. A 1303

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Figure 6. Mannofullerenes show greatly enhanced antiadhesion activity against E. coli. Maximum adhesion force (a, c) and representative force profiles (b, d) obtained by recording multiple force−distance curves between FimH cells and mannose substrates in the absence or in the presence of heptylmannoside 2 (a, b) or bis-mannofullerene 1 (c, d) at increasing concentration. Given the behavior observed in Figure 5c, sets of only 20 curves were recorded for each cell in each condition. Data from seven independent experiments are shown for each condition. The cartoons are not to scale.

already decreased substantially the adhesion force (Figure 6c,d). At 10 μM mannofullerenes, the adhesion force was strongly reduced (128 pN vs 936 pN, Figure 6c,d), and the adhesion probability was lower (68% vs 100%), two effects which were never achieved with monovalent mannoside 2. So our single-cell data demonstrate stronger blocking activity for mannofullerenes compared to monovalent heptylmannoside sugars (10 μM mannofullerenes vs 200 μM mannose). Thus, even if the fullerene concentration is substituted by the “effective mannose concentration” (by multiplying the fullerene concentration with the number n of mannose units), fullerene glycoconjugates show stronger blocking capacities than monomers. We therefore conclude that mannofullerenes inhibit bacterial adhesion via the multivalent presentation of carbohydrates rather than a trivial concentration effect. This is consistent with earlier ITC and surface plasmon resonance (SPR) data showing that fullerene mannoconjugates have low nanomolar affinities toward FimH and can accommodate up to seven adhesins.11 We expect that the strong antiadhesion activity of globular glycoconjugates result from two factors, i.e., multiple high affinity interactions with FimH and the selfassociation or aggregation of pili, as also suggested by our SMFS data. Conclusions. Because many bacterial pathogens have become resistant to antibiotics, there is a strong need to develop novel means of fighting bacterial infections. As many bacteria attach to host cells through protein−carbohydrate interactions, the use of high-affinity multivalent carbohydrate compounds to block bacterial adhesins is a promising approach in antiadhesion therapy. The advantage of antiadhesive compounds over antibiotics is that they clear rather than kill bacteria, meaning the development of resistant mutants is reduced. We have shown that AFM-based nanoscopy, virtually

prominent feature is that the adhesion profiles showed multiple rupture and plateau events (Figure 5d), indicating that separating the bacteria from the mannose surface leads to the unfolding of pili. The minimal plateau force observed at long distance was 86 ± 15 pN, thus smaller than in SMFS experiments, but in the range of the force reported for the unfolding of individual pili.30,31 This emphasizes the influence of the experimental set up on the mechanical response of piliated bacteria: while mannose-tips in SMFS always stretch multiple pili in parallel, separation of whole bacteria from mannose surfaces in SCFS leads to the unfolding of individual pili. Another interesting observation is that the mean adhesion force decreased progressively when multiple curves (>100) were recorded with the same cell (Figure 5, parts c and d). This effect was not observed on glass substrates (Figure 5c), indicating that it depends specifically on FimH-mannose interactions. We suggest that multiple stretching of the same pili through FimH-mannose bonds leads to their denaturation, thereby decreasing the overall adhesiveness of the cell surface. This phenomenon may play a role in host colonization. When subjected to prolonged shear stress, bacterial cells may become less adhesive, therefore spread and colonize new sites. Do mannofullerenes inhibit single-cell adhesion forces? Figure 6a,b shows that addition of 1 μM soluble monovalent heptylmannose 2 had no effect on the maximal bacterialmannose adhesion forces (data obtained with 7 different cells using different substrates; only the first 20 curves were considered in these measurements given the behavior discussed above, Figure 5c). Increasing the sugar concentration to 10, 100, and 200 μM lead to a moderate decrease of the adhesion force, and did not change the adhesion probability (100%). By contrast, addition of 1 μM of 20-valent mannofullerene 1 1304

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Barbara, CA) with oxide-sharpened microfabricated Si3N4 cantilevers with a nominal spring constant of 0.01 N m−1. One hundred microliters of an overnight-cultured cell suspension were brought in contact with freshly cleaved mica substrates mounted on steel pucks. The samples were incubated for 2 h at 37 °C, gently rinsed in three successive baths of ultrapure water (Elga, purelab water) and allowed to dry at 30 °C overnight. Single-Molecule Force Spectroscopy. Bacteria were immobilized by mechanical trapping into a polycarbonate porous membrane (Millipore) with a pore size similar to the cell size. After the cell suspension was filtered, the filter was gently rinsed in TBS buffer, carefully cut (1 cm × 1 cm), attached to a steel sample puck using double-faced adhesive tape. The mounted sample was transferred to the AFM liquid cell while avoid dewetting prior to force measurements. Force curves using functionalized tips were recorded on a Nanoscope VIII Multimode AFM (Bruker Corporation) at room temperature in TBS. A single cell was first localized using a silicon nitride tip. Adhesion and rupture length histograms were obtained by recording 32 × 32 force distance curves on areas of 500 nm × 500 nm on the bacterial surface. All curves were recorded with a maximum applied force of 250 pN, with a retraction speed of 2 μm s−1 and a contact time of 100 ms. Single-Cell Force Spectroscopy. For single cell force spectroscopy, cell probes were prepared using a recently developed protocol that combines colloidal probe cantilevers and a bioinspired polydopamine wet adhesive.32,36 Briefly, silica microspheres (6.1 μm diameter, bangs laboratories) were attached on triangular-shaped tipless cantilevers (NP-O10 Microlevers, Bruker Corporation) using UV-curable glue (NOA 63, Norland Edmund Optics). The cantilevers were then immersed for 1 h in a 10 mM Tris buffer solution (pH 8.5) containing 4 mg mL−1 dopamine hydrochloride (99%, Sigma) and dried with N2 flow. Single bacteria were then attached onto polydopamine-coated colloidal probes using a Bioscope Catalyst AFM (Bruker Corporation). To this end, overnight bacterial cultures were diluted to 1:100 in the corresponding medium, and 50 μL of the diluted suspension was deposited in a glass Petri dish containing mannose substrates. A colloidal probe was brought into contact with an isolated bacterium for 3 min, and the obtained cell probe was then transferred over the solid substrate for force measurements. SCFS measurements were performed at room temperature using a Bioscope catalyst AFM (Bruker Corporation). The spring constant of the cantilevers were determined by the thermal noise method. Multiples force curves were recorded on various spots of the substrate using a maximum applied force of 250 pN, a contact time of 100 ms and a retraction speed of 2 μm s−1. For blocking experiments, thiolterminated heptyl α-D-mannoside 2 and thiol-mannofullerenes 1 were injected into the AFM chamber at the desired concentration 30 min prior force measurements.

applicable to any microbial pathogen, is a versatile tool to quantify the adhesion forces between multivalent glycoconjugates and their target adhesins, and to assess their antiadhesion activity directly on living bacteria. SMFS revealed that heptylmannoside monomers bind to FimH on type 1 pili, and that pulling on these bonds leads to constant force plateaus originating from the force-induced uncoiling (unfolding) of the helical quaternary structure of the pili. Bis-mannofullerenes displaying 20 peripheral mannose residues showed greatly enhanced adhesion toward FimH, demonstrating that the multivalent presentation of sugars enables to achieve high-affinity interactions with multiple pili. SCFS showed that, compared to monovalent heptylmannoside, mannofullerene exhibits remarkably strong antiadhesion activity. This behavior cannot be explained by a simple concentration effect, but rather by the multivalent high affinity interactions of glycoconjugates with FimH. Compared to ITC and SPR, force nanoscopy enables the label-free analysis of glycoconjugates directly on live cells. The next step will be to increase the throughput of single-cell AFM assays, which could be achieved using the recently developed Fluidic force microscopy (FluidFM) technology. With FluidFM, many cells can be probed in a short time frame, meaning that statistically relevant data can be obtained within a few hours.34 Methods. Bacterial Strains and Strain Culture. We used two E. coli strains, i.e., E. coli UTI89 WT and the FimH UTI89 strain engineered for continuous fimbriation. Both strains were cultivated overnight in lysogeny broth (LB, Sigma) at 37 °C with shaking at 200 rpm. Synthesis of Mannoconjugates (See Supporting Information). The disulfide-containing bis-mannofullerene (Figure 1c) was prepared by two sequential CuAAC reactions between the multivalent scaffold 3, displaying 10 azide groups and one TMS−alkyne, with azide functionalized heptylmannoside 4 and then bis-azide pegylated linker 5 possessing a central disulfide bond. The thiol-terminated alkyl mannoside 2 (Figure 1d) was obtained using standard procedures from D-mannose. The synthetic scheme developed for the synthesis of heptyl-α-Dmannose by Gouin and Kovensky35 was followed: the sugar was activated at the anomeric position by a trichloroacetimidate and coupled to the corresponding primary alcohol using BF3·Et2O as a promotor. KSAc was then used to introduce a thioacetate functional group into the molecule, which was deprotected under Zemplén conditions to afford the final pure glycoside. Functionalization of Tips and Substrates. To prepare mannose-coated surfaces, glass coverslips were coated by electron beam thermal evaporation with a 5 nm thick Cr layer followed by a 30 nm thick Au layer. Gold surfaces were immersed overnight in a solution of 0.5 mM thiol-terminated heptyl α-D-mannoside 2 diluted in ultrapure water and rinsed in water. To obtain functionalized tips, gold-coated cantilevers (OMLC-TR4, Olympus Ltd., Tokyo, Japan, nominal spring constant ∼0.02 N m−1) were immersed for 12 h in a 1 mM solution of either thiol-terminated heptyl α-D-mannoside 2 or thiol−mannofullerene 1. Prior to tip immersion, 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) solution was added to the thiol solutions (50% vol) for reduction of the disulfide bond of the mannofullerene. Atomic Force Microscopy Imaging. AFM contact mode images were obtained in air at room temperature using a Nanoscope VIII Multimode AFM (Bruker Corporation, Santa



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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b04689. Detailed description of the methods and synthesis and characterization of mannoconjugates (PDF) 1305

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(10) Touaibia, M.; Wellens, A.; Shiao, T. C.; Wang, Q.; Sirois, S.; Bouckaert, J.; Roy, R. Mannosylated G(0) Dendrimers with Nanomolar Affinities to Escherichia coli FimH. ChemMedChem 2007, 2, 1190−1201. (11) Durka, M.; Buffet, K.; Iehl, J.; Holler, M.; Nierengarten, J.-F.; Taganna, J.; Bouckaert, J.; Vincent, S. P. The functional valency of dodecamannosylated fullerenes with Escherichia coli FimH–towards novel bacterial antiadhesives. Chem. Commun. 2011, 47, 1321−1323. (12) Hahn, E.; Wild, P.; Hermanns, U.; Sebbel, P.; Glockshuber, R.; Häner, M.; Taschner, N.; Burkhard, P.; Aebi, U.; Müller, S. A. Exploring the 3D molecular architecture of Escherichia coli type 1 pili. J. Mol. Biol. 2002, 323, 845−857. (13) Iehl, J.; Nierengarten, J. F. A Click−Click Approach for the Preparation of Functionalized [5:1]-Hexaadducts of C60. Chem. - Eur. J. 2009, 15, 7306−7309. (14) Fortgang, P.; Maisonhaute, E.; Amatore, C.; Delavaux-Nicot, B.; Iehl, J.; Nierengarten, J. F. Molecular Motion Inside an Adsorbed [5:1] Fullerene Hexaadduct Observed by Ultrafast Cyclic Voltammetry. Angew. Chem., Int. Ed. 2011, 50, 2364−2367. (15) Muñoz, A.; Sigwalt, D.; Illescas, B. M.; Luczkowiak, J.; Rodriguez-Perez, L.; Nierengarten, I.; Holler, M.; Remy, J.-S.; Buffet, K.; Vincent, S. P.; Rojo, J.; Delgado, R.; Nierengarten, J. F.; Martín, N. Nat. Chem. 2015, 8, 50−57. (16) Bouckaert, J.; Li, Z.; Xavier, C.; Almant, M.; Caveliers, V.; Lahoutte, T.; Weeks, S. D.; Kovensky, J.; Gouin, S. G. Heptyl α- DMannosides Grafted on a β-Cyclodextrin Core To Interfere with Escherichia coli Adhesion: An In Vivo Multivalent Effect. Chem. - Eur. J. 2013, 19, 7847−7855. (17) Gobbo, P.; Workentin, M. S. Improved methodology for the preparation of water-soluble maleimide-functionalized small gold nanoparticles. Langmuir 2012, 28, 12357−12363. (18) Gobbo, P.; Novoa, S.; Biesinger, M. C.; Workentin, M. S. Interfacial strain-promoted alkyne-azide cycloaddition (I-SPAAC) for the synthesis of nanomaterial hybrids. Chem. Commun. 2013, 49, 3982−3984. (19) Nierengarten, J.-F.; Iehl, J.; Oerthel, V.; Holler, M.; Illescas, B. M.; Muñoz, A.; Martín, N.; Rojo, J.; Sánchez-Navarro, M.; Cecioni, S.; Vidal, S.; Buffet, K.; Durka, M.; Vincent, S. P. Fullerene sugar balls. Chem. Commun. 2010, 46, 3860−3862. (20) Durka, M.; Buffet, K.; Iehl, J.; Holler, M.; Nierengarten, J.-F.; Vincent, S. P. The Inhibition of Liposaccharide Heptosyltransferase WaaC with Multivalent Glycosylated Fullerenes: A New Mode of Glycosyltransferase Inhibition. Chem. - Eur. J. 2012, 18, 641−651. (21) Buffet, K.; Gillon, E.; Holler, M.; Nierengarten, J.-F.; Imberty, A.; Vincent, S. P. Fucofullerenes as tight ligands of RSL and LecB, two bacterial lectins. Org. Biomol. Chem. 2015, 13, 6482−6492. (22) Abellán-Flos, M.; Tanç, M.; Supuran, C. T.; Vincent, S. P. Exploring carbonic anhydrase inhibition with multimeric coumarins displayed on a fullerene scaffold. Org. Biomol. Chem. 2015, 13, 7445− 7451. (23) Bryan, M. C.; Fazio, F.; Lee, H.-K.; Huang, C.-Y.; Chang, A.; Best, M. D.; Calarese, D. A.; Blixt, O.; Paulson, J. C.; Burton, D.; Wilson, I. A.; Wong, C.-H. Covalent display of oligosaccharide arrays in microtiter plates. J. Am. Chem. Soc. 2004, 126, 8640−8641. (24) Huang, C.-Y.; Thayer, D. A.; Chang, A. Y.; Best, M. D.; Hoffmann, J.; Head, S.; Wong, C.-H. Carbohydrate Microarray for Profiling the Antibodies Interacting with Globo H Tumor Antigen. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 15−20. (25) El-Kirat-Chatel, S.; Beaussart, A.; Vincent, S. P.; Abellán Flos, M.; Hols, P.; Lipke, P. N.; Dufrêne, Y. F. Forces in yeast flocculation. Nanoscale 2015, 7, 1760−1767. (26) Bouckaert, J.; Berglund, J.; Schembri, M.; De Genst, E.; Cools, L.; Wuhrer, M.; Hung, C.-S.; Pinkner, J.; Slättegard, R.; Zavialov, A.; Choudhury, D.; Langermann, S.; Hultgren, S. J.; Wyns, L.; Klemm, P.; Oscarson, S.; Knight, S. D.; De Greve, H. Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin. Mol. Microbiol. 2005, 55, 441−455. (27) Dupres, V.; Menozzi, F. D.; Locht, C.; Clare, B. H.; Abbott, N. L.; Cuenot, S.; Bompard, C.; Raze, D.; Dufrene, Y. F. Nanoscale

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Corresponding Authors

*(Y.D.) E-mail: [email protected]. *(S.P.V.) E-mail: [email protected]. Present Addresses ⊥

CNRS, Laboratoire Interdisciplinaire des Environnements Continentaux, LIEC, UMR 7360, Vandoeuvre-lès-Nancy, F54501, France. ∥ Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour l’Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France. # CNRS, Laboratoire de Chimie Physique et Microbiologie pour l’Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F54600, France. Author Contributions

A.B. and M.A.F. contributed equally. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Work at the Université catholique de Louvain was supported by the National Fund for Scientific Research (FNRS), the FNRSWELBIO under Grant No. WELBIO-CR-2015A-05, the Université catholique de Louvain (Fonds Spéciaux de Recherche), the Federal Office for Scientific, Technical and Cultural Affairs (Interuniversity Poles of Attraction Programme), and the Research Department of the Communauté française de Belgique (Concerted Research Action). Y.F.D. is a Research Director of the FNRS. Work at the Université de Namur was supported by People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme “DyNano” FP7/2007-2013, under REA Grant Agreement No. 289033.



REFERENCES

(1) Ofek, I.; Hasty, D. L.; Sharon, N. Anti-adhesion therapy of bacterial diseases: prospects and problems. FEMS Immunol. Med. Microbiol. 2003, 38, 181−191. (2) Sharon, N. Carbohydrates as future anti-adhesion drugs for infectious diseases. Biochim. Biophys. Acta, Gen. Subj. 2006, 1760, 527− 537. (3) Shoaf-Sweeney, K. D.; Hutkins, R. W. Adherence, anti-adherence, and oligosaccharides preventing pathogens from sticking to the host. Adv. Food Nutr. Res. 2008, 55, 101−161. (4) Shmuely, H.; Ofek, I.; Weiss, E. I.; Rones, Z.; Houri-Haddad, Y. Cranberry components for the therapy of infectious disease. Curr. Opin. Biotechnol. 2012, 23, 148−152. (5) Krachler, A. M.; Orth, K. Targeting the bacteria-host interface. Virulence 2013, 4, 284−294. (6) Aronson, M.; Medalia, O.; Schori, L.; Mirelman, D.; Sharon, N.; Ofek, I. Prevention of colonization of the urinary tract of mice with Escherichia coli by blocking of bacterial adherence with methyl alphaD-mannopyranoside. J. Infect. Dis. 1979, 139, 329−332. (7) Lindhorst, T. K.; Kieburg, C.; Krallmann-Wenzel, U. Inhibition of the type 1 fimbriae-mediated adhesion of Escherichia coli to erythrocytes by multiantennary alpha-mannosyl clusters: the effect of multivalency. Glycoconjugate J. 1998, 15, 605−613. (8) Lindhorst, T. K.; Dubber, M. Octopus glycosides: multivalent molecular platforms for testing carbohydrate recognition and bacterial adhesion. Carbohydr. Res. 2015, 403, 90−97. (9) Touaibia, M.; Roy, R. Glycodendrimers as anti-adhesion drugs against type 1 fimbriated E. coli uropathogenic infections. Mini-Rev. Med. Chem. 2007, 7, 1270−1283. 1306

DOI: 10.1021/acs.nanolett.5b04689 Nano Lett. 2016, 16, 1299−1307

Letter

Nano Letters mapping and functional analysis of individual adhesins on living bacteria. Nat. Methods 2005, 2, 515−520. (28) Alsteens, D.; Garcia, M. C.; Lipke, P. N.; Dufrene, Y. F. Forceinduced formation and propagation of adhesion nanodomains in living fungal cells. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 20744−20749. (29) Andre, G.; Kulakauskas, S.; Chapot-Chartier, M. P.; Navet, B.; Deghorain, M.; Bernard, E.; Hols, P.; Dufrene, Y. F. Imaging the nanoscale organization of peptidoglycan in living Lactococcus lactis cells. Nat. Commun. 2010, 1, 27. (30) Forero, M.; Yakovenko, O.; Sokurenko, E. V.; Thomas, W. E.; Vogel, V. Uncoiling mechanics of Escherichia coli type I fimbriae are optimized for catch bonds. PLoS Biol. 2006, 4, e298. (31) Miller, E.; Garcia, T.; Hultgren, S.; Oberhauser, A. F. The mechanical properties of E. coli type 1 pili measured by atomic force microscopy techniques. Biophys. J. 2006, 91, 3848−3456. (32) Helenius, J.; Heisenberg, C. P.; Gaub, H. E.; Muller, D. J. Singlecell force spectroscopy. J. Cell Sci. 2008, 121, 1785−1791. (33) Beaussart, A.; El-Kirat-Chatel, S.; Herman, P.; Alsteens, D.; Mahillon, J.; Hols, P.; Dufrene, Y. F. Single-cell force spectroscopy of probiotic bacteria. Biophys. J. 2013, 104, 1886−1892. (34) Guillaume-Gentil, O.; Potthoff, E.; Ossola, D.; Franz, C. M.; Zambelli, T.; Vorholt, J. A. Force-controlled manipulation of single cells: from AFM to FluidFM. Trends Biotechnol. 2014, 32, 381−388. (35) Gouin, S. G.; Wellens, A.; Bouckaert, J.; Kovensky, J. Synthetic multimeric heptyl mannosides as potent antiadhesives of uropathogenic Escherichia coli. ChemMedChem 2009, 4, 749−755. (36) Beaussart, A.; El-Kirat-Chatel, S.; Sullan, R. M. A.; Alsteens, D.; Herman, P.; Derclaye, S.; Dufrêne, Y. F. Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy. Nat. Protoc. 2014, 9, 1049−1055.

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