Mannose Surfaces Exhibit Self-Latching, Water Structuring, and

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Mannose surfaces exhibit self-latching, water-structuring, and resilience to chaotropes: Implications for pathogen virulence Hashanthi K. Abeyratne-Perera, and Preethi L. Chandran Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01006 • Publication Date (Web): 17 Aug 2017 Downloaded from http://pubs.acs.org on August 21, 2017

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Mannose surfaces exhibit self-latching, water-structuring, and resilience to chaotropes: Implications for pathogen virulence Hashanthi K. Abeyratne-Perera 1, Preethi L. Chandran PhD 2,1 1 2

Biochemistry and Molecular Biology Department, Chemical Engineering Department,

Howard University, Washington DC.

Corresponding author Preethi L. Chandran, PhD Assistant Professor Department of Chemical Engineering, College of Engineering and Architecture, Howard University Department of Biochemistry and Molecular Engineering, College of Medicine, Howard University Address: 1011 LK Downing Hall 2300 6th Street, NW, Howard University Washington, DC 20059. Email: [email protected] Phone: 202-806-4595

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ABSTRACT: Several viral and fungal pathogens, including HIV, SARS, Dengue, Ebola and Cryptococcus neoformans, display a preponderance of mannose residues on their surface, particularly during the infection cycle or in harsh environments. The innate immune system, on the other hand, abounds in mannose receptors which recognize mannose residues on pathogens and trigger their phagocytosis. We pose the question if there is an advantage for pathogens to display mannose on their surface, despite these residues being recognized by the immune system. The surface properties and interactions of opposing monolayers of mannobiose (disaccharide of mannose) were probed using Atomic Force Spectroscopy. Unlike its diastereoisomer lactose, mannobiose molecules exhibited lateral packing interactions that manifest on the surface scale as a self-recognizing latch. A break-in force is required for opposing surfaces to penetrate and a break-out (or self-adhesion force) of similar magnitude is required for penetrated surfaces to separate. A hierarchy of self-adhesion forces was distinguished as occurring at the single residue (~25pN), cluster (~250pN), monolayer (~1.1nN), and supra-monolayer level. The break-in force and break-out force appear resilient to the presence of simple chaotropes that attenuate a layer of structured-water around the mannose surface. The layer of structured water otherwise extends to distances several times longer than a mannobiose residue indicating a long-range propagation of the latter’s hydrogen bonding. The span of the structured water increases with the velocity of an approaching surface, similar to shear-thickening, but fissures at higher approach velocities. Our studies suggest that mannose residues could guide interpathogen interactions, such as in biofilms, and serve as a moated fortress for pathogens to hide behind to resist detection and harsh environments.


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INTRODUCTION Mannose residues are extensively present on the surface of pathogenic viruses and fungi, and are centrally involved in the biology of pathogen virulence and host immune response 1, 2. The fungal cell wall contains only a limited selection of sugars and mannose is a major component of the cell wall3. In both viruses and fungi, mannoses occur as much as 20-50% and are distributed towards the outer surface 3, 4. The mannose content in the cell wall is critical for fungal viability, and is part of the pathogen associated molecular patterns (PAMP) recognized by receptors in the cell-mediated immune pathway 3, 5. In addition, mannose is extensively present in the polysaccharide capsule surrounding the cell wall of pathogenic fungi which associates with fungal virulence 6. For instance, mannose constitutes one- to two- thirds of the polysaccharide capsule in Cryptococcus neoformans, a fungus which causes meningitis and opportunistic infection in immune-compromised individuals 7. High-mannose patches are also present on the envelope of viruses belonging to the filovirus family which includes lethal strains like Ebola 8, Marburg 8, and Dengue 9 that cause hemorrhagic fever. The HIV virus has a ‘glycan shield’ which prevents antibody detection of the viral proteins, and mannose occurs extensively on its surface 10,15. Highly-mannosylated regions have also been identified on the surface of the coronavirus SARS 11. With the rising interest in immune-directed therapies, delivery vehicles with surface-coats of mannose are being developed for targeting dendritic cells and macrophages which have surface receptors for mannose 12, 13, 14. Interestingly mannose receptors (MRs) themselves are highly glycosylated and contain mannosylated glycans on their surfaces 15, 16.

The mannose content of pathogens has typically been discussed in the context of their recognition by mannose-binding lectins (MBLs). These are mannose receptors that circulate in the blood stream or are present on immune cells such as, macrophages 17 and dendritic cells 18, and can serve to intercept pathogens at points of entry into the body. MBL binding triggers opsonization, and activates the innate pathway of the immune response that operates in the immediate hours and days following an infection 18, 19

. In fact increasing the amount of circulating MBLs was found clinically effective for neutralizing

highly mannosylated pathogens, like Ebola, and for developing subsequent immunity 8, 20. We pose the question of whether the mannose on pathogens serves a role that extends beyond communicating with the host immune system via lectins. For instance, do self-specific carbohydrate-carbohydrate interactions occur between mannosylated surfaces, and confer biophysical advantages for pathogen virulence and interactions?


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Carbohydrate-Carbohydrate interactions (CCIs) are a class of self-interactions observed between the acidic glycans of proteoglycans 21, 22, 23 and glycosphingolipids 24 typically expressed on cell surfaces. The self-specific character distinguishes these interactions from the non-specific associations common between glycosylated surfaces. For instance, CCI forces between opposing glycans of the same type were found to be three times stronger than those between glycans of different alleles 25. It is proposed that CCI is facilitated by the repetitive hydrogen-bonding, ionic-bridges, and other weak intermolecular forces induced between ordered clustering of glycans 26, 27, 28. The significance of CCIs as an interaction force lies in its net strength which results when single residue-residue adhesions (~20 - 40 pN) 21 are magnified by the multiplicity and multivalency of glycan presentation on cells, to levels that can bear the weight of several cells 29. Since initial findings that CCIs mediate self-recognition in sponge cells 30, CCIs have been reported to occur during biological processes like fertilization, embryogenesis, cell development and metastasis 31, 32. Mannose residues appear to occupy the outer reaches of the glycan shells that cover pathogens, and hence are popular targets for lectins and carbohydrate receptors. It is not clear if mannosylated surfaces offer specific advantages for pathogen hydration, biofilm formation, and immune-evasion 33, leading to their evolutionary positioning on the surface of polysaccharide capsules. Also, CCIs between mannose residues have never been reported, and these interactions could confer biophysical advantages for pathogen virulence, pathogen-pathogen interaction, and host-pathogen interaction. The goal of our study is to investigate the biophysical properties of mannobiosylated monolayers, which can be considered as models of the high mannose patches on fungal and viral pathogens. We specifically interrogate the selfinteractions between monolayers of mannobiose using Atomic Force Spectroscopy. Mannobiose, a disaccharide of mannose (alpha 1-4 linkage), can be regarded as the simplest mimic of a mannosylated polymer.

EXPERIMENTAL SECTION Synthesis of 4-azido-2,3,5,6-tetrafluoro-N-(2-mercaptoethyl) benzamide or ATFMB linker 30 mg of 4-Azido-2, 3, 5, 6-tetrafluorobenzene, succinimidyl ester or ATFB, SE (TCI America, Inc., PA) and molar excess of 2-mercaptoethylamine or cysteamine (Sigma-Aldrich Inc., St. Louis MO) were reacted in a 10ml of 1:1 acetone: HEPES at pH 8.5 solution on ice for 2 hours. The volume of the reaction mixture was then increased up to 40ml with HEPES buffer, and 4-azido-2,3,5,6-tetrafluoro-N-(2mercaptoethyl) benzamide (ATFMB) was isolated as a precipitate after centrifugation. The supernatant had a strong UV-Vis absorbance peak at 290nm wavelength due to the ‘leaving group’ hydroxylsuccinimide. The precipitate was washed three times with HEPES buffer and ninhydrin assay 34 was


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performed on each wash to track the removal of unreacted cysteamine. The synthesis was confirmed by Fourier Transform Infrared Spectroscopy or FTIR (PerkinElmer spectrum 100). Anchoring mannobiose on gold surfaces via ATFMB linkers Gold-coated AFM disks (Ted Pella Inc., Redding, CA) and probes (NPG-10, Bruker Inc., Billerica, MA) were rinsed with piranha solution (3:1, H2SO4: H2O2), followed by generous washing with MilliQ water, and drying under a N2 stream. The cleaned disks and probes were incubated for 12-15 hours in a saturated solution of freshly synthesized ATFMB dissolved in 1:1 acetone:ethanol to form self-assembledmonolayers. The surfaces were rinsed with ethanol and transferred to a clean petri-dish with 200 µl of 20 mg/ml alpha -1,4- mannobiose solution, and irradiated with UV (254nm wavelength) from a 8 Watt UV lamp (Pierce Thermofisher Scientific Inc., Waltham, WA) for 10 minutes. The distance between the UV lamp and the specimen was maintained approximately at 3cm. Under these conditions, mannobiose residues crosslink with the aryl-azide group at the free end of the assembled linkers. The functionalized AFM disks and probes were rinsed with MilliQ water to remove unattached sugars. The same procedure was used for attaching lactose and alpha -1,2 mannobiose to gold surfaces. X-ray photoelectron spectroscopy (XPS) Linker only and linker+mannobiose monolayers were assembled on gold-coated silicon wafers for XPS surface analysis with a Kratos Axis 165 x-ray photoelectron spectrometer (Kratos, Manchester, England) operating in hybrid mode with monochromatic aluminum x-rays (240W). Survey and high-resolution spectra were gathered at the pass energies 160 eV and 40 eV, respectively and the binding energies were calibrated to the reference hydrocarbon C 1s peak at 284.8 eV. Data were analyzed with CASA XPS software and peaks fitted with 30% Lorentzian 70% Gaussian peak shape after subtraction of a linear background. All the quantifications used relative sensitivity factors from the Kratos vision library. Due to the labile nature of the azide functional group, high-resolution N 1s spectra were acquired from ATFMBattached gold surfaces (10 scans) before the survey spectra. Contact angle analysis The static contact angle was measured with a Phoenix 150 goniometer (Surface & Electro-Optics Corporation, South Korea) using the standard sessile drop method, where a drop of water hanging from a syringe is brought in to contact with the surface and allowed to spread 35. The contact angle on either side of the drop at equilibrium was determined with the Surfaceware9 software available on the instrument. The average of three surface measurements is reported for each surface type.


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Functionalization of gold surfaces with Concanavalin A 10 mg of lyophilized Con A from Canavalia ensiformis or Jack bean (Sigma-Aldrich Inc., St. Louis MO) was reacted with 3 mg of 3,3-dithiobis (sulfosuccinimidyl propionate) or DTSSP (Sigma-Aldrich Inc., St. Louis MO) in PBS for one hour 36. 25mM Tris (2-carboxyethyl) phosphine or TCEP (Sigma-Aldrich Inc., St. Louis MO) was then added for one hour to reduce the disulfide bonds in DTSSP. All reactions were carried out in an ice bath. The thiol-functionalized ConA molecules were isolated by filter centrifugation (25kDa MW cut off, Merck Millipore Inc., Billerica, MA). The isolated protein was stored in PBS with 10mM CaCl2. Gold-coated AFM surfaces were incubated with the protein isolate for 24 hours and rinsed with PBS before force-spectroscopy measurements. Force Spectroscopy Force spectroscopy was performed with a Multimode/PicoForce system (NS-V controller, Bruker Nanosurfaces, Inc., Santa Barbara, CA) in a fluid cell. Gold-coated NPG-10 cantilevers (Bruker Inc., Billerica, MA) were functionalized, and their deflection sensitivity determined by indenting on a plain gold surface. The spring constant (k) of the cantilever was determined using the thermal tuning method. The indentation velocity was maintained between 100-150 nm/s. Experiments were initially performed in pure water contained within the fluid cell, and which was systematically supplemented with incremental amounts of NaCl to understand how salt affects the surface properties. The data was analyzed with an inhouse Matlab code and points corresponding to the force contact, mannobiose break-in, peak adhesion, and last-ruptures were selected. At least 300 force curves were analyzed for each experiment, and histograms of the data are presented.

RESULTS AND DISCUSSION Mannobiose anchoring to gold surface The mannobiose was anchored to gold surfaces via a linker molecule (ATFMB or 4-azido-2,3,5,6tetrafluoro-N-(2-mercaptoethyl) benzamide) that has a thiol functional group at one end and an aryl-azide on the other. The linkers self-assemble on gold surfaces via the thiol end, and the sugar residues were photochemically linked to the aryl-azide end 37, 38. The reaction scheme for generating anchored mannobiose is shown in Fig. 1. The ATFMB linker was synthesized by reacting ATFB, SE with excess cysteamine and isolated as a precipitate (Fig. 1). The leaving group and unreacted cysteamine separate to the aqueous phase and are removed by repeated washing. The synthesis and purification of ATFMB was confirmed with FTIR spectroscopy (Fig. 2A).


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The FTIR spectrum of the reactant cysteamine had the characteristic S-H bond stretch (2080 cm-1) and primary amine stretches (2434 cm-1 and 2503 cm-1); and the reactant ATFB, SE had prominent N3 (2125 cm-1) and carbonyl (1650 cm-1) bond signals (Fig. 2A, green line and orange line respectively). The purified product, ATFMB exhibited a N-H stretch at 3291cm-1 along with the azide and carbonyl signals (Fig. 2A, black line). The expected thiol signal was masked by the stronger azide signal in the vicinity. ATFMB linkers self–assemble on gold substrates via the thiol- terminal 39, 40 (Fig. 2B top panel). The formation of the ATFMB monolayer was accompanied by a change in surface contact angle to 46± 2°, which is significantly different than the 60° – 70° contact angle typical for clean gold surfaces 41, 42 (Fig. 2B, middle panel). N3 F

O O N

O

N3

C F

F

O

44°0C C

F

+

SH

F

F

H 2N

Cysteamine

Acetone: HEPES at pH 8.5 Acetone:HEPES 1 : 1

F

F C

1:1, pH 8.5

ATFB, SE

NH

HS

HO HO

HOH OH H HOH O OH H OH OH OH O H H H HO H OHOH H HO H H HOO O HOH HO H H H H HH OOH H H OH HO H OHOH O HOHHO O HO H H HO OH HO H O O HOO H O H H H H HO H H HO H H HO H H H OH NH NH NH NH H F F F F F F F F F F

F

S

F

F F

F

F

C O

C O

C O

C O

NH

NH

NH

NH

S

S

Au

S

O

ATFM B

Acetone:EtoH Acetone:EtoH 1:1 1 : 1 HOH H H

H

N3

N3

OH O

HO HO

O

H HOH OH O

HO

H H

H

H OH

UVUV 254nm 254 nm

N3

N3

F

F F

F F

F F

F

F F

F F

F F

S

S

S

F

C O NH

C O NH

C O NH

F

C O NH

S

Au

Figure 1: The reaction scheme for carbohydrate functionalization on gold. ATFB, SE and cysteamine were reacted in 1:1 acetone: HEPES at pH 8.5 to synthesize ATFMB, which was isolated as a precipitate in an aqueous media. Gold-coated disks and AFM probes were incubated with a saturated solution of 1:1 acetone: EtoH containing ATFMB to produced self-assembled monolayers of the linker. The monolayer was then covered with mannobiose solution and UV-irradiated to attach the sugar residues to the free aryl-azide end of the ATFMB monolayer.

Exposed aryl-azide groups on the assembled ATFMB linkers were irradiated with UV light (254 nm) in a solution containing free mannobiose (Fig. 1). When irradiated, aryl-azides convert to highly-reactive nitrenes, which insert into the C-H bonds of the mannobiose creating a covalent linkage 37, 38 (depicted in Fig. 1, lower panel). At the macroscale, mannobiose anchoring was verified by the change in the contact angle of the ATFMB-coated surface from 46± 2° to the more hydrophilic 12± 2° (Fig. 2B, middle panel). At the nanoscale, mannobiose anchoring and its availability for interactions were verified by probing with ConA, a lectin with high affinity for mannose 38. The interaction force curves had the signature negative peaks and ruptures similar to protein binding and release in Force Spectroscopy (Fig. 2B, lower panel,


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right image). Surfaces with assembled linkers alone did not show such interaction patterns with ConA (Fig. 2B, lower panel, left).

A

Confirmation of bifunctional linker synthesis

B

Confirmation of mannobiose anchoring

+ ATFB, SE

Cysteamine Hydroxysuccinimide ATFMB

UV

Mannobiose-anchored surface

Linker-attached surface

Cysteamine S-H

H-N-H

Contact angle= 460

T%

0

Contact angle= 120

N3 N-H

N3

ATFMB

C=O 1200

1700

2200

2700

Wavelength

3200

Approach Retract

2 1 0 -1

0

-2

50

100

150

200

Separation (nm)

-3

3700

cm-1

Approach Retract

2

Force (nN)

C=O

0

3

3

ATFB,SE Force (nN)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 0 -1

0

50

100

150

200

Separation (nm)

-2 -3

Mannobiose - ConA interaction

Linker - ConA interaction

Figure 2. Validation of bifunctional linker synthesis and mannobiose anchoring to gold surfaces. (A) (Top) Reaction mechanism between ATFB, SE and cysteamine to produce ATFMB. (Bottom) FTIR absorption spectrum for cysteamine (green), ATFB, SE (orange), and the reaction precipitate ATFMB (black). (B) (Top) Reaction scheme showing covalent attachment of mannobiose to the linker molecules assembled on gold surfaces. (Middle) Surface contact angle becomes more hydrophilic following mannobiose anchoring. (Bottom) Interaction of ConA coated AFM tips with linker- and mannobiose- anchored surfaces. The appearance of negative force peaks and release in the retraction curve indicates strong ConA binding with the mannobiosylated surface but not with the linker surface.

Sample

Atomic %

F/C

N 1s

C 1s

O 1s

S 2p

F 1s

Au 4f

XPS

Theoretical

A. ATFMB (linker) on Au

5.4 ± 0.1

29.2 ± 0.3

6.2 ± 0.7

1.9 ± 0.4

7.3 ± 2

50 ± 0.5

0.3 ± 0.1

0.4

B. Mannobiose + linker on Au

4.6± 0.9

35.1 ± 2.2

7.2 ± 0.8

1.6 ± 0.2

6 ± 1.3

45.4 ± 1

0.2 ± 0

0.2

Table 1: Relative surface-elemental composition from gold surfaces with ATFMB linker and mannobiose+linker monolayers obtained using XPS.

The assembly of linkers and the attachment of mannobiose on gold surfaces was further verified by surveying the surface elemental composition with XPS (Table 1). Both the survey and high-resolution spectra were consistent with the expected composition of linker-assembled and mannobiose-attached gold


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surfaces depicted in Fig. 2B. In particular, the F/C ratio for the two surfaces corresponded to the theoretical values expected from their respective chemical structures (Table 1). The high-resolution N1s spectra of the surfaces with ATFMB linkers had three peaks at binding energies corresponding to the latter’s amide group and the two nitrogen species (i.e., two end nitrogens and one middle nitrogen) in its terminal azide group (Suppl. Fig. 1A). As expected from the chemical composition of the ATFMB linkers, a 1:1 ratio was observed for the contribution from the amide and azide peaks (Suppl. Fig. 1A). A 1:2 ratio was observed for the contribution from the two nitrogen species in the azide functional group (Suppl. Figs. 1A). These characteristics of the N 1s spectra of ATFMB were also reported by Zorn et al 43 for similar linkers. When ATFMB linkers are reacted with mannobiose, the azide conversion to N-H is evident from the disappearance of the azide N1s peaks (Suppl. Fig. 1C)

Interaction between two opposing mannobiose monolayers The set up in Atomic Force Spectroscopy (AFM) for measuring the interactions between two mannobiose monolayers is shown in Fig. 3 (panel A). As the mannobiose-coated AFM tip moves towards a mannobiose-coated surface and then retracts, the cantilever bearing the tip deflects upward or downward depending on the monolayers’ resistance to indenting or separating (Fig. 3). The cantilever deflection (D) is related to the resisting force (F) via the spring constant; i.e. F = kD. In the case of opposing mannobiosylated monolayers, the approach and retraction force curves have distinct patterns, which give insight into the nature of their interactions (Fig. 3).


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Figure 3. Force curves and schematic of the interaction between two mannobiose monolayers. Representative force curves obtained in Atomic Force Spectroscopy when a mannobiose-coated tip approaches a mannobiose-coated surface and then retracts. The Y-axis shows the force (nN) experienced by the approaching and retracting cantilever, X-axis shows the separation between the cantilever and the surface. The force curves exhibit signature patterns of ‘water structuring’, break-through into mannobiose monolayers, strong (‘peak’) adhesion at the monolayer interface, and last rupture forces from remnant interactions. The top panel illustrates the potential interaction configurations in regions A- F of the force curves based on the separation between opposing monolayers and the cantilever resistance.

A mannobiose + linker layer on the AFM tip or surface is expected to have a length in the approximate range of 1.8 - 2.5 nm (~0.8 in the short axis and ~1.5 nm in the long axis expected for mannobiose by extrapolating those of glucose 44, and ~ 1nm for the stretched linker 45, 46. However, during the approach phase, a resistive force was observed developing at distances 5- 50 nanometers beyond the thickness of two monolayers (point B on the force curve). Espinosa-Marzal et al. 47 observed similar ‘long-distance’ resistance from glycoproteins adsorbed in a surface force apparatus and attributed it to the ‘structuring’ of water molecules by the surface-bound glycans. While we refer to this solvent layer that resists indentation as ‘structured water’, it has alternatively been referred to as ‘solvation’ or ‘hydration’ layer 48, 49, 50.


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As shown in Fig. 3, when the two approaching surfaces contact each other, a modest force build up occurs at near-constant separation (point C to D), followed by a step release in both the force and separation (point E). The build-up and release occurs two times, which could be coincident with the sequential interpenetration of the mannose tiers in the two monolayers (Panels D to F in Fig. 3). The step decrease in surface separation accompanying each force release is ~0.4 nm, which is on the order of a single mannose dimension along the short axis (46). In other words, two mannobiose monolayers do not compress uniformly like an elastic material, but by the interpenetration of their mannose tiers. The build-up and release of force with each interpenetration indicates that energy is required to break existing interactions within a mannose tier, but which is released when stabilizing interactions are restored after interpenetration. We refer to the first penetration force at the interface between the monolayers as the ‘mannobiose breakthrough force’ (Fig. 3B). During the retraction of the mannobiosylated surfaces, a peak negative force develops close to the pullout distance between the two monolayers (panel G in Figure 3), indicating a strong self-adhesion between the two layers. The ‘peak adhesion’ force gets released in one large step, and is followed by one to three smaller releases of remnant adhesion force. We record the last release of adhesion force as the ‘last rupture force’ (Fig. 3, panel H). The above experiment was performed with a relatively soft AFM cantilever (k ~ 0.06 N/m), but the patterns were still retained in experiments with stiffer cantilevers (k ~ 0.1 – 0.6 N/m) (Fig. 4). Linkerlinker (ATFMB-ATFMB) surfaces did not show water structuring, break-in, adhesion, and other features (Suppl. Fig 2), indicating these features arise from the mannobiose monolayer. However mannobiose monolayers anchored by thiolated-PEG linkers, instead of cysteamine, displayed interpenetration, selfadhesion, and water-structuring behavior (Suppl. Fig. 3), indicating that these properties are independent of linker chemistry. The experiment in Fig. 3 was performed on mannobiose monolayers in water medium without supplemented salt. The effect of salt and other characteristics of the water-structuring and peak adhesion properties are discussed in the sections below.

Water structuring properties of a mannobiosylated surface The approach of the mannobiosylated cantilever was resisted at distances further away from the mannobiosylated surface. Marzal et al. 47 reported a similar long-range resistance arising from glycancoated surfaces, due to the glycan hydrogen-bonding interactions ordering the water network around. We report the length of the water-structuring layer as the distance between the ‘contact’ (where resistance to approach first appears) and ‘mannobiose break-in’ (where the mannobiose monolayers contact) point


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(Fig. 3). The length of the structured water layer extended as far as 50 nm, with a mode of ~30 nm (Figs. 4A, D). The latter is in the range reported by Marzal et al. 47 for alpha-1-acid and antitrypsin glycans. The medium was supplemented with 150mM and 300mM of NaCl. With salt addition, the point of ‘contact’ moved towards the ‘mannobiose break-in’ point (Figs. 4A-C). The mode of water-structuring distances decreased from ~30 nm to ~1.2 nm in 150mM NaCl and to ~0.6 nm in 250 mM NaCl respectively (Figs. 4D-F). Salt ions are chaotropic, and their ion-dipole interactions are stronger and interfere with the hydrogen-bonding interactions between water molecules 51. Thus the above salt effect confirms that the long-distance resistance is due to a structuring of the water network induced by the anchored mannobiose.

Figure 4: Effect of salt on the water-structuring that develops between two mannobiose monolayers after 30 min incubation. (A-C) Representative AFS force curves showing the ‘contact point’ (i.e., the surface separation where the first resistive force appears) moving closer to the break-in point with increasing salt concentration. (D-F) Frequency distributions of the water-structuring span showing it significantly decreasing with increasing salt. Finescale distributions are shown in insets. (G-I) At low salt conditions, the water-structuring distance and the mannobiose break-through force correlate positively, but the correlation disappears at higher salt conditions with the water-structuring, but not the break-in force, becoming significantly reduced.


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If the solvent water-structuring is facilitated by the mannobiose residues, we would expect it to increase with the density and packing of the mannobiose monolayer 47. The latter are captured in the mannobiose break-in force. As shown in Fig. 4G in water medium, the water-structuring distance correlates strongly with the mannobiose break-in force (R2 = 0.9033). However, in the presence of salt ions, the correlation becomes weak because, while the water-structuring layer is severely reduced, the mannobiose break-in force is not (Figs. 4H, I). In fact, the distribution of break-in forces was not significantly altered by the salt supplementation (Suppl. Fig. 4). It appears that while the mannobiose monolayer imposes the waterstructuring effect, the disturbance of the latter by chaotropic agents does not significantly feedback into the integrity of its packing. We observed that the structured water layer develops over time as the sample rested in its experimental setup. For instance, the distribution of water-structuring distance was significantly less at 15 min compared to after 30 min (Fig. 5A). Accordingly, all water-structuring data presented for analysis were collected after resting the monolayers for at least 30 min (Fig. 4 and Figs. 5B- 5D). Since the structured water network forms over time, we would also expect it to rearrange over time to accommodate a moving body (i.e. the AFM tip). In other words, a faster-moving body would experience a higher resistance from structured-water due to the inertia involved in rearranging the water molecules. As expected, the waterstructuring distance increased with the approach velocity of an uncoated AFM tip (Fig. 5B). However, at approach velocities of 200 nm/sec and above, the cantilever deflection jumped to negative values in sections of the structured-water layer, indicating sudden drops in the hydrodynamic drag resisting tip approach (Figs. 5C, 5D). Marzal et al. reported similar jumps between approaching glycan-coated surface due to the possible sloughing off of layers of structured water which is unable to rearrange to accommodate the moving probe 47. It appears that there is a non-instantaneous relaxation time inherent to the rearrangements of the structured water, which manifests as biphasic dependence between the frictional drag and velocity of an approaching body. At velocities slower than the relaxation rate, the frictional drag increased with approach velocity; but at velocities faster than the relaxation rate, the frictional drag decreased with approach velocity due to possible ‘fracturing’ of the structured water layer.


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Figure 5: Time-dependent characteristics of the structured-water layer (A) The water-structuring distance between opposing mannobiose monolayers (on AFM tip and sample surface) measured within 15 min in plain water. The water-structuring distance increases to the levels in Fig. 4C by ~30 min. (B) The length of the water structuring layer on a mannobiose monolayer increases with the approach rate (i.e. the ramp rate) of an uncoated tip. (C) Representative force curve obtained when an uncoated AFM tip approaches a mannobiose monolayer at a higher ramp rate (200nm/sec). The cantilever deflection does not increase monotonically as it moves through the structured water layer but jumps to negative values indicating loss of hydrodynamic resistance through sections of the structured water, due to the latter possibly fracturing off. (D) Multiple approach curves showing the jump features in Fig. 5C.

Surface adhesion properties of opposing mannobiose monolayers. Strong negative peaks of adhesive forces were observed close to separations where retracting mannobiose layers are expected to pull out (position G in Fig. 3). Figure 6A shows the frequency distribution of the peak adhesive forces at different salt concentrations. In water, the distribution was multimodal with maxima appearing every ~ 1.2 – 1.5 nN intervals in the range between 0.2 and 6nN (Fig. 6A, maxima indicated by arrows). However, with salt supplementation, the distribution around the higher maxima decreased with values centering around the first maxima of ~1.1nN (Fig. 6A, red arrows).


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The distribution of last rupture forces was obtained for different salt concentrations (Fig. 6B). We note an increased incidence of zero rupture forces with increasing salt concentration. The frequency increased from 1% of all analyzed curves in water to 21% of analyzed curves in 150mM, and 51% in 250mM supplemented NaCl, respectively. The values of the non-zero rupture forces, however, were distributed normally, around a single mean of 0.3nN, which was not significantly affected by salt concentration. The distribution of last-rupture forces was analyzed at a finer scale around 250 pN. Interestingly, the finer values exhibited periodic clustering at every 25 +/- 3 pN in both water and salt medium (Fig. 6B, insets). This pattern was consistently reproduced in >1000 force-distance curves obtained using multiple AFM probes and on multiple days, suggesting that 25 +/- 3 pN could be the smallest unit of force due to single mannose-mannose unbinding. Our results distinguish four levels of mannose interaction. One is at the single mannose-mannose level, which has rupture forces of ~25 +/- 3 pN (Fig. 6B insets). The rupture values are in the range reported for single residue interactions in Lewisx trisaccharides (20 +/- 4pN)

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and sulfated disaccharides (30 +/-

6pN) 52. A second level of adhesion can be distinguished in the values of last-rupture forces that are centered around 250 pN, which could be due to the last-surviving cluster adhesions between the surfaces (Fig. 6B). The mannobiose cluster forces are within the range of adhesion forces reported for other glycan clusters (ex. 100 – 400 pN) for sulfated disaccharides 53 and 5-174 pN for trisaccharide Lewisx determinants (Gal1->4 [Fuc1->3] ->GlcNAc) 28.A third level of adhesion (peak adhesion) appears to be consistently centered as a prominent peak about 1.1nN (Fig. 6A, red arrows). Interestingly it has values similar to the mannobiose break-in force (Suppl. Fig. 4), and could possibly be due to the de-penetration or ‘break-out’ of the two monolayers. It is possible that the values of the break-in and break-out forces are determined by the initial area of contact between the monolayers on the AFM tip and surface, as opposed to a clustering preference of mannobiose residues. These three levels of mannose interactions (and the monolayer break-in force) are relatively unaffected by salt and its chaotropic effect. However, a fourth level of mannose adhesion was observed as higher multiples of the basal 1.1nN peak-adhesion (Fig. 6A, blue arrows). Similar to water-structuring, this force was attenuated by salt supplementation. It is possible that this fourth-level force involves larger swaths of monolayers that were brought into contact when the conical AFM tip continued indenting towards the surface. Like proteins, these higher-order forces appear stabilized or organized by hydrogen-bonding interactions with the solvent interface 54, 55.


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Figure 6: Effect of salt on peak adhesive and last-rupture forces (A) Frequency distribution of peak adhesive forces measured at the surface of two retracting alpha-1,4 mannobiose monolayers in medium with increasing salt concentrations. Values of adhesive forces centered around 1.1 nN, and frequency of higher values was diminished when the salt content was increased. (B) Frequency distribution of the last-rupture or final remnant adhesive forces between retracting mannobiose monolayers. The distribution of force values did not change with increasing salt, though the incidence of zero rupture force increased. The insets show the fine-scale distribution of the rupture forces between 200 and 300 pN. The force values clustered at intervals of 25 ± 3 pN (blue arrows), which could be the force due to the rupture of a single mannose-mannose adhesion.

We note that longer and polymers of mannose (as opposed to monolayers of mannobiose) also displayed self-adhesion forces with the traditional saw-tooth profile of force release occurring as interactions between mannose polymers were released (Suppl. Fig. 5).


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Adhesion of lactose-lactose and lactose-mannobiose surfaces In order to understand if the above self-adhesion and water-structuring properties were innate to mannobiose monolayers or could be reproduced in related disaccharides, we examined the interactions between lactose-lactose and mannobiose-lactose surfaces. Lactose is a diastereoisomer of mannobiose (Fig. 7A). Monolayers of lactose exhibited water-structuring similar to mannobiose layers (Fig. 7B, left), which was expected since water-structuring is a generic feature of oligosaccharides 47. However both lactose-lactose and lactose-mannobiose layers exhibited significantly lower ranges of self-adhesion force (~0-15 pN for lactose-lactose, ~0-360 pN for lactose-mannobiose, compared to ~0.2-5.5nN for mannobiose-mannobiose surfaces) and interpenetration behavior (Fig. 7B). The self-adhesion and interpenetration behavior appears specific to the structure of mannobiose and not solely determined by an oligosaccharide’s ability to structure water. The higher values of mannobiose-mannobiose self-interaction suggest that mannobiose surfaces can drive a self-recognition behavior, with the dissociation rate for mannobiose-mannobiose surfaces being significantly lower than that between mannose and other residues.

A Lactose (Galactose (β1à4) Glucose)

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Figure 7: Lactose-Lactose and Mannobiose-Lactose interactions (A) Chemical structure of alpha-1,4-mannobiose which is a disaccharide of mannose residues (left) and that of lactose molecule which is a disaccharide of glucose and galactose, and a diastereomer of mannobiose. (B) Approach


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force curves (blue) for lactose-lactose interaction (left) and lactose-mannobiose interaction (right) are smooth and do not show significanr monolayer interpenetration; whereas retraction curves (red) show significantly reduced to no self-adhesion forces (insets). Both surfaces exhibit significant water-structuring manifesting as a resistance to indentation (AFM ‘contact point’ in Fig. 7B) that appears well before the surfaces make true contact.

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Figure 8: Effect of free disaccharides on the interaction between opposing mannobiose monolayers. (A) Frequency distribution of peak adhesion forces and water-structuring (inset) between opposing mannobiose monolayers measured in water, and when supplemented with 6mM and 9mM free mannobiose, followed by rinsing with water to measure recovery. (B) Frequency distribution of peak adhesion forces and water-structuring (inset) between opposing mannobiose monolayers measured in water, and when supplemented with 6mM and 9mM free lactose, followed by rinsing with water to measure recovery. Interaction measurements were performed after 30 mins of incubation to allow free carbohydrates to settle and the water structuring layer to develop.


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Competitive inhibition by free disaccharide supplementation We tested if adding free mannobiose residues lowered the self-adhesion between mannobiose monolayers by competing for the same ‘adhesion sites’. Figure 8A summarizes the effect of mannobiose supplementation on the water-structuring and adhesion forces between opposing mannobiose surfaces. As discussed earlier the adhesion forces of mannobiose were distributed multimodally with peaks occurring at multiples of ~1.1nN (Fig. 8A). With increasing free mannobiose in the medium, there was progressive muting of the higher peaks, with values coalescing around the basal peak at ~1.1nN (Fig. 8A). There was also a significant decrease in water-structuring (Fig. 8A, inset). The latter is expected since free oligosaccharide can act like chaotropes; the solvation spheres of the diffusing disaccharide interfere with the hydrogen bonding in the structured water. In fact, the combined observations of [i] attenuated waterstructuring, [ii] attenuated fourth-level adhesion (i.e. the higher adhesion peaks), and [iii] unaffected third-level adhesion (i.e. basal adhesion peak and monolayer break-out force) are reminiscent of those obtained observed following salt supplementation (Fig. 6). It appears the supplemented mannobiose is unable to penetrate the monolayer and competitively inhibit the self-interactions. Instead it influences monolayer biophysics in its role as a chaotropic solute interfering with the solvent network. This finding was tested by supplementing the medium with free lactose (Fig. 8B). Lactose would be a chaotrope disaccharide like mannobiose, but one which does not interact with the mannobiose monolayer (Fig. 7B). Lactose supplementation also muted water structuring and fourth-level adhesions, with adhesions coalescing around the basal peak of 1.1nN (Fig. 8B). Rinsing of the free oligosaccharides in both cases restored the water-structuring and the fourth-level adhesive interactions. The similarity in the effects of supplementing NaCl, lactose and mannobiose, indicate that free mannobiose was not competitively inhibiting the lateral interactions within a monolayer, but was acting as a generic chaotrope of solvent interactions. Interestingly the packing interactions between mannobiose moieties within the monolayer (as captured by the monolayer break-out and break-in force) are found relatively resilient to the action of modest chaotropes.

Mannose residues are found on the outer surface of the glycan shields of several virulent pathogens and are recognized by front-line players of the immune system. We pose the question if the mannose preponderance affords a biophysical advantage to pathogens, despite the residues being the recognition target of the host immune system. Using Atomic Force Spectroscopy we interrogated the interactions of a monolayer of mannobiose (disaccharide of mannose) as the simplest 3D model of a mannosylated glycan patch or shield. The layer was generated by photochemically-linking mannobiose to the exposed arylazide end-groups of a self-assembled monolayer; and verified by FTIR analysis of the anchoring linker, as


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well as by the significant change in contact angle, ConA binding, and indentation profile of the mannobiosylated monolayer compared to the monolayer with unreacted azide-groups or to the surface without monolayer. The approach of mannobiose monolayers was resisted at distances much larger than the contact distance of the two monolayers. The resistance appears to arise from an ordering of the water arrangement imposed by the mannobiose surface, and its correlated with density and organization of monolayer packing as captured via the mannobiose break-through force. We refer to this long-distance resistance, previously observed with other glycans, as water structuring 47. The water-structuring resistance ranged from 5 – 50nm in water, but was reduced by freely diffusing salt ions and oligosaccharides, both of which are chaotropic agents and disturb the hydrogen-bonding network of structured water (Figs. 4 and 8). The reduction in water-structuring by free oligosaccharides confirms that this long-distance resistance is not arising from unanchored molecules that had settled on the surface. There appears to be a noninstantaneous relaxation time associated with the generation and rearrangement of the structured-water network. The water-structuring layer organizes over the span of ~30 minutes and imposes a biphasic resistance to an approaching body. Initially, an approaching body experiences drag resistance farther and farther from the surface as its velocity increases (Fig. 5B). However, at higher approach velocities, the drag resistance drops in sections of the approach and the approaching body ‘jumps’ towards the surface. This could be due to the fissuring of sections of structured-water which are unable to rearrange at the rate of the moving body. Espinosa-Marzal et al. 47 reported similar jumps by surfaces approaching a glycan layer. The ability to induce water structuring at physiological ionic strength may have biological significance. The water structuring in the immediate vicinity of the surface may be important for retaining a hydrated layer around the pathogens and creating an ‘exclusion zone’ 56, 57.The hydrogen-bonding network could also serve to orient approaching ligands for binding to receptors. An interesting effect of the waterstructuring layer is that it could amplify differences in the approach velocity of particles; it would further slowdown the slow-moving particles (by posing an increased frictional resistance) and speed up faster moving ones (by possibly cleaving off to create zones of depleted friction). In fact, one would expect this biphasic drag to preferentially exclude or attract larger particles based on their speed; larger particles, such as cells or antibodies, will require a greater restructuring of the solvent network, and therefore experience larger frictional drags or larger jumps. At physiological salt concentrations, 2.5 nm is about the length of the structured water layer we detect with our experimental setup. We note that the fine detection of the beginning of the structured-water resistance is impeded in our experiments by the noise in


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the AFM signal; our reported distance may therefore be smaller than that experienced by cells and macromolecules diffusing in the vicinity of the glycan layer. Some cell-surface glycans exhibit self-specific adhesions referred to as Carbohydrate-Carbohydrate interactions (CCIs). Mannobiose monolayers also exhibit self-specific adhesions, but with additional rich features (Fig. 3) not discussed in other reports. As the two mannobiosylated surfaces contact each other, we observe a force build-up at constant separation, followed by both a force release and reduction in separation (Fig. 3). Instead of being uniformly compressed, opposing mannobiose monolayers appear to penetrate. Penetration occurs via the breaking of existing bonds and reforming with new bonds in a mannose tier, which manifests as a force build up and release. These observations indicate that there are lateral packing interactions between the mannose in a monolayer, and about 1.1 nN of force needs to be supplied to break through it (Suppl. Fig. 4). ~1.1nN is also the amount of basal force required to break out from the interactions (Fig.6). The consistency in the distribution of mannose break-in forces even in the presence of salt (Suppl. Fig. 4) could reflect either a clustering preference among mannose residues or simply the initial area of contact between the AFM tip and surface. Similar interpenetration has not been reported in other glycans. Mannose residues do not exhibit such interpenetration when paired with another sugar residue like lactose. The stress-induced and self-specific interpenetration of mannobiosemannobiose residues can manifest as a self-latching dynamics between mannose-rich patches or shields on pathogens under high-shear situations. Researchers have described protein-based catch bonds in bacteria and leukocytes that get strengthened under tensile and shear forces 58. Our results suggest a possible carbohydrate-based ‘latch’ dynamics between pathogens that is facilitated by mannose patches under high-force conditions. The mannose self-adhesive interactions appear facilitated by its structure and not just by its ability to foster hydrogen-bonding interactions. Lactose, a diastereoisomer of mannobiose that also structures water, did not exhibit self-adhesion (Fig. 7). Mannobiose-lactose surfaces also exhibited significantly lower adhesion forces. The difference between homotypic and heterotypic adhesion could translate into a self-selecting and self-sorting behavior between glycosylated entities bearing mannose patches and mannose polymers (Suppl. Fig.5). For instance, mannose receptors in lungs bear terminal mannose residues, whereas mannose receptors in other organs have sialic acid residues 15; it remains to be studied if there is a pathogen-recognition imperative underlying these distributions. Also, it has been reported that the level of high-mannose surface glycans are elevated during breast cancer progression; which begets the question if these polymers facilitate a self-recognition behavior during cancer metastasis 59


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An interesting observation was that the hydrogen-bonding interactions of mannose that promote waterstructuring were not coupled to its hydrogen-bonding interactions that promote its self-adhesion. The former were disrupted by diffusing ions (Figs 4D-F) and disaccharides (Fig 8), whereas the mannosemannose interactions at the molecular-, cluster-, and monolayer- level (‘break-in’ and basal ‘break-out’), were relatively unaffected by the chaotropic effect of the supplemented salt and sugars. It appears that a mannose patch could afford some level of protection from chaotropes to cell surfaces. Chaotropes did affect a higher fourth-level of force that appears to involve larger patches of the monolayer, possibly ordered or routed through the solvent network. Finally, we note that our analysis has not considered the osmotic effects resulting from the added ions or disaccharides. However, a solvent efflux due to hyperosmotic effects is expected to shrink mannose packing and alter interaction strengths, which we did not observe to a significant degree (Fig. 6). We speculate that a mannosylated surface could afford several advantages to a pathogen adapting to a harsh environment and becoming virulent. The Cryptococcus neoformans, for instance, develops a capsule, which has >70% mannose content in conditions of nutrient or pH limitation 60.They also develop the capsule upon infecting a host, at which point the capsule appears to help them evade the immune system; pathogens with smaller capsules are rapidly cleared by the immune system 61, 62. It is possible that the mannose distribution on pathogens could have offered evolutionary advantages for their survival, leading to the elaborate system of mannose lectins developed by the host immune system. CONCLUSIONS Several viruses and pathogen develop capsules which are rich in surface mannose during infection or when exposed to harsh conditions. We performed force spectroscopy on a mannobiose monolayer to understand the physical properties conferred by such a surface. Strong hydrogen-bonding interactions between the mannobiose layer and the solvent manifest as long-range forces that oppose approaching particles, but which disrupt at higher approach velocities and get attenuated at higher salt concentrations. The strong hydrogen-bonding between mannobiose residues manifest as a resistance to the interpenetration of opposing monolayers, and upon penetration resist the break out of the two layers. Salt concentrations do not attenuate a basal level of interpenetration and self-adhesion forces. These findings suggest that developing a mannose-rich capsule could offer advantages for pathogen survival and virulence.

Acknowledgements


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We are grateful to all Howard University faculty and lab managers who generously gave access and assistance with instrumentation for this work, particularly Dr. J.W. Mitchell, Dr. Oladapo Bakare, Mr. Andy Hai-ting, Mr. James Griffin, Mr. Crawford Taylor and Mr. Anthony Gomez. We also thank faculty and researchers who graciously commented on the manuscript and engaged in insightful discussions at various stages of the project: Dr. Matthew George Jr., Dr. W. Malcolm Byrnes, Dr. Eric Walters, Dr. Tina Brower-Thomas, Dr. Saswati Basu, Dr. Oladapo Bakare, Dr. Anna Allen, and Dr. Lystranne Maynard-Smith. We are also grateful to Dr. Karen Gaskell of the University of Maryland Surface Analysis Center for assistance with X-Ray Photoelectron Spectroscopy. This work was financially supported by the National Science Foundation under grant no. 1407891 awarded to Dr. Preethi Chandran and by a mini-grant awarded to Preethi Chandran under NSF grant no. 1208880 (PI: Dr. Sonya Smith)


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REFERENCES 1. Doering, T. L. A unique α-1, 3 mannosyltransferase of the pathogenic fungus Cryptococcus neoformans. Journal of bacteriology 1999, 181 (17), 5482-5488. 2. Ibuki, M.; Kovacs-Nolan, J.; Fukui, K.; Kanatani, H.; Mine, Y. β 1-4 mannobiose enhances Salmonella-killing activity and activates innate immune responses in chicken macrophages. Veterinary immunology and immunopathology 2011, 139 (2), 289-295. 3. Romani, L. Immunity to fungal infections. Nat Rev Immunol 2004, 4 (1), 11-24. 4. Levitz, S. M. Innate Recognition of Fungal Cell Walls. PLoS Pathog 2010, 6 (4), e1000758. 5. Meyer-Wentrup, F.; Cambi, A.; Figdor, C. G.; Adema, G. J. Detection of Fungi by Mannose-based Recognition Receptors. In Immunology of Fungal Infections; Springer, 2007, pp 293-307. 6. Rodrigues, M. L.; Nimrichter, L.; Cordero, R. J. B.; Casadevall, A. Fungal Polysaccharides: Biological Activity Beyond the Usual Structural Properties. Frontiers in Microbiology 2011, 2, 171. 7. Cottrell, T. R.; Griffith, C. L.; Liu, H.; Nenninger, A. A.; Doering, T. L. The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis. Eukaryotic cell 2007, 6 (5), 776-785. 8. Michelow, I. C.; Lear, C.; Scully, C.; Prugar, L. I.; Longley, C. B.; Yantosca, L. M.; Ji, X.; Karpel, M.; Brudner, M.; Takahashi, K. High-dose mannose-binding lectin therapy for Ebola virus infection. Journal of Infectious Diseases 2011, 203 (2), 175-179. 9. Miller, J. L.; M deWet, B. J.; Martinez-Pomares, L.; Radcliffe, C. M.; Dwek, R. A.; Rudd, P. M.; Gordon, S. The mannose receptor mediates dengue virus infection of macrophages. PLoS Pathog 2008, 4 (2), e17. 10. Ji, X.; Gewurz, H.; Spear, G. T. Mannose binding lectin (MBL) and HIV. Molecular immunology 2005, 42 (2), 145-152. 11. Krokhin, O.; Li, Y.; Andonov, A.; Feldmann, H.; Flick, R.; Jones, S.; Stroeher, U.; Bastien, N.; Dasuri, K. V.; Cheng, K. Mass Spectrometric Characterization of Proteins from the SARS Virus A Preliminary Report. Molecular & Cellular Proteomics 2003, 2 (5), 346-356. 12. Park, I. Y.; Kim, I. Y.; Yoo, M. K.; Choi, Y. J.; Cho, M.-H.; Cho, C. S. Mannosylated polyethylenimine coupled mesoporous silica nanoparticles for receptor-mediated gene delivery. International journal of pharmaceutics 2008, 359 (1), 280-287. 13. Kim, T. H.; Nah, J. W.; Cho, M.-H.; Park, T. G.; Cho, C. S. Receptor-mediated gene delivery into antigen presenting cells using mannosylated chitosan/DNA nanoparticles. Journal of nanoscience and nanotechnology 2006, 6 (9-1), 2796-2803. 14. Kelly, C.; Jefferies, C.; Cryan, S.-A. Targeted liposomal drug delivery to monocytes and macrophages. Journal of drug delivery 2010, 2011. 15. Su, Y.; Bakker, T.; Harris, J.; Tsang, C.; Brown, G. D.; Wormald, M. R.; Gordon, S.; Dwek, R. A.; Rudd, P. M.; Martinez-Pomares, L. Glycosylation influences the lectin activities of the macrophage mannose receptor. Journal of Biological Chemistry 2005, 280 (38), 3281132820. 16. Martinez-Pomares, L. The mannose receptor. Journal of leukocyte biology 2012, 92 (6), 1177-1186.


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17. Schlesinger, L. Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors. The Journal of Immunology 1993, 150 (7), 2920-2930. 18. Sallusto, F.; Cella, M.; Danieli, C.; Lanzavecchia, A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. The Journal of Experimental Medicine 1995, 182 (2), 389-400. 19. Turner, M. W. The role of mannose-binding lectin in health and disease. Molecular Immunology 2003, 40 (7), 423-429. 20. Ji, X.; Olinger, G. G.; Aris, S.; Chen, Y.; Gewurz, H.; Spear, G. T. Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization. Journal of General Virology 2005, 86 (9), 2535-2542. 21. Bucior, I.; Scheuring, S.; Engel, A.; Burger, M. M. Carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition. J Cell Biol 2004, 165 (4), 529-37. 22. Cui, L.; Johkura, K.; Yue, F.; Ogiwara, N.; Okouchi, Y.; Asanuma, K.; Sasaki, K. Spatial distribution and initial changes of SSEA-1 and other cell adhesion-related molecules on mouse embryonic stem cells before and during differentiation. Journal of Histochemistry & Cytochemistry 2004, 52 (11), 1447-1457. 23. Song, Y.; Withers, D. A.; Hakomori, S.-i. Globoside-dependent adhesion of human embryonal carcinoma cells, based on carbohydrate-carbohydrate interaction, initiates signal transduction and induces enhanced activity of transcription factors AP1 and CREB. Journal of Biological Chemistry 1998, 273 (5), 2517-2525. 24. Hakomori, S.-i. Carbohydrate-carbohydrate interaction as an initial step in cell recognition. Pure and applied chemistry 1991, 63 (4), 473-482. 25. Garcia-Manyes, S.; Bucior, I.; Ros, R.; Anselmetti, D.; Sanz, F.; Burger, M. M.; Fernandez-Busquets, X. Proteoglycan mechanics studied by single-molecule force spectroscopy of allotypic cell adhesion glycans. Journal of Biological Chemistry 2006, 281 (9), 5992-5999. 26. Spillmann, D. Carbohydrates in cellular recognition: from leucine-zipper to sugar-zipper? Glycoconjugate journal 1994, 11 (3), 169-171. 27. Popescu, O.; Checiu, I.; Gherghel, P.; Simon, Z.; Misevic, G. Quantitative and qualitative approach of glycan-glycan interactions in marine sponges. Biochimie 2003, 85 (1), 181-188. 28. Tromas, C.; Rojo, J.; de la Fuente, J. M.; Barrientos, A. G.; García, R.; Penadés, S. Adhesion forces between Lewisx determinant antigens as measured by atomic force microscopy. Angewandte Chemie International Edition 2001, 40 (16), 3052-3055. 29. Popescu, O.; Checiu, I.; Gherghel, P.; Simon, Z.; Misevic, G. N. Quantitative and qualitative approach of glycan-glycan interactions in marine sponges. Biochimie 2003, 85 (1–2), 181-188. 30. Misevic, G. N.; Burger, M. Carbohydrate-carbohydrate interactions of a novel acidic glycan can mediate sponge cell adhesion. Journal of Biological Chemistry 1993, 268 (7), 49224929. 31. Eggens, I.; Fenderson, B. A.; Toyokuni, T.; Hakomori, S.-i. A role of carbohydratecarbohydrate interaction in the process of specific cell recognition during embryogenesis and organogenesis: a preliminary note. Biochemical and biophysical research communications 1989, 158 (3), 913-920.


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32. Rojo, J.; Morales, J. C.; Penadés, S. Carbohydrate-carbohydrate interactions in biological and model systems. In Host-Guest Chemistry; Springer, 2002, pp 45-92. 33. Zaragoza, O.; Rodrigues, M. L.; De Jesus, M.; Frases, S.; Dadachova, E.; Casadevall, A. The capsule of the fungal pathogen Cryptococcus neoformans. Advances in applied microbiology 2009, 68, 133-216. 34. Curtis KA, M. D., Millard P, Basu S, Horkar F, Chandran PL. Unusual Salt and pH Induced changes in polyethylenimine solution. PLoS One 2016, 11 (9). 35. Malaisamy, R.; Lepak, L.; Spencer, M.; Jones, K. L. Surface modification of porous alumina membranes by collagen layers: Performance and characterization. Separation and Purification Technology 2013, 115, 114-122. 36. Nia, H. T.; Ortiz, C.; Grodzinsky, A. Aggrecan: approaches to study biophysical and biomechanical properties. Glycosaminoglycans: Chemistry and Biology 2015, 221-237. 37. Madwar, C.; Chu Kwan, W.; Deng, L.; Ramström, O.; Schmidt, R.; Zou, S.; Cuccia, L. A. Perfluorophenyl Azide Immobilization Chemistry for Single Molecule Force Spectroscopy of the Concanavalin A/Mannose Interaction. Langmuir 2010, 26 (22), 16677-16680. 38. Wang, X.; Ramström, O.; Yan, M. A photochemically initiated chemistry for coupling underivatized carbohydrates to gold nanoparticles. Journal of materials chemistry 2009, 19 (47), 8944-8949. 39. Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Selfassembled monolayers of thiolates on metals as a form of nanotechnology. Chemical reviews 2005, 105 (4), 1103-1170. 40. Bain, C. D.; Whitesides, G. M. Molecular-level control over surface order in selfassembled monolayer films of thiols on gold. Science 1988, 240 (4848), 62-63. 41. Hoypierres, J.; Dulong, V.; Rihouey, C.; Alexandre, S.; Picton, L.; Thébault, P. Two Methods for One-Point Anchoring of a Linear Polysaccharide on a Gold Surface. Langmuir 2015, 31 (1), 254-261. 42. Miodek, A.; Regan, E. M.; Bhalla, N.; Hopkins, N. A. E.; Goodchild, S. A.; Estrela, P. Optimisation and Characterisation of Anti-Fouling Ternary SAM Layers for Impedance-Based Aptasensors. Sensors 2015, 15 (10), 25015-25032. 43. Zorn, G.; Liu, L.-H.; Árnadóttir, L.; Wang, H.; Gamble, L. J.; Castner, D. G.; Yan, M. Xray photoelectron spectroscopy investigation of the nitrogen species in photoactive perfluorophenylazide-modified surfaces. The Journal of Physical Chemistry C 2013, 118 (1), 376-383. 44. Lourvanij, K. Partial Dehydration of Glucose to Oxygenated Hydrocarbons in MolecularSieving Catalysts. 1995. 45. Speight, J. G. Lange's handbook of chemistry; McGraw-Hill New York2005; Vol. 1. 46. Costain, C. C.; Stoicheff, B. P. Microwave spectrum, molecular structure of vinyl cyanide and a summary of CC, CH bond lengths in simple molecules. The Journal of Chemical Physics 1959, 30 (3), 777-782. 47. Espinosa-Marzal, R. M.; Fontani, G.; Reusch, F. B.; Roba, M.; Spencer, N. D.; Crockett, R. Sugars communicate through water: oriented glycans induce water structuring. Biophysical journal 2013, 104 (12), 2686-2694. 48. Vedamuthu, M.; Singh, S.; Robinson, G. W. Properties of liquid water: origin of the density anomalies. The Journal of Physical Chemistry 1994, 98 (9), 2222-2230. 49. Chaplin, M. A proposal for the structuring of water. Biophysical chemistry 2000, 83 (3), 211-221.


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50. Ebbinghaus, S.; Kim, S. J.; Heyden, M.; Yu, X.; Heugen, U.; Gruebele, M.; Leitner, D. M.; Havenith, M. An extended dynamical hydration shell around proteins. Proceedings of the National Academy of Sciences 2007, 104 (52), 20749-20752. 51. Hribar, B.; Southall, N. T.; Vlachy, V.; Dill, K. A. How ions affect the structure of water. Journal of the American Chemical Society 2002, 124 (41), 12302-12311. 52. De Souza, A. C.; Ganchev, D. N.; Snel, M. M.; van der Eerden, J. P.; Vliegenthart, J. F.; Kamerling, J. P. Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge Microciona prolifera. Glycoconjugate journal 2009, 26 (4), 457-465. 53. Lorenz, B. r.; Álvarez de Cienfuegos, L.; Oelkers, M.; Kriemen, E.; Brand, C.; Stephan, M.; Sunnick, E.; Yüksel, D.; Kalsani, V.; Kumar, K. Model system for cell adhesion mediated by weak carbohydrate–carbohydrate interactions. Journal of the American Chemical Society 2012, 134 (7), 3326-3329. 54. Cacace, M.; Landau, E.; Ramsden, J. The Hofmeister series: salt and solvent effects on interfacial phenomena. Quarterly reviews of biophysics 1997, 30 (03), 241-277. 55. Tarek, M.; Tobias, D. Role of protein-water hydrogen bond dynamics in the protein dynamical transition. Physical Review Letters 2002, 88 (13), 138101. 56. Zheng, J.-m.; Chin, W.-C.; Khijniak, E.; Pollack, G. H. Surfaces and interfacial water: evidence that hydrophilic surfaces have long-range impact. Advances in colloid and interface science 2006, 127 (1), 19-27. 57. Pollack, G. Cells, gels and the engines of life. 2001. 58. Forero, M.; Thomas, W. E.; Bland, C.; Nilsson, L. M.; Sokurenko, E. V.; Vogel, V. A catch-bond based nanoadhesive sensitive to shear stress. Nano Letters 2004, 4 (9), 1593-1597. 59. De Leoz, M. L. A.; Young, L. J.; An, H. J.; Kronewitter, S. R.; Kim, J.; Miyamoto, S.; Borowsky, A. D.; Chew, H. K.; Lebrilla, C. B. High-mannose glycans are elevated during breast cancer progression. Molecular & Cellular Proteomics 2011, 10 (1), M110. 002717. 60. Vartivarian, S. E.; Anaissie, E. J.; Cowart, R. E.; Sprigg, H. A.; Tingler, M. J.; Jacobson, E. S. Regulation of cryptococcal capsular polysaccharide by iron. Journal of Infectious Diseases 1993, 167 (1), 186-190. 61. Blackstock, R.; Buchanan, K. L.; Adesina, A. M.; Murphy, J. Differential Regulation of Immune Responses by Highly and Weakly Virulent Cryptococcus neoformansIsolates. Infection and immunity 1999, 67 (7), 3601-3609. 62. Blackstock, R.; Murphy, J. W. Secretion of the C3 component of complement by peritoneal cells cultured with encapsulated Cryptococcus neoformans. Infection and immunity 1997, 65 (10), 4114-4121.

Table of Content


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

Page 28 of 34

Page 29 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Table of Content


Confirmation of bifunctional linker synthesis

Confirmation of mannobiose anchoring

Page 30 of 34

+ Cysteamine Hydroxysuccinimide ATFMB

UV

1200

Mannobiose-anchored surface

Linker-attached surface

Cysteamine S-H

H-N-H

Contact angle= 460 ± 20

Contact angle= 120 ± 20 3

3

ATFB,SE N3 N-H

N3

ATFMB

C=O 1700

2200

2700

Wavelength cm-1

3200

Force (nN)

C=O

Approach Retract

2 1 0 -1 -2 -3

0

50

100

150

Approach Retract

2

200

Separation (nm)

3700 ACS Paragon Plus Environment Linker - ConA interaction

Force (nN)

SE

T%

1 2 3 4 5 6 7 ATFB, 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Langmuir

1 0 -1 -2

0

50

100

150

200

Separation (nm)

-3

Mannobiose - ConA interaction

Page 31 of 34

Force (nN)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26A 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

AFM cantilever AFM tip Linker (~1.1nm) Mannobiose (~1.7nm) Water structuring AFM surface B

3

C

E

D

F

Mannobiose break through force

H

G

Approach

2

Retract F E D Water C structuring B

1

A 0 0

5

10

-1 -2

G

H

Peak adhesive force

Separation (nm)


15 Last rupture force

20

Langmuir

5

10 15 20 25 30 Separation (nm)

-2

0

0

-2

0

5

10 15 20 25 30

Separation (nm)

Frequency

2

0.8

Force (nN)

F 1

1

Mannobiose break through force (nN)

42.6

32.6

Water structuring distance (nm)

C 3 Contact point

42.6

0

0.2 2.5

0

0.4

H

0.3 0.2 0.1 0

0.6 0.4

I

0.2 0

2.5

Force (nN)

Contact point

1

Frequency

0.6

2

-1

0.3 0.2 0.1 0

Mannobiose break through force (nN)

E 0.8

B3

150mM NaCl


32.6

Separation (nm)

0.4 1.2 2 2.8 3.6 4.4

-3

22.6

0.0

-2

22.6

10 15 20 25 30

0.2 0.6 1 1.4 1.8 2.2

5

12.6

-1 0

0.1

12.6

0

0.2

2.5

Contact point

Frequency

Retract

1

-1

G

D0.3



Manobiose break through force (nN)

Approach

3 2

Force (nN)

0mM NaCl

A

250mM NaCl

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 34

2 1.5 1 0.5 0

y = 0.0381x R² = 0.9033

0 10 20 30 40 50 Water structuring distance (nm) 2 1.5 1 0.5

y = 0.0046x + 1.0254 R² = 0.02551

0 0 10 20 30 40 50 Water structuring distance (nm) 2 1.5

y = 0.0077x + 0.5965 R² = 0.06399

1 0.5 0 0 10 20 30 40 50 Water structuring distance (nm)

B

0.2 0.1 0

2.5 7.6 12.6 17.6 22.6 27.6 32.6 37.6 42.6 47.6

Frequency

0.3

50

Avg. water structuring length (nm)

A 0.4

C 2.0

Approach Loss of cantilever deflection indicating loss of hydrodynamic resistance contact point

1.0 0.5

20 10 0 20

0

50 100 Separation (nm)

150

50

100

Ramp rate (nm/s)

1 0 -1

0

-2 -3

0.0 -0.5

30

D 2

Ramp rate = 200 nm/sec

1.5

40

-10


Force (nN)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Force (nN)

Page 33 of 34

200

-4 -5


50

100

Separation (nm) Sample approach curves at ramp rate = 200 nm/sec

Langmuir

Force (pN)

0.95

0.85

285

280

263

241

219

0.95

0.85

0.75

0.65

0.55

0.45

Peak adhesive forces (nN)

0.35

0

0.2 0.8 1.4 2.0 2.6 3.2 3.8 4.4 5.0 5.6

0.25

0

Force (pN)

0.1

0.15

0.05

0.2

0.05

0.1

0.75

0.65

0

0.15

Last rupture forces (nN) 0.3

0.2 0.1 0.0

0.1

0.65

0.55

0.45

0.2 0.8 1.4 2.0 2.6 3.2 3.8 4.4 5.0 5.6 Peak adhesive forces (nN)

0

0.35

0

Force (pN)

0.25

0.05

Last rupture forces (nN)


0.95

0.1

0.85

Frequency

0.2

219 230 241 255 263 280 285

0.15

In 250mM NaCl 0.4 0.3

0.75

In 250mM NaCl

0.15

0.2

0.3

In 150mM NaCl 0.2 0.1

0.3

In 150mM NaCl Frequency

Frequency

0.2

Last rupture forces (nN)

210

0.2 0.8 1.4 2.0 2.6 3.2 3.8 4.4 5.0 5.6 Peak adhesive forces (nN)

0.55

0

0

0.45

0.1

0.35

0.05

0.2

0.15

0.1

0.05

Frequency

Frequency

0.15

0.3 0.2 0.1 0

In water

210 218 222 225 249 250 257 277 280 289

0.3

In water

0.05

0.2

B

0.25

A

Frequency

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 34

Mannose Surfaces Exhibit Self-Latching, Water Structuring, and

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