Multivalent Affidendrons with High Affinity and Specificity towards

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Biological and Medical Applications of Materials and Interfaces

Multivalent Affidendrons with High Affinity and Specificity towards Staphylococcus aureus as Versatile Tools for Modulating Multicellular Behaviors Petar Vukojicic, Ghislaine Behar, Maun H. Tawara, Marcos FernandezVillamarin, Frédéric Pecorari, Eduardo Fernandez-Megia, and Barbara Mouratou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05702 • Publication Date (Web): 23 May 2019 Downloaded from http://pubs.acs.org on May 27, 2019

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ACS Applied Materials & Interfaces

Multivalent Affidendrons with High Affinity and Specificity Versatile

towards Staphylococcus Tools

for

Modulating

aureus

as

Multicellular

Behaviors Petar Vukojicic,1,2 Ghislaine Béhar,1 Maun H. Tawara,2 Marcos Fernandez-Villamarin,2 Frédéric Pecorari,1 Eduardo Fernandez-Megia,*,2 and Barbara Mouratou*,1 1. CRCINA, INSERM, CNRS, Université d'Angers, Université de Nantes, Nantes, France. 2. Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS) and Departamento de Química Orgánica, Universidade de Santiago de Compostela, Jenaro de la Fuente s/n, 15782 Santiago de Compostela, Spain.

ACS Paragon Plus Environment

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ABSTRACT

Multivalency is a widely occurring natural phenomenon often exploited in nanotechnology to enhance biorecognition. We report the preparation and characterization of versatile, multivalent Affitin-dendrimer conjugates (Affidendrons) showcased by a set targeting Staphylococcus aureus (S. aureus), an opportunistic pathogen causing numerous hospital- and community-acquired infections. Affitins are small affinity proteins characterized by higher stability and lower costeffective production than antibodies. The strategy presented provides a platform for the rational design of multivalent nanodevices that, retaining the ability of Affitins to recognize their target with high specificity, achieve a largely enhanced affinity. Affidendrons with precisely designed size and valency have been exploited to modulate complex multicellular behaviors of S. aureus, such as agglutination and biofilm formation. Agglutination assays showed that Affidendrons rapidly cross-link S. aureus strains with high bacterial cell selectivity. Moreover, remarkably low concentrations of Affidendrons were able to effectively prevent biofilm formation. Overall, Affidendrons represent a promising platform for the rapid and selective pathogen identification, infection imaging, and theranostic applications.

KEYWORDS: Affitins, Affidendrons, conjugation, GATG dendrimers, Staphylococcus aureus


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INTRODUCTION Multivalency is widely encountered throughout nature as a hallmark of enhanced affinity and a tool to modulate recognition and binding. The multivalent presentation of ligands and receptors results in much higher binding affinities and specificities than monovalent interactions.1,2 This phenomenon is often exploited in nanotechnology where multivalent scaffolds allow for decoration with multiple copies of targeting moieties such as antibodies,3,4 peptides,5,6 sugars7,8 or other small molecules.9,10 The resulting multivalent nanostructures have proven useful for imaging, diagnostic and therapeutic purposes,3-5, 7-9 as well as bacterial clustering11 and cell activation.12 Since the rational design of multivalent nanoparticles enables high precision over molecular and cellular phenomena, versatility and control of size, structure, surface functionalization and target specificity are desired. Interestingly, these properties of targeted nanoparticles also determine their pharmacokinetics and pharmacodynamics in vivo.13,14 Among multivalent nanoplatforms, dendrimers are especially attractive for various biomedical uses. 8,11,13 They are monodisperse, unimolecular nanoparticles that introduce small-molecule level of control into the design of nanostructures.15 Besides, both particle size and number of branches can be controlled by the generation of dendrimers. We chose Gallic Acid-Triethylene Glycol (GATG) dendrimers as a platform for creating highly customizable targeted nanoparticles due to their straightforward synthesis, high tunability and abundance of clickable azide groups on their surface.16 Traditionally, antibodies have been the most widely used targeting moieties, since they achieve high specificities and affinities for a variety of targets.3,4,17-19 Lately, alternative scaffolds have been sought to combine these properties with a much improved stability, smaller size and cost-


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effective production.20 Affitins, derived from small archaeal Sul7d proteins, such as Sac7d, consisting of only 66 amino acids, combine these requirements.21-23 Affitins blend a straightforward procedure for target-driven selection of new binders,21,23 and a flexible, smart design with pre-selected specificity for potentially any molecular target. This has resulted in a wide range of available Affitins.21,22,24-26 We have previously described the generation of Affitin C5 that recognizes the staphyloccocal protein A (SpA)26 - a protein widely expressed on the surface of S. aureus that plays important roles in bacterial virulence, immune evasion mechanisms, bacterial self-aggregation and biofilm formation.27,28 We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones with a nanomolar range affinity.26 Here, we report a versatile strategy for decorating dendrimers with Affitins, which allows for finetuning of the size, multivalency, surface functionality and recognition properties of the resulting Affidendron conjugates. Affidendrons retain the ability of Affitins to bind SpA both on a protein level and on the surface of bacteria with an affinity several orders of magnitude higher. Furthermore, we demonstrate the ability of Affidendrons to initiate a highly specific agglutination of S. aureus, in a concentration-dependent manner, as well as their properties to inhibit biofilm formation.

RESULTS AND DISCUSSION GATG dendrimers of third and fourth generation (2[G3]-N3 and 2[G4]-N3),29-31 containing 54 and 162 terminal azides, were engineered for water-solubility and fluorescently labeled to facilitate quantification and visualization of final products. To this end, a one-pot three-step synthetic route was devised comprising of an initial reduction of a third of the terminal azides to primary amines


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via Staudinger reaction, followed by fluorescent labeling with fluorescein isothiocyanate (FITC). Finally, since cationic dendrimers have been shown to cause unspecific bacterial clustering32-34 and cytotoxicity,35,36 we used glutaric anhydride to replace positively charged amines with negatively charged carboxylates. Water-soluble, fluorescent 2[G3]-FITC and 2[G4]-FITC were obtained in very good yields (Table 1; synthesis and characterization described in the SI; Figures S1-S8). Table 1. Surface functionalization of 2[G3]-FITC / 2[G4]-FITC. Average number of peripheral functions N3

CO2Na

Fluorescein

Total

2[G3]-FITC

37

15

2

54

2[G4]-FITC

113

44

5

162

Affitin C5 that targets SpA domain A with high specificity and affinity (KD = 108 nM)26 was selected for conjugation to 2[G3]-FITC and 2[G4]-FITC. To this end, we first introduced by sitedirected mutagenesis a unique cysteine residue at the C-terminus of C5 (C5-SH) as chemical handle for orthogonal conjugation to the dendrimers (see SI for details; Figures S9-S14). A bifunctional maleimide-cyclooctyne linker was then connected via Michael addition to afford a clickable Affitin that was readily conjugated to 2[G3]-FITC and 2[G4]-FITC via strain-promoted azide–alkyne cycloaddition (SPAAC).37,38 The Mansfield-Tomalia-Rakesh equation39 predicted a maximum of 25 and 50 Affitins (Nmax) to be accommodated on the dendritic surface of 2[G3]FITC and 2[G4]-FITC respectively based on their relative radii (ca 4.5 nm for 2[G3]-FITC, 7.5 nm for 2[G4]-FITC,31 and 1.4 nm for C5-SH40). Accordingly, two degrees of SPAAC labeling


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were pursued for each dendrimer generation accounting for half, and a quarter of their Nmax. Four Affidendrons, differing in average valency (N, determined by MALDI and UV) and size (13-22 nm by DLS) were obtained with a 70-95% conjugation efficiency. Their purity and monodispersity were verified by SEC and DLS, which showed the expected increase in size with generation and N: 2[G3]-[C5]low (N=6, 13 nm), 2[G3]-[C5]high (N=10, 15 nm), 2[G4]-[C5]low (N=12, 17 nm), and 2[G4]-[C5]high (N=27, 22 nm) (Figures 1 and S15-S22; see SI for details; Table S1). Interestingly, the presence of remaining azides on the surface of Affidendrons entitles them for facile subsequent functionalizations.

Figure 1. A) Schematic representation of Affidendrons 2[Gn]-[C5]L (n: dendrimer generation, L: Affitin valency, low or high) reflecting the average number of Affitins per dendrimer (N). B) DLS size distribution, C) 10% SDS-PAGE, and D) SEC analysis.


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We analyzed Affidendrons by surface plasmon resonance (SPR) using a biotinylated SpA domain A immobilized to a streptavidin chip to provide a low target density surface (25 resonance units, RU). Kinetic data was obtained for 2[G3]-[C5]low using our previously described approach,41 where analysis of the early association and late dissociation phases of the sensorgrams allow to assess the binding efficiency and contribution of binding modes of multivalent analytes (Figures 2 and S23, Table 2; see SI for details).

Figure 2. SPR sensorgrams measuring the interaction of Affitin C5 and Affidendrons (6.25-100 nM for 2[G3]-[C5]low and 2[G3]-[C5]high, 1.56-12.5 nM for 2[G4]-[C5]low and 2[G4]-[C5]high) with immobilized SpA domain A. RU, response units.


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Table 2. Binding data from separate SPR kinetic analysis of C5 and 2[G3]-[C5]low: kon (×106 M-1 s-1), koff (×10-4 s-1) and KD (nM). kon

koff

KD

C5

0.95

979

103

2[G3]-[C5]low

2.50

4.30

0.17

kon-high, koff-high, and KD-high for 2[G3]-[C5]low based on early association and late dissociation phase analysis.

These data were compared to the kinetics of monomeric C5 binding, analyzed by a 1:1 Langmuir model. We observed approximately three orders of magnitude improvement in KD for 2[G3][C5]low compared to the monomeric binding - largely due to a much slower dissociation rate. This suggests that the Affidendron can simultaneously bind at least two SpA domain A on the chip surface, confirming the efficient chelating character of the design. The other three Affidendrons with a larger N valency did not show any observable dissociation in the later phases of the sensorgrams in agreement with an almost irreversible binding to the target (Figure 2). Still, the rate of dissociation at early dissociation times (given by the percentage of SPR signal decay observed during 300 s, Table S2) provided an insight for their comparison. As expected, stronger binding resulted from the higher generation and N values: 24% decay for 2[G3]-[C5]low, 10% for 2[G3]-[C5]high, and only 2% for both 2[G4]-[C5]low and 2[G4]-[C5]high. To further demonstrate the enhanced binding affinity achieved by the multivalent presentation of Affitins, we performed a competition labeling experiment of S. aureus (Newman strain) between Affitin C5 (5 µM; fluorescently labeled with Alexa Fluor 647) and the highest valency Affidendrons 2[G3]-[C5]high (500 nM) and 2[G4]-[C5]high (50 nM). As seen in Figure 3, Affidendrons far outcompete the monomer for binding to the cell surface of S. aureus. Good


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colocalization is observed between the fluorescein channel, where signal coming from the conjugates is observed, and the bright-field showing the position of the cells. Conversely, very faint signal coming from the Alexa 647 channel, originating from C5, is observed when monomeric C5 is used. These results suggest that Affidendrons represent a promising novel type of reagents for infection imaging.

Figure 3. A) Fluorescence microscopy detection of S. aureus Newman strain. Competition between 500 nM 2[G3]-[C5]high and 5 µM C5-Alexa 647 (upper row); 50 nM 2[G4]-[C5]high and 5 µM C5-Alexa 647 (lower row).


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Bacterial self-clustering and agglutination triggered by external agents are important processes in controlling their complex multicellular behavior.42 Furthermore, agglutination is widely used in slide assays as a simple and rapid identification method of S. aureus.43 In order to examine the usefulness of Affidendrons as agglutination agents specific for SpA-expressing S. aureus strains, an assay was performed11 where bacterial clusters were directly observed under the microscope and their average relative size determined. In addition, further insight into the extent and dynamics of agglutination was obtained via a precipitation assay,28 where the drop in optical density (OD, due to precipitation of clusters) was monitored over time. To this end, the S. aureus strains ST25 and Newman expressing SpA were considered along with three negative controls: S. aureus Newman ΔSpA mutant, Gram-positive S. epidermidis, and Gram-negative E. coli DH5α Iq. As for the Affidendrons, the higher valency 2[G3]-[C5]high and 2[G4]-[C5]high were challenged with monomeric Affitin C5.


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Figure 4. A) Representative brightfield images showing bacterial agglutination. B) Precipitation assays showing the drop in OD600 due to agglutination throughout time (error bars – SD from three replicates). Bacteria ST25 (left) and Newman (right) treated with: PBS500 (control, cyan), C5 (1 μM, red), 2[G3]-[C5]high (500 nM, yellow), and 2[G4]-[C5]high (50 nM, blue).

Figures 4 and S24 show that massive agglutination was only observed in samples combining Affidendrons and SpA-expressing S. aureus strains (Table 3). The absence of agglutination in the SpA-deficient strains indicates that the effect is driven by the underlying highly specific AffitinSpA binding. Similar outcome emerged when attending to the precipitation assay (Figures 4B and S25). The more accentuated agglutination and precipitation effects observed for ST25 strain compared to Newman [average relative size twice that of largest Newman clusters (Table 3); agglutination of ST25 achieved at lower Affidendron concentrations (Table 3); largest extent of precipitation: 85% for ST25 vs 62% for Newman (Table S3)] point to possibly different local densities or accessibility of SpA on the membrane of both strains.

Table 3. Average relative cluster sizes of bacteria as determined by agglutination assay. Agglutination agent Bacterial strain C5 2[G3]-[C5]high 2[G4]-[C5]high 1 µM 500 nM 50 nM 5 nM 50 nM 5 nM S. aureus ST25 1.0 0.8 0.5 0.2 Newman 0.4 0.5 Newman ΔSpA S. epidermidis E. coli DH5α Iq -

(-) no agglutination observed


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Staphylococcal biofilms are the most frequent cause of biofilm-associated infections, including those related to wounds, valve endocarditis and devices. Traditionally, biofilm formation is divided into three main phases: initial attachment, maturation, which consists of accumulation and channel formation, and dissemination.44 It has been shown that SpA promotes the development of S. aureus biofilm. Since blocking SpA inhibits biofilm formation,28,45 we wonder whether Affidendrons might efficiently be used as staphylococcal biofilm inhibition agents. Accordingly, a biofilm inhibition assay was performed using a standard microtiter dish test based on crystal violet staining. Low, non-agglutinating concentrations of Affidendrons showed significant biofilm inhibition (p < 0.001 for 2[G3]-[C5]high and p < 0.01 for 2[G4]-[C5]high) in S. aureus (Newman strain) by > 70% (Figure 5). Affidendron [G3]-[C5]high (at a concentration of 50 nM) and [G4]-[C5]high (at a concentration of 5 nM) were able to inhibit biofilm formation to 8.2 ± 1.9% and 17.5 ± 9.6% of the untreated bacteria, respectively. Conversely, small and non-statistical differences in biofilm formation were seen for S. epidermidis, which was used as a positive control due to the robust biofilms it usually forms. Our results showed that strong, multivalent SpA binding was necessary for biofilm inhibition.


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Figure 5. Biofilm formation in the presence and absence of Affidendrons. Images of representative wells from the crystal violet biofilm assay are shown. Untreated bacteria were used as controls (100%). Error bars – SD from three replicates. (**) marks significant difference between treated and untreated bacteria at p

Multivalent Affidendrons with High Affinity and Specificity towards

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