Selective Biofilm Inhibition through Mucin-Inspired Engineering of Silk Glycopolymers

Abstract

Mucins are key components of innate immune defense and possess remarkable abilities to manage pathogenic microbes while supporting beneficial ones and maintaining microbial homeostasis at mucosal surfaces. Their unique properties have garnered significant interest in developing mucin-inspired materials as novel therapeutic strategies for selectively controlling pathogens without disrupting the overall microbial ecology. However, natural mucin production is challenging to scale, driving the need for simpler materials that reproduce mucin’s bioactivity. In this work, we generated silk-based glycopolymers with different monosaccharides (GalNAc, GlcNAc, NeuNAc, GlcN, and GalN) and different grafting densities. Using the oral cavity as a model system, we treated in vitro cultures of pathogenic Streptococcus mutans and commensal Streptococcus sanguinis with our glycopolymers, finding that silk-tethered GalNAc uniquely prevented biofilm formation without affecting overall bacterial growth of either species. This relatively simple material reproduced mucin’s virulence-neutralizing effects while maintaining biocompatibility. These mucin-inspired materials represent a valuable tool for preventing infection-related harm and offer a strategy for the domestication of pathogens in other environments.

Graphical Abstract

INTRODUCTION

Mucins are essential glycoproteins in the human mucosal defense system and play a crucial role in regulating microbial interactions at epithelial surfaces. These complex biomolecules possess the remarkable ability to modulate the behavior of potentially pathogenic microorganisms while fostering an environment conducive to beneficial microbes, thereby maintaining microbial homeostasis. As principal structural components of mucus—a biopolymer matrix that covers all wet epithelial surfaces of the human body—mucins demonstrate a unique capacity to domesticate opportunistic pathogens,^1,2 acting as a protective layer against mechanical damage and microbial infections.² In saliva, mucins form an intricate three-dimensional network surrounding resident microbes, facilitating direct interactions that influence microbial behavior (Figure 1a).

Figure 1.

Processed silk provides a scaffold for mucin-inspired antivirulence biomaterials. (a) Cryogenic scanning electron microscopy (cryoSEM) images showing the three-dimensional network formed by mucins in saliva, with S. mutans spiked in, surrounding, and directly interacting with oral microbes. Two representative images are shown. Scale bar: 500 nm. (b) Native mucin is characterized by highly glycosylated STP (serine, threonine, proline) domains. Mucin O-glycans are added onto the hydroxyl groups of serine and threonine residues (80% of the total mucin mass). (c) S. mutans forms biofilms on tooth-like surfaces by adhering to EPSs synthesized from sucrose. The mucin polymer reduces the existing biofilm while preventing further biofilm formation. (d) Extraction of SF protein from B. mori silkworm by removal of sericin protein (degumming) and dissolution in a lithium bromide solution (bottom panel). The top panel presents the different structural hierarchies and versions of SF polymers during extraction. (e) Amino acid composition of the heavy-chain of SF.

The polymeric structure of mucins is characterized by dense oligosaccharide branches (glycans) that protrude from a protein backbone and constitute 50–80% of the mucin mass (Figure 1b).³ These mucin-associated glycans are initiated by N-acetyl galactosamines (GalNAc) O-linked to serine or threonine, with extending structures formed from N-acetyl glucosamine (GlcNAc), galactose, l-fucose and N-acetyl neuraminic acid (NeuNAc) residues.⁴

Recent work has found that upon sensing mucin glycans, many opportunistic pathogens suppress their virulence pathways.^5–7 An oral pathogen, Streptococcus mutans,⁸ is largely found asymptomatically in healthy individuals but causes cavities in people with reduced salivary mucus production.^9,10 This virulence primarily arises when S. mutans is in its biofilmforming phenotype, where it produces a thick extracellular matrix that adheres strongly to smooth tooth surfaces and releases acidic metabolic byproducts (Figure 1c).^8,11,12 MUC5B, the mucin most abundant in healthy saliva, prevents S. mutans from forming biofilms by altering bacterial gene expression.^7,13,14 This dynamic, which is mirrored in other mucosal environments,^5,6 presents mucin as a new tool for preventing and potentially treating microbial infections.

Despite its potential to manage problematic pathogens, mucin’s structural complexity has limited its development for practical applications. The high molecular weight of its backbone and complex glycosylation patterns¹ make it difficult to produce mucin through heterologous expression. Additionally, harvesting mucin from animal sources or human cell lines is not yet scalable.¹⁵ There is a need for defined and scalable mucin-inspired biomaterials that can be systematically investigated for structure–function relationships. While there have been major advances in polymer and protein chemistry, few biomaterials mimic mucin’s functions and properties. So far, researchers have focused on creating mucin-mimetic materials that replicate various structural features, focusing on glycosylation,¹⁶ polymer length,¹⁷ or cross-linking domains.¹⁸ However, in our approach, we prioritized the bioactivity that reduces virulence over reproduction of the exact mucin structure.

RESULTS AND DISCUSSION

We adopted silk fibroin (SF), a protein derived from Bombyx mori silkworm, as the backbone of our glycopolymers because of its biocompatibility, stability (at physiological pH), and ease of aqueous processability (Figure 1d).^19–22 SF’s diverse amino acid profile enables selective chemical installation of glycan motifs at varying grafting densities, making it an ideal scaffold for mucin motifs (Figure 1e).²³ Importantly, as most SF amino acids are inert, chemistry can be selectively performed on reactive serine residues (Figure 1e). Using this knowledge and motivated by our previous methods tethering amino sugars to SF,²³ we developed a three-step protocol in which the reducing end of biologically relevant acetyl-sugars is covalently conjugated to SF (Figure 2a).

Figure 2.

Out of a set of mucin-inspired silk glycopolymers, GalNAc-modified silk polymers (SF(S)-GalNAc) reduced biofilm formation in S. mutans without killing. (a) Stepwise synthesis of different modification chemistries performed on regenerated SF to covalently append different monosaccharides to obtain (b) silk glycopolymers. (c) Similar to the mucin MUC5B, SF(S)-GalNAc reduced the number of cells in the biofilm without reducing the overall size of the bacterial population, while SF and soluble GalNAc had no effect. Silk glycopolymers were tested at 4.5 wt %, and sugar-only solutions (specifically GalNAc, GlcNAc, NeuNAc, Gal, and Glc) were tested at 0.9 wt %, as the silk glycopolymers are expected to be about one-fifth sugar by mass (Figure S15). MUC5B was tested at 0.5 wt %. (d) SF and most SF-sugar polymers did not affect the overall growth of S. mutans. However, SF(S)-NeuNAc was toxic to S. mutans, mirroring the effect of soluble NeuNAc, which is also toxic at the concentration tested. (e) Binding assays showed that S. mutans adhered to polystyrene surfaces but did not bind strongly to MUC5B, SF, or SF(S)-GalNAc. MUC5B reduced biofilm formation, but SF did not, suggesting that the primary mechanism by which SF(S)-GalNAc reduces biofilm formation is not competitive inhibition of bacterial binding to surfaces. (f) The amount of biofilm biomass was measured using crystal violet, which stains live cells, cellular debris, and EPSs produced by S. mutans. SF(S)-GalNAc treatment significantly reduced the amount of S. mutans biofilm biomass, indicating an altered microbial behavior. SF-(S)-NeuNAc also reduced the total polysaccharides produced, likely because it was toxic to S. mutans. Conversely, SF(S)-GlcNAc showed an increase in polysaccharide production, likely because GlcNAc is a building block in peptidoglycans. In (c–f), heatmaps show the geometric mean. In (d, e), the log₁₀ transformation of CFUs is plotted. Significant differences from media controls were determined with a Kruskal–Wallis test followed by an uncorrected Dunn’s test (* indicates p < 0.05).

Inspired by mucin’s display of sugar motifs, we synthesized five silk-based glycopolymers by covalently incorporating different simple sugars, including GalNAc, GlcNAc, NeuNAc, galactosamine (GalN), and glucosamine (GlcN), onto SF. In this process, we used SF harvested after 60 min of extraction (disperse MW: ~80–100 kDa),²⁴ though the SF polymer chain length can be easily varied with different extraction times (Figure 1d).²⁵ Briefly, the serine residues of SF were carboxylated to yield SF(S)-COOH (Figure 2a). SF(S)-COOH was coupled to a sugar (GalN, GlcN) or an ethylene diamine (EDA) linker, resulting in SF(S)-sugar or SF(S)-EDA. The free amine of SF(S)-EDA was conjugated to the carboxylic acid residues of sugars such as GalNAc or NeuNAc, providing SF(S)-GalNAc and SF(S)-NeuNAc. Through this synthesis, we produced SF(S)-GalNAc, SF(S)-GlcNAc, SF(S)-NeuNAc, SF(S)-GalN, and SF(S)-GlcN, respectively (Figure 2a,b). Nuclear magnetic resonance, primary amine quantification, zeta potential, and degree of sugar substitution studies confirmed successful sugar incorporation (Figures S1–S15). Fourier transform infrared spectroscopy revealed no significant change between the β-sheet in SF and the SF glycopolymers (29–32%) (Figure S16) as previously reported.²³ We synthesized mucin-inspired glycopolymers by chemically tethering sugars to SF with varying densities and linkage chemistries. This method can be generalized to branched sugars, thereby providing a blueprint for synthesizing future generations of mucin-inspired glycopolymers.

We utilized the oral cavity as a model system for investigating how mucin-inspired glycopolymers disrupt bacterial pathogenicity. Because the harm done by S. mutans largely depends on biofilm formation, we performed in vitro assays of this phenotype to assess the potential pathogenicity of these bacteria. Most antibiofilm coatings reduce the overall bacterial population.²⁶ In contrast, mucin can reduce fractional biofilms without killing bacteria,^5,6,13 avoiding potential negative growth effects on commensals. Thus, we sought a polymer that could reproduce mucin’s ability to reduce S. mutans biofilm formation without limiting growth.

We assayed the biofilm formation and growth of S. mutans in a 96-well plate assay under biofilm-promoting conditions. After 6 h, we quantified the suspended cells and remaining biofilm by a colony-forming unit (CFU) assay. In the medium-only control, 90% of the S. mutans cells were in a biofilm with the rest unadhered or suspended. Addition of native mucin (0.5% MUC5B) reduced the fractional biofilm formation to 30% (Figure 2c), without affecting the total growth (Figure 2d).

When we tested our polymer set in this assay (4.5 wt %), SF(S)-GalNAc and SF(S)-NeuNAc induced significant changes in the bacterial populations. When incubated with SF(S)-GalNAc, only 2% of cells remained in the biofilm with 98% suspended (Figure 2c, Figure S17). In comparison, neither SF nor the equivalent amount of free GalNAc alone (0.9%, as the polymers are about one-fifth of the sugar by mass, Figure S15) influenced fractional biofilm formation. Further, SF(S)-GalNAc reduced biofilm formation without reducing the total population size, while SF(S)-NeuNAc and soluble NeuNAc showed a strong killing effect (Figure 2d, Figure S18), with a moderate decrease in the fractional biofilm to 40%. SF(S)-GlcNAc, SF(S)-GalN, and SF(S)-GlcN did not influence the S. mutans fractional biofilm formation or growth. Thus, sugar identity is a key parameter in mimicking mucin bioactivity, and grafted GalNAc has a unique ability to reprogram S. mutans behavior.

To determine whether mucin and SF(S)-GalNAc inhibit biofilm formation by preventing initial bacterial binding, we examined bacterial adhesion to surfaces coated with mucin, SF, or SF glycopolymers. As previously reported,²⁷ S. mutans did not bind to MUC5B (Figure 2e). Interestingly, SF-coated surfaces were equally effective in repelling initial S. mutans adsorption but did not reduce the level of biofilm formation, indicating that reduced binding is insufficient to prevent biofilm formation. S. mutans did not show increased binding to SF(S)-GalNAc compared with the negative control, although the bacteria did bind to SF(S)-GalNAc more than to unmodified SF (Figure 2e, Figure S19). We observed a similar trend for SF(S)-NeuNAc. These results suggest that SF(S)-GalNAc does not prevent biofilm formation solely by acting as a physical barrier and that S. mutans may interact with the GalNAc or NeuNAc moieties displayed on the SF.

Because SF(S)-GalNAc did not appear to act as a physical barrier, we hypothesized that it may disrupt biofilm maturation. After initial surface attachment, S. mutans uses sucrose to synthesize exopolysaccharides (EPSs), embedding the bacteria in an extracellular matrix and forming a mature biofilm. Using crystal violet, we stained S. mutans biofilms and quantified the total biomass, including bacterial cells and EPS (Figure 2f). MUC5B, SF, and SF(S)-GalNAc reduced the biomass by 77, 24, and 98%, respectively, indicating that SF(S)-GalNAc induced the bacteria to produce less EPSs. Similarly, MUC5B, SF, and SF(S)-GalNAc, but not free GalNAc, reduced expression of some glucosyltransferases and glucan-binding proteins associated with biofilm formation, suggesting that alterations in gene expression account for some of the alteration in the phenotype (Figure S20). In contrast, SF(S)-GlcNAc induced an increase in bacterial biomass, even though it did not increase cell growth. Although GlcNAc is converted into peptidoglycan in the cell wall of S. mutans,²⁸ since free GlcNAc has no effect on biomass formation, the bacteria appear to alter phenotype based on the presentation of tethered GlcNAc by an unknown mechanism. The killing of S. mutans by SF(S)-NeuNAc corresponded to a reduced total bacterial biomass. Together, these data confirm that SF(S)-GalNAc alters the bacterial phenotype.

To visualize this shift in community structure caused by SF(S)-GalNAc, we stained S. mutans biofilm cultures with SYTO9 and performed confocal microscopy (Figure 3a). Normally, without any treatment, the bacteria tightly adhere to the surface; however, when treated with SF(S)-GalNAc or MUC5B, the population remained suspended. Cultures treated with the SF polymer showed no change in the community structure (Figure 3a). This microscopy reveals SF(S)-GalNAc’s ability to shift S. mutans away from a biofilmformation phenotype. To further understand the interactions between our silk materials and the microbes, we employed cryogenic scanning electron microscopy (cryoSEM) as applied in Figure 1a. This technique allowed us to examine the structural relationships in detail under near-native conditions and determine if the silk material engages in similar interactions with the embedded microbes, as seen with native saliva (Figure 1a). Our data show that both SF and SF(S)-GalNAc networks intensively envelop S. mutans cells (Figure 3b,c). Interestingly, despite the similar physical encapsulation observed in both networks, only SF(S)-GalNAc prevents biofilm formation. This suggests that the presence of GalNAc results in a qualitatively different interaction with S. mutans compared with silk alone, beyond mere physical entrapment. These findings underscore the importance of the GalNAc moiety in modulating S. mutans behavior. The similarity between the SF(S)-GalNAc and native salivary mucin interactions with S. mutans (Figure 1a) supports our hypothesis that changes in S. mutans virulence arise not just from physical entrapment but through specific biochemical interactions with presented glycans, mirroring the proposed mechanism in native saliva. Therefore, the SF(S)-GalNAc network appears to induce a specific response in S. mutans that inhibits biofilm formation.

Figure 3.

Biofilm-reducing efficiency of silk glycopolymers was specific to GalNAc grafting and generalizable to commensal S. sanguinis. (a) Confocal microscopy of an in vitro culture of S. mutans showing a dense biofilm close to the surface. When treated with SF(S)-GalNAc or MUC5B, S. mutans bacteria no longer attached to the surface, while unmodified SF and GalNAc showed minimal effects. Scale bar: 50 μm. (b) CryoSEM image of S. mutans embedded in a 2.5 wt % SF network. (c) CryoSEM image of S. mutans embedded in a 2.5 wt % SF(S)-GalNAc network. Both images demonstrate intensive envelopment of bacterial cells by the respective network materials. (d) At 4.5 wt %, SF(S)-GalNAc reduced biofilm formation of S. sanguinis strains JFP36 and 10556, without affecting their growth. Heatmaps show the geometric mean: significant differences from media controls were determined with a Kruskal–Wallis test followed by an uncorrected Dunn’s test (* indicates p < 0.05). (e) The biofilm-reducing effect of SF(S)-GalNAc was concentration-dependent. As the polymer concentration increased, a more significant reduction in the biofilm was observed. (f) The low-density GalNAc (SF(D,E)-GalNAc) reduced biofilm formation but required polymer higher dosage than SF(S)-GalNAc. All experiments were performed with at least three biological triplicates. In (e, f), bars represent the geometric mean, and error bars show the geometric error.

Having established that SF(S)-GalNAc could effectively prevent biofilm formation in S. mutans, we assayed how the polymer affected S. sanguinis, a commensal oral bacterial species that competes with S. mutans.¹³ We found that SF(S)-GalNAc had a similar effect on S. sanguinis as S. mutans, reducing biofilm formation without altering the total growth of S. sanguinis (Figure 3d, Figure S23). Thus, SF(S)-GalNAc reproduced mucins’ natural protective effect by preventing the biofilm formation of an opportunistically pathogenic oral microbe (S. mutans), without harming commensal organisms (S. sanguinis).

To probe the effect of grafting density on biofilm reduction, we examined how tethering configurations influenced efficacy of the GalNAc silk glycopolymers. We designed a new glycopolymer, SF(D, E)-GalNAc, that had a 6–8 times lower GalNAc substitution compared to parent SF(S)-GalNAc (Supporting Information, Figures S13–S16, S21–S22).²³ SF(S)-GalNAc and SF(D,E)-GalNAc both reduced biofilm formation in a concentration-dependent manner (Figure 3e, f) without killing S. mutans. At 4.5 polymer wt %, SF(D,E)-GalNAc and SF(S)-GalNAc reduced biofilm formation by 83% and 96% respectively, indicating that a higher sugar grafting density led to increased neutralization potency, albeit with nonlinear dosing-dependence. Notably, water contact angle measurements revealed that SF(D,E)-GalNAc is moderately more hydrophobic than SF(S)-GalNAc (Figures S27), suggesting that hydrophilicity as well as increased sugar density could have contributed to the relatively higher efficacy of SF(S)-GalNAc.

These assays demonstrate that SF-GalNAc polymers can disrupt the S. mutans biofilm. To test the material properties and host cell biocompatibility of our polymer set, we used rheometry (4.5 wt % polymer solutions in ultrapure water). The SF and derivatives demonstrated shear thinning behavior, with a viscosity of 2–6 mPa s⁻¹ at a shear rate of 90 s⁻¹, similar to the viscosity of healthy saliva at 3–5 mPa s⁻¹ for the same rate (Figure S24).^29–31 We assessed polymer cytocompatibility based on the metabolic activity of L929 human murine fibroblast cell monolayers incubated with test solutions for 24 h. The relative metabolic activity was 60–80% for cells incubated in sugar solutions compared with that for SF alone and ~65–85% for sugar-conjugated silk solutions (Figure S25). We also assessed the cytocompatibility of water-insoluble films fabricated from 4.5 wt % glycopolymer solutions (Figure S26a). Extracts from aminated silk films did not cause a significant reduction in metabolic activity compared with the negative control, except for SF(S)-GalNAc and SF(S)-GlcNAc, which caused a reduction of ~20% relative to SF (Figure S26b). Bright field imaging showed that cells from all test groups displayed a well-spread, confluent morphology 24 h after incubation in the extract solutions (Figure S26c). Together these results indicate that silk-based polymers are largely biocompatible.

Here, we demonstrated that S. mutans biofilm formation decreased upon treatment with SF(S)-GalNAc. Previously we demonstrated that soluble GalNAc was able to partially reproduce some of mucin’s effects on virulence gene expression, while other mucin monosaccharide sugars had no effect.⁷ These results bolster the conclusion that S. mutans responds to GalNAc by inducing a change in cell and community phenotypes. However, no known cell-surface proteins interact with GalNAc, so the mechanism of this interaction is still uncertain.

Perhaps even more surprising than the biofilm-reducing effects of SF(S)-GalNAc were the unexpected phenotypes induced by the other polymers tested. For instance, SF(S)-GlcNAc did not affect fractional biofilm formation and even increased the level of biofilm maturation. Given that GlcNAc and GalNAc differ only by stereochemistry at the C4 position, these opposing effects on the phenotype highlight the specificity of bacterial sensing mechanisms. Additionally, it supports the conclusion that SF(S)-GalNAc is not working through any physical effects, as the material properties of the two polymer solutions are similar (Figure S24). In addition, we were surprised by the toxicity of SF(S)-NeuNAc, which proved to be highly effective at actively killing S. mutans bacteria. While soluble NeuNAc, also known as sialic acid, is likely toxic due to dropping the pH of the cultures, our SF(S)-NeuNAc polymers were buffered to neutral pH. We hypothesize that SF(S)-NeuNAc acts by disrupting membrane integrity and increasing permeability, a mechanism common to antibacterial polymers³² and also human milk oligosaccharides.³³

In summary, we have identified a silk-based antivirulent mucin-mimetic biomaterial that renders an opportunistic pathogen docile. These mucin-inspired materials are structurally different from native mucin and present simpler sugars, yet they exceed its ability to prevent S. mutans biofilm formation. These findings can serve as a platform for translation to other opportunistic pathogens tamed by mucin.^5,6 This virulence-neutralizing behavior was specific and selective depending on the sugar identity and glycopolymer concentration. Synthesis of these mucin-inspired silk glycopolymers leverages aqueous chemistry that is robust, can be generalized to different silks, molecular weights, and other sugar types (identity, compositions, and complex glycans), and can be scaled as needed. Future studies can explore the impact of the backbone and linker structure on the exposure and subsequent activity of the sugars presented. Importantly, our materials draw on the strengths of natural mucosal protective barriers by redomesticating an opportunistic pathogen without detrimentally affecting commensal organisms. We expect our findings to aid in addressing virulent pathogens that are becoming increasingly more resistant. We also anticipate that these silk glycopolymers can also be used as a mucin-inspired biomaterial platform to prevent or treat microbial infections in other microbiomes. Further, these mucin substitutes will also find applications as additives to skin and oral-care products, dietary supplements, agricultural applications, as coatings for different biomedical devices, food (to reduce spoilage), and antibiotic adjuvants or alternatives compounding its potential broader utility.