(C-90) Synthetic mucin analogs based on silk biomaterial: synthesis, biophysical characterization, and their microbial interactions

Introduction:: According to the World Health Organization (WHO), antibiotic resistance (AbR), is one of the top 10 threats to global health, responsible for millions of deaths every year. The prevalence of AbR is in part due to the emergence and spread of drug-resistant infections as well as the availability, poor quality and clinical misuse of antibiotics. With the global proliferation of antibiotic resistance, new strategies to prevent and treat harmful infections are urgently needed. Here, inspiration can be drawn from mucin, a high molecular weight glycoprotein found in our body that can prevent virulent phenotypes by reprogramming microbial behavior. However, the scalable biosynthesis of natural mucin is a major challenge, prompting efforts to generate synthetic analogues of mucin. Here, leveraging insights into how bacteria sense mucin glycans in native environment, regulatory glycans were covalently introduced as pendant groups onto the silk protein backbones using different modification methods, to yield silk glycopolymers.

Materials and Methods:: Using different chemistries, five different sugar molecules i.e., (N-acetyl Galactosamine (GalNAc), N-acetyl glucosamine (GlcNAc), sialic acid (NeuNAc), galactosamine (GalN) and glucosamine (GlcN) were incorporated with different substitution efficiencies. The materials were prepared by using different silk protein modification chemistries such as serine carboxylation and carbodiimide coupling to selectively incorporate the sugar molecules on the serine, aspartic and glutamic acid residues of silk. The resulting glycopolymers were characterized by using different analytical techniques such as nuclear magnetic resoncance (NMR), primary amine quantification by 2, 4, 6, trinitrobenzene sulfonic acid (TNBSA) assay, zeta potential and Fourier transform infrared (FTIR) spectroscopy. In addition, the cytotoxicity of the glycopolymers was assessed with murine fibroblast cells.


Results, Conclusions, and Discussions:: Oral cavity was used as a model to test the ability of the silk glycopolymers to combat pathogenicity of Streptococcus mutans (s. mutans) which is responsible for biofilm formation. The glycopolymers were tested at 4.5 wt%. Among the glycopolymers, 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. Under similar experimental conditions, neither SF nor free GalNAc alone influenced fractional biofilm formation. In addition, SF(S)-GalNAc reduced biofilm formation without reducing the total bacterial population while SF(S)-NeuNAc and soluble NeuNAc exhibited a killing effect, with a moderate decrease in the fractional biofilm. Other glycopolymers such as SF(S)-GlcNAc, SF(S)-GalN, and SF(S)-GlcN did not influence 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. We also demonstrated that such antimicrobial effect depends on the glycopolymer concentration, glycan identity and density, with pathogen-specific impact depending on the specific polymer. These biomaterials represent new tools for controlling microbial infections without breeding resistance and may find applications as antifoulants, antibiotic substitutes and tissue and oral disinfectants.


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