Mucins are large gel-forming polymers inside the mucus barrier that inhibit the yeast-to-hyphal transition of Candida albicans, a key virulence trait of this important human fungal pathogen. However, the molecular motifs in mucins that inhibit filamentation remain unclear despite their potential for therapeutic interventions. Here, we determined that mucins display an abundance of virulence-attenuating molecules in the form of mucin O-glycans. We isolated and cataloged >100 mucin O-glycans from three major mucosal surfaces and established that they suppress filamentation and related phenotypes relevant to infection, including surface adhesion, biofilm formation and cross-kingdom competition between C. albicans and the bacterium Pseudomonas aeruginosa. Using synthetic O-glycans, we identified three structures (core 1, core 1 + fucose and core 2 + galactose) that are sufficient to inhibit filamentation with potency comparable to the complex O-glycan pool. Overall, this work identifies mucin O-glycans as host molecules with untapped therapeutic potential to manage fungal pathogens.

All animals except sponges produce mucus. Across the animal kingdom, this hydrogel mediates surface wetting, viscosity, and protection against microbes. The primary components of mucus hydrogels are mucins-high molecular weight O-glycoproteins that adopt extended linear structures. Glycosylation is integral to mucin function, but other characteristics that give rise to their advantageous biological activities are unknown. We postulated that the extended conformation of mucins is critical for their ability to block microbial virulence phenotypes. To test this hypothesis, we developed synthetic mucin mimics that recapitulate the dense display of glycans and morphology of mucin. We varied the catalyst in a ring-opening metathesis polymerization (ROMP) to generate substituted norbornene-derived glycopolymers containing either cis- or trans-alkenes. Conformational analysis of the polymers based on allylic strain suggested that cis- rather than trans-poly(norbornene) glycopolymers would adopt linear structures that mimic mucins. High-resolution atomic force micrographs of our polymers and natively purified Muc2, Muc5AC, and Muc5B mucins revealed that cis-polymers adopt extended, mucin-like structures. The cis-polymers retained this structure in solution and were more water-soluble than their trans-analogs. Consistent with mucin's linear morphology, cis-glycopolymers were more potent binders of a bacterial virulence factor, cholera toxin. Our findings highlight the importance of the polymer backbone in mucin surrogate design and underscore the significance of the extended mucin backbone for inhibiting virulence.

The antimicrobial and therapeutic efficacy of bacteriophages is currently limited, mostly due to rapid emergence of phage-resistance and the inability of most phage isolates to bind and infect a broad range of clinical strains. Here, we discuss how phage therapy can be improved through recent advances in genetic engineering. First, we outline how receptor-binding proteins and their relevant structural domains are engineered to redirect phage specificity and to avoid resistance. Next, we summarize how phages are reprogrammed as prokaryotic gene therapy vectors that deliver antimicrobial 'payload' proteins, such as sequence-specific nucleases, to target defined cells within complex microbiomes. Finally, we delineate big data- and novel artificial intelligence-driven approaches that may guide the design of improved synthetic phage in the future.

The rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic "phagebodies." We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model. We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.

Binding of small molecules to mucus membranes in the body has an important role in human health, as it can affect the diffusivity and activity of any molecule that acts in a mucosal environment. The binding of drugs and of toxins and signaling molecules from mucosal pathogens is of particular clinical interest. Despite the importance of mucus-small molecule binding, there is a lack of data revealing the precise chemical features of small molecules that lead to mucus binding. We developed a novel equilibrium dialysis assay to measure the binding of libraries of small molecules to mucin and other mucus components, substantially increasing the throughput of small molecule binding measurements. We validated the biological relevance of our approach by quantifying binding of the antibiotic colistin to mucin, and showing that this binding was associated with inhibition of colistin's bioactivity. We next used a small molecule microarray to identify 2,4-diaminopyrimidine as a mucin binding motif and confirmed the importance of this motif for mucin binding using equilibrium dialysis. Furthermore, we showed that, for molecules with this motif, binding to mucins and the mucus-associated biopolymers DNA and alginate is modulated by differences in hydrophobicity and charge. Finally, we showed that molecules lacking the motif exhibited different binding trends from those containing the motif. These results open up the prospect of routine testing of small molecule binding to mucus and optimization of drugs for clinically relevant mucus binding properties. © 2019 American Chemical Society.

Mucus, a biopolymer hydrogel that covers all wet epithelia of the body, is a potential site for infection by pathogenic bacteria. Mucus can bind small molecules and influence bacterial physiology, two factors that may affect the efficacy of antibiotics. In spite of this, the impact of mucus on antibiotic activity has not been thoroughly characterized. We examined the activity of polymyxin and fluoroquinolone antibiotics against the opportunistic pathogen Pseudomonas aeruginosa in native mucus and purified mucin biopolymer environments. We found that mucus reduces the effectiveness of polymyxins and fluoroquinolones against P. aeruginosa. Mucin biopolymers MUC5AC, MUC2, and MUC5B are primary contributors to this reduction. Our findings highlight that the biomaterial environmental context should be considered when evaluating antibiotics in vitro. © 2019 American Chemical Society.

Creating a selective gel that filters particles based on their interactions is a major goal of nanotechnology, with far-reaching implications from drug delivery to controlling assembly pathways. However, this is particularly difficult when the particles are larger than the gel's characteristic mesh size because such particles cannot passively pass through the gel. Thus, filtering requires the interacting particles to transiently reorganize the gel's internal structure. While significant advances, e.g., in DNA engineering, have enabled the design of nano-materials with programmable interactions, it is not clear what physical principles such a designer gel could exploit to achieve selective permeability. We present an equilibrium mechanism where crosslink binding dynamics are affected by interacting particles such that particle diffusion is enhanced. In addition to revealing specific design rules for manufacturing selective gels, our results have the potential to explain the origin of selective permeability in certain biological materials, including the nuclear pore complex.

Mucus is a biological gel that lines all wet epithelia in the body, including the mouth, lungs, and digestive tract, and has evolved to protect the body from pathogenic infection. However, microbial pathogenesis is often studied in mucus-free environments that lack the geometric constraints and microbial interactions in physiological three-dimensional mucus gels. We developed fluid-flow and static test systems based on purified mucin polymers, the major gel-forming constituents of the mucus barrier, to understand how the mucus barrier influences bacterial virulence, particularly the integrity of Pseudomonas aeruginosa biofilms, which can become resistant to immune clearance and antimicrobial agents. We found that mucins separate the cells in P. aeruginosa biofilms and disperse them into suspension. Other viscous polymer solutions did not match the biofilm disruption caused by mucins, suggesting that mucin-specific properties mediate the phenomenon. Cellular dispersion depended on functional flagella, indicating a role for swimming motility. Taken together, our observations support a model in which host mucins are key players in the regulation of microbial virulence. These mucins should be considered in studies of mucosal pathogenesis and during the development of novel strategies to treat biofilms.

Mucus is a hydrogel that exhibits complex selective permeability, permitting the passage of some particles while restricting the passage of other particles including important therapeutics. In this review, we discuss biochemical mechanisms underlying mucus penetration and mucus binding, emphasizing the importance of steric, electrostatic, and hydrophobic interactions. We discuss emerging techniques for engineering nanoparticle surface chemistries for mucus penetration as well as recent advances in tuning mucus interactions with small molecule, peptide, or protein therapeutics. Finally, we highlight recent work suggesting that mucus permeability can serve as a biomarker for disease and physiological states such as pregnancy.