The majority of viruses within the gut are obligate bacterial viruses known as bacteriophages (phages). Their bacteriotropism underscores the study of phage ecology in the gut, where they modulate and coevolve with gut bacterial communities. Traditionally, these ecological and evolutionary questions were investigated empirically via in vitro experimental evolution and, more recently, in vivo models were adopted to account for physiologically relevant conditions of the gut. Here, we probed beyond conventional phage–bacteria coevolution to investigate potential tripartite evolutionary interactions between phages, their bacterial hosts, and the mammalian gut mucosa. To capture the role of the mammalian gut, we recapitulated a life-like gut mucosal layer using in vitro lab-on-a-chip devices (to wit, the gut-on-a-chip) and showed that the mucosal environment supports stable phage–bacteria coexistence. Next, we experimentally coevolved lytic phage populations within the gut-on-a-chip devices alongside their bacterial hosts. We found that while phages adapt to the mucosal environment via de novo mutations, genetic recombination was the key evolutionary force in driving mutational fitness. A single mutation in the phage capsid protein Hoc—known to facilitate phage adherence to mucus—caused altered phage binding to fucosylated mucin glycans. We demonstrated that the altered glycan-binding phenotype provided the evolved mutant phage a competitive fitness advantage over its ancestral wild-type phage in the gut-on-a-chip mucosal environment. Collectively, our findings revealed that phages—in addition to their evolutionary relationship with bacteria—are able to evolve in response to a mammalian-derived mucosal environment.
Multivalent protein−glycan interactions are widespread in biology and are vital for initial recognition in cell−cell communication, host immune regulation, and viral and bacterial infection. Recently, fluorescent nanomaterials such as quantum dots (QDs) have been identified as a capable scaffold for multivalent carbohydrate immobilization. Carbon dots (CDs) are essentially heavy-metal-free QDs, offering a low-toxicity alternative for bioimaging and biosensing applications. Herein, we report a simple and versatile route for linker functionalization of lactose with (3-glycidyloxypropyl)trimethoxysilane (GOPTS) for conjugation to various CDs. CDs derived by thermal treatment of polyethylenimine (PEI) with citric acid and lactose, as the glycan, were employed in a number of detailed biological applications and evaluations. The self-assembled glycan monolayer (SAGM) method described here resulted in a high yield of lactose-conjugated CDs. Specific interactions of our lactose-coated CDs with cells, lectin microarrays, and as internalized bioimaging nanolights were demonstrated, with intracellular localization in a variety of cell lines observed. This present study offers a facile, one-pot, green, and low-cost synthesis of fluorescent multivalent nanoparticles that can be applied in the study of diverse interactions across the glycointeractome.
The interaction of carbohydrate-binding proteins (CBPs) with their corresponding glycan ligands is challenging to study both experimentally and computationally. This is in part due to their low binding affinity, high flexibility, and the lack of a linear sequence in carbohydrates, as exists in nucleic acids and proteins. We recently described a function-prediction technique called SPOT-Struc that identifies CBPs by global structural alignment and binding-affinity prediction. Here we experimentally determined the carbohydrate specificity and binding affinity of YesU (RCSB PDB ID: 1oq1), an uncharacterized protein from Bacillus subtilis that SPOT-Struc predicted would bind high mannose-type glycans. Glycan array analyses however revealed glycan binding patterns similar to those exhibited by fucose (Fuc)-binding lectins, with SPR analysis revealing high affinity binding to Lewisx and lacto-N-fucopentaose III. Structure based alignment of YesU revealed high similarity to the legume lectins UEA-I and GS-IV, and docking of Lewisx into YesU revealed a complex structure model with predicted binding affinity of −4.3 kcal/mol. Moreover the adherence of B. subtilis to intestinal cells was significantly inhibited by Lex and Ley but by not non-fucosylated glycans, suggesting the interaction of YesU to fucosylated glycans may be involved in the adhesion of B. subtilis to the gastrointestinal tract of mammals.
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