ABSTRACT
Molecules interfering with lipid bilayer function exhibit strong antiviral activity against a broad range of enveloped viruses, with a lower risk of resistance development than that for viral protein-targeting drugs. Amphipathic peptides are rich sources of such membrane-interacting antivirals. Here, we report that influenza viruses were effectively inactivated by M2 AH, an amphipathic peptide derived from the M2 protein of the influenza virus. Although overall hydrophobicity (
Subject(s)
Antiviral Agents/pharmacology , Cell Membrane/drug effects , Influenza A Virus, H1N1 Subtype/drug effects , Peptides/pharmacology , Viral Matrix Proteins/chemistry , Amino Acid Sequence , Animals , Antiviral Agents/chemical synthesis , Cell Membrane/chemistry , Cell Membrane/virology , Dogs , Hydrophobic and Hydrophilic Interactions , Influenza A Virus, H1N1 Subtype/growth & development , Influenza A Virus, H1N1 Subtype/ultrastructure , Inhibitory Concentration 50 , Lipid Bilayers/chemistry , Liposomes/chemistry , Madin Darby Canine Kidney Cells , Peptides/chemical synthesis , Structure-Activity Relationship , Viral Load/drug effectsABSTRACT
2'-Fucosyllactose (2'-FL) is the most abundant milk oligosaccharide in human breast milk and it has several benefits for infant health. The quantification of 2'-FL in breast milk or in samples from other sources generally requires lengthy analyses. These methods cannot be used to simultaneously detect 2'-FL in numerous samples, which would be more time-efficient. In this study, two genes, namely α1,2-fucosidase from Xanthomonas manihotis and l-fucose dehydrogenase from Pseudomonas sp. no. 1143, were identified, cloned and overexpressed in E. coli. The recombinant enzymes were produced as 6â¯×â¯His-tagged proteins and were purified to homogeneity using Ni2+ affinity chromatography. The purified α1,2-fucosidase and l-fucose dehydrogenase are monomers with molecular masses of 63â¯kDa and 36â¯kDa, respectively. Both enzymes have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37⯰C, making it possible to develop a coupled enzyme reaction in a single buffer system for the quantitative determination of 2'-FL in a large number of samples simultaneously. This method can be used to quantify 2'-FL in infant formulas and in samples collected from different phases of the biotechnological production of this oligosaccharide. Furthermore, the method is applicable for the rapid screening of active variants during the development of microbial strains producing 2'-FL.
Subject(s)
Enzyme Assays , Infant Formula/chemistry , Milk, Human/chemistry , Trisaccharides/analysis , Carbohydrate Dehydrogenases/chemistry , Humans , Infant , Infant, Newborn , Pseudomonas/metabolism , Xanthomonas axonopodis/metabolism , alpha-L-Fucosidase/chemistryABSTRACT
2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide and is important for infant nutrition and health. Because 2'-FL has potential as a functional ingredient in advanced infant formula and as a prebiotic in various foods, a cost-effective method for 2'-FL production is desirable. α1,2-Fucosyltransferase (α1,2-FT) is one of the key enzymes enabling the microbial biosynthesis of this complex sugar. However, the α1,2-FTs reported so far for the whole-cell biosynthesis of 2'-FL originate from pathogens, posing a potential hurdle for approval as a food production method depending on countries. In this study, 10 α1,2-FT genes from bacteria of biosafety level one were identified, and the main features of the deduced amino acid sequences were characterized. Four codon-optimized α1,2-FT genes were synthesized and introduced into Escherichia coli ΔL M15 strain containing the plasmid pBCGW encoding guanosine 5'-diphosphate-l-fucose biosynthetic enzymes. Among the four genes, 2'-FL was produced only by the α1,2-FT from Thermosynechococcus elongatus (Te2FT). Bifidobacterium thermacidophilum α1,2-FT (Bt2FT) showed high expression but was not active in E. coli ΔL M15. The other two α1,2-FTs were not expressed to a detectable level. During batch flask fermentation of Te2FT-expressing E. coli ΔL M15 cells, 0.49 g/L 2'-FL was obtained after 72 h of induction. This is comparable to the values previously reported for α1,2-FTs from Helicobacter pylori and Bacteroides fragilis.
Subject(s)
Escherichia coli/genetics , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Trisaccharides/biosynthesis , Bacterial Proteins/genetics , Bacteroides fragilis/enzymology , Bacteroides fragilis/metabolism , Bifidobacterium/genetics , Bifidobacterium/metabolism , Cyanobacteria/enzymology , Cyanobacteria/genetics , DNA, Bacterial , Escherichia coli/metabolism , Fermentation , Gene Expression Regulation, Bacterial , Helicobacter pylori/enzymology , Helicobacter pylori/metabolism , Milk, Human , OligosaccharidesABSTRACT
Membrane-disrupting agents that selectively target virus versus host membranes could potentially inhibit a broad-spectrum of enveloped viruses, but currently such antivirals are lacking. Here, we develop a nanodisc incorporated with a decoy virus receptor that inhibits virus infection. Mechanistically, nanodiscs carrying the viral receptor sialic acid bind to influenza virions and are co-endocytosed into host cells. At low pH in the endosome, the nanodiscs rupture the viral envelope, trapping viral RNAs inside the endolysosome for enzymatic decomposition. In contrast, liposomes containing a decoy receptor show weak antiviral activity due to the lack of membrane disruption. The nanodiscs inhibit influenza virus infection and reduce morbidity and mortality in a mouse model. Our results suggest a new class of antivirals applicable to other enveloped viruses that cause irreversible physical damage specifically to virus envelope by viruses' own fusion machine. In conclusion, the lipid nanostructure provides another dimension for antiviral activity of decoy molecules.