ABSTRACT
Alginate lyases are potential agents for disrupting alginate-rich Pseudomonas biofilms in the infected lungs of cystic fibrosis patients but there is as yet no clinically approved alginate lyase that can be used as a therapeutic. We report here the endolytic alginate lyase activity of a recombinant Cellulophaga algicola alginate lyase domain (CaAly) encoded by a gene that also codes for an N-terminal carbohydrate-binding module, CBM6, and a central F-type lectin domain (CaFLD). CaAly degraded both polyM and polyG alginates with optimal temperature and pH of 37°C and pH 7, respectively, with greater preference for polyG. Recombinant CaFLD bound to fucosylated glycans with a preference for H-type 2 glycan motif, and did not have any apparent effect on the enzyme activity of the co-associated alginate lyase domain in the recombinant protein construct, CaFLD_Aly. We assessed the potential of CaAly and other alginate lyases previously reported in published literature to inhibit biofilm formation by a clinical strain, Pseudomonas aeruginosa MCC 2081. Of all the alginate lyases tested, CaAly displayed most inhibition of in vitro biofilm formation on plastic surfaces. We also assessed its inhibitory ability against P. aeruginosa 2081 biofilms formed over a monolayer of A549 lung epithelial cells. Our study indicated that CaAly is efficacious in inhibition of biofilm formation even on A549 lung epithelial cell line monolayers.
Subject(s)
Anti-Bacterial Agents/administration & dosage , Bacterial Proteins/administration & dosage , Biofilms/drug effects , Flavobacteriaceae/enzymology , Polysaccharide-Lyases/administration & dosage , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , A549 Cells , Biofilms/growth & development , Humans , Polysaccharide-Lyases/metabolism , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/isolation & purificationABSTRACT
The typical F-type lectin domain (FLD) has an L-fucose-binding motif [HX(26)RXDX(4)R/K] with conserved basic residues that mediate hydrogen bonding with alpha-L-fucose. About one-third of the nonredundant FLD sequences in the publicly available databases are "atypical"; they have motifs with substitutions of these critical residues and/or variations in motif length. We addressed the question if atypical FLDs with substitutions of the critical residues retain lectin activity by performing site-directed mutagenesis and assessing the glycan-binding functions of typical and atypical FLDs. Site directed mutagenesis of an L-fucose-binding FLD from Streptosporangium roseum indicated that the critical His residue could be replaced by Ser and the second Arg by Lys without complete loss of lectin activity. Mutagenesis of His to other naturally substituting residues and mutagenesis of the first Arg to the naturally substituting residues, Lys, Ile, Ser, or Cys, resulted in loss of lectin activity. Glycan binding analysis and site-directed mutagenesis of atypical FLDs from Actinomyces turicensis, and Saccharomonospora cyanea confirmed that Ser and Thr can assume the L-fucose-binding role of the critical His, and further suggested that the residue in this position is dispensable in certain FLDs. We identified, by sequence and structural analysis of atypical FLDs, a Glu residue in the complementarity determining region, CDR5 that compensates for a lack of the critical His or other appropriate polar residue in this position. We propose that FLDs lacking a typical FLD sequence motif might nevertheless retain lectin activity through the recruitment of other strategically positioned polar residues in the CDR loops. © 2018 IUBMB Life, 71(3):385-397, 2019.
Subject(s)
Fucose/chemistry , Lectins/chemistry , Polysaccharides/chemistry , Actinobacteria/chemistry , Actinobacteria/metabolism , Actinomycetaceae/chemistry , Actinomycetaceae/metabolism , Amino Acid Motifs , Binding Sites , Cloning, Molecular , Erythrocytes/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Fucose/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Hemagglutination Inhibition Tests , Humans , Lectins/genetics , Lectins/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Polysaccharides/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
BACKGROUND: The yeast Saccharomyces boulardii is used worldwide as a probiotic to alleviate the effects of several gastrointestinal diseases and control antibiotics-associated diarrhea. While many studies report the probiotic effects of S. boulardii, no genome information for this yeast is currently available in the public domain. RESULTS: We report the 11.4 Mbp draft genome of this probiotic yeast. The draft genome was obtained by assembling Roche 454 FLX + shotgun data into 194 contigs with an N50 of 251 Kbp. We compare our draft genome with all other Saccharomyces cerevisiae genomes. CONCLUSIONS: Our analysis confirms the close similarity of S. boulardii to S. cerevisiae strains and provides a framework to understand the probiotic effects of this yeast, which exhibits unique physiological and metabolic properties.
