title: Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence.
abstract: The carbohydrate-rich coating of human tissues and cells provide a first point of contact for colonizing and invading bacteria. Commensurate with N-glycosylation being an abundant form of protein glycosylation that has critical functional roles in the host, some host-adapted bacteria possess the machinery to process N-linked glycans. The human pathogen Streptococcus pneumoniae depolymerizes complex N-glycans with enzymes that sequentially trim a complex N-glycan down to the Man3GlcNAc2 core prior to the release of the glycan from the protein by endo-β-N-acetylglucosaminidase (EndoD), which cleaves between the two GlcNAc residues. Here we examine the capacity of S. pneumoniae to process high-mannose N-glycans and transport the products. Through biochemical and structural analyses we demonstrate that S. pneumoniae also possesses an α-(1,2)-mannosidase (SpGH92). This enzyme has the ability to trim the terminal α-(1,2)-linked mannose residues of high-mannose N-glycans to generate Man5GlcNAc2. Through this activity SpGH92 is able to produce a substrate for EndoD, which is not active on high-mannose glycans with α-(1,2)-linked mannose residues. Binding studies and X-ray crystallography show that NgtS, the solute binding protein of an ABC transporter (ABCNG), is able to bind Man5GlcNAc, a product of EndoD activity, with high affinity. Finally, we evaluated the contribution of EndoD and ABCNG to growth of S. pneumoniae on a model N-glycosylated glycoprotein, and the contribution of these enzymes and SpGH92 to virulence in a mouse model. We found that both EndoD and ABCNG contribute to growth of S. pneumoniae, but that only SpGH92 and EndoD contribute to virulence. Therefore, N-glycan processing, but not transport of the released glycan, is required for full virulence in S. pneumoniae. To conclude, we synthesize our findings into a model of N-glycan processing by S. pneumoniae in which both complex and high-mannose N-glycans are targeted, and in which the two arms of this degradation pathway converge at ABCNG.
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.
authors.forename: Melissa
authors.initials: M
authors.lastname: Robb
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.
authors.forename: Joanne K
authors.initials: JK
authors.lastname: Hobbs
authors.affiliation: Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America.
authors.forename: Shireen A
authors.identifier: 0000-0002-0135-7349
authors.initials: SA
authors.lastname: Woodiga
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.
authors.forename: Sarah
authors.identifier: 0000-0001-6821-0256
authors.initials: S
authors.lastname: Shapiro-Ward
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.
authors.forename: Michael D L
authors.initials: MD
authors.lastname: Suits
authors.affiliation: Michael Smith Laboratories and Department of Chemistry, University of British Columbia, 2185 East Mall, Vancouver, British Columbia, Canada.
authors.forename: Nicholas
authors.initials: N
authors.lastname: McGregor
authors.affiliation: Michael Smith Laboratories and Department of Chemistry, University of British Columbia, 2185 East Mall, Vancouver, British Columbia, Canada.
authors.forename: Harry
authors.initials: H
authors.lastname: Brumer
authors.affiliation: Department of Infection, Immunity & Inflammation, University of Leicester, Leicester, United Kingdom.
authors.forename: Hasan
authors.initials: H
authors.lastname: Yesilkaya
authors.affiliation: Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America.
authors.forename: Samantha J
authors.identifier: 0000-0003-2213-5822
authors.initials: SJ
authors.lastname: King
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.
authors.forename: Alisdair B
authors.initials: AB
authors.lastname: Boraston
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D004195:Disease Models, Animal; D006026:Glycoside Hydrolases / Q000378:metabolism*; D054884:Host-Pathogen Interactions / Q000502:physiology*; D051379:Mice; D011008:Pneumococcal Infections / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013296:Streptococcus pneumoniae / Q000378:metabolism / Q000472:pathogenicity*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D011134:Polysaccharides; D006026:Glycoside Hydrolases