A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans

Yasmine Fathy Mohamed, Nichollas E. Scott, Antonio Molinaro, Carole Creuzenet, Ximena Ortega, Ganjana Lertmemongkolchai, Michael M. Tunney, Heather Green, Andrew M. Jones, David DeShazer, Bart J. Currie, Leonard J. Foster, Rebecca Ingram, Cristina De Castro, Miguel A. Valvano

    Research output: Contribution to journalArticleResearchpeer-review

    Abstract

    The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan-specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

    Original languageEnglish
    Pages (from-to)13248-13268
    Number of pages21
    JournalJournal of Biological Chemistry
    Volume294
    Issue number36
    Early online date26 Jul 2019
    DOIs
    Publication statusPublished - 6 Sep 2019

    Fingerprint

    Burkholderia
    Glycosylation
    Machinery
    Polysaccharides
    Burkholderia cenocepacia
    Proteins
    Genes
    Burkholderia mallei
    Burkholderia Infections
    Trisaccharides
    Biosynthesis
    Bioelectric potentials
    Bioinformatics
    Gene Knockout Techniques
    Rhizosphere
    Recombinant Proteins
    Lipopolysaccharides
    Multigene Family
    Infection
    Computational Biology

    Cite this

    Fathy Mohamed, Y., Scott, N. E., Molinaro, A., Creuzenet, C., Ortega, X., Lertmemongkolchai, G., ... Valvano, M. A. (2019). A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans. Journal of Biological Chemistry, 294(36), 13248-13268. https://doi.org/10.1074/jbc.RA119.009671
    Fathy Mohamed, Yasmine ; Scott, Nichollas E. ; Molinaro, Antonio ; Creuzenet, Carole ; Ortega, Ximena ; Lertmemongkolchai, Ganjana ; Tunney, Michael M. ; Green, Heather ; Jones, Andrew M. ; DeShazer, David ; Currie, Bart J. ; Foster, Leonard J. ; Ingram, Rebecca ; De Castro, Cristina ; Valvano, Miguel A. / A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans. In: Journal of Biological Chemistry. 2019 ; Vol. 294, No. 36. pp. 13248-13268.
    @article{65bb02e8a342441f8cda54838b5342b5,
    title = "A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans",
    abstract = "The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan-specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.",
    keywords = "bacteria, Burkholderia, cystic fibrosis, galleria mellonella, glanders, glycosylation, immunogenicity, melioidosis, nuclear magnetic resonance (NMR), phenotypic arrays",
    author = "{Fathy Mohamed}, Yasmine and Scott, {Nichollas E.} and Antonio Molinaro and Carole Creuzenet and Ximena Ortega and Ganjana Lertmemongkolchai and Tunney, {Michael M.} and Heather Green and Jones, {Andrew M.} and David DeShazer and Currie, {Bart J.} and Foster, {Leonard J.} and Rebecca Ingram and {De Castro}, Cristina and Valvano, {Miguel A.}",
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    language = "English",
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    Fathy Mohamed, Y, Scott, NE, Molinaro, A, Creuzenet, C, Ortega, X, Lertmemongkolchai, G, Tunney, MM, Green, H, Jones, AM, DeShazer, D, Currie, BJ, Foster, LJ, Ingram, R, De Castro, C & Valvano, MA 2019, 'A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans', Journal of Biological Chemistry, vol. 294, no. 36, pp. 13248-13268. https://doi.org/10.1074/jbc.RA119.009671

    A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans. / Fathy Mohamed, Yasmine; Scott, Nichollas E.; Molinaro, Antonio; Creuzenet, Carole; Ortega, Ximena; Lertmemongkolchai, Ganjana; Tunney, Michael M.; Green, Heather; Jones, Andrew M.; DeShazer, David; Currie, Bart J.; Foster, Leonard J.; Ingram, Rebecca; De Castro, Cristina; Valvano, Miguel A.

    In: Journal of Biological Chemistry, Vol. 294, No. 36, 06.09.2019, p. 13248-13268.

    Research output: Contribution to journalArticleResearchpeer-review

    TY - JOUR

    T1 - A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans

    AU - Fathy Mohamed, Yasmine

    AU - Scott, Nichollas E.

    AU - Molinaro, Antonio

    AU - Creuzenet, Carole

    AU - Ortega, Ximena

    AU - Lertmemongkolchai, Ganjana

    AU - Tunney, Michael M.

    AU - Green, Heather

    AU - Jones, Andrew M.

    AU - DeShazer, David

    AU - Currie, Bart J.

    AU - Foster, Leonard J.

    AU - Ingram, Rebecca

    AU - De Castro, Cristina

    AU - Valvano, Miguel A.

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    N2 - The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan-specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

    AB - The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan-specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

    KW - bacteria

    KW - Burkholderia

    KW - cystic fibrosis

    KW - galleria mellonella

    KW - glanders

    KW - glycosylation

    KW - immunogenicity

    KW - melioidosis

    KW - nuclear magnetic resonance (NMR)

    KW - phenotypic arrays

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    U2 - 10.1074/jbc.RA119.009671

    DO - 10.1074/jbc.RA119.009671

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    EP - 13268

    JO - Journal of Biological Chemistry

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