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

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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 Burkholderia 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 in 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 confirm the production of glycoproteins with similar glycans in the Burkholderia species B. thailandensis, B. gladioli, and B. pseudomallei. Further, 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, B. multivorans, B. pseudomallei, or B. 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
JournalThe Journal of biological chemistry
Early online date26 Jul 2019
DOIs
Publication statusEarly online date - 26 Jul 2019

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Burkholderia
Glycosylation
Machinery
Polysaccharides
Burkholderia cenocepacia
Proteins
Genes
Burkholderia Infections
Trisaccharides
Malleus
Biosynthesis
Bioelectric potentials
Bioinformatics
Gene Knockout Techniques
Rhizosphere
Recombinant Proteins
Multigene Family
Infection
Computational Biology
Gram-Negative Bacteria

Cite this

Mohamed, Yasmine Fathy ; 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: The Journal of biological chemistry. 2019.
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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 Burkholderia 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 in 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 confirm the production of glycoproteins with similar glycans in the Burkholderia species B. thailandensis, B. gladioli, and B. pseudomallei. Further, 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, B. multivorans, B. pseudomallei, or B. 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.",
author = "Mohamed, {Yasmine Fathy} 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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A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans. / Mohamed, Yasmine Fathy; 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: The Journal of biological chemistry, 26.07.2019.

Research output: Contribution to journalArticle

TY - JOUR

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

AU - Mohamed, Yasmine Fathy

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.

PY - 2019/7/26

Y1 - 2019/7/26

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 Burkholderia 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 in 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 confirm the production of glycoproteins with similar glycans in the Burkholderia species B. thailandensis, B. gladioli, and B. pseudomallei. Further, 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, B. multivorans, B. pseudomallei, or B. 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 Burkholderia 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 in 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 confirm the production of glycoproteins with similar glycans in the Burkholderia species B. thailandensis, B. gladioli, and B. pseudomallei. Further, 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, B. multivorans, B. pseudomallei, or B. 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.

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DO - 10.1074/jbc.RA119.009671

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JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

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