Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway

ABSTRACT Porphyromonas gingivalis and Tannerella forsythia use the type IX secretion system to secrete cargo proteins to the cell surface where they are anchored via glycolipids. In P. gingivalis, the glycolipid is anionic lipopolysaccharide (A-LPS), of partially known structure. Modified cargo prot...

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Autores principales:	Paul D. Veith, Mikio Shoji, Richard A. J. O’Hair, Michael G. Leeming, Shuai Nie, Michelle D. Glew, Gavin E. Reid, Koji Nakayama, Eric C. Reynolds
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Porphyromonas gingivalis Tannerella forsythia glycoprotein type IX secretion system lipopolysaccharide Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/fb654876e30d46e7ac470725d551babe
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spelling	oai:doaj.org-article:fb654876e30d46e7ac470725d551babe2021-11-15T16:19:09ZType IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway10.1128/mBio.01497-202150-7511https://doaj.org/article/fb654876e30d46e7ac470725d551babe2020-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01497-20https://doaj.org/toc/2150-7511ABSTRACT Porphyromonas gingivalis and Tannerella forsythia use the type IX secretion system to secrete cargo proteins to the cell surface where they are anchored via glycolipids. In P. gingivalis, the glycolipid is anionic lipopolysaccharide (A-LPS), of partially known structure. Modified cargo proteins were deglycosylated using trifluoromethanesulfonic acid and digested with trypsin or proteinase K. The residual modifications were then extensively analyzed by tandem mass spectrometry. The C terminus of each cargo protein was amide-bonded to a linking sugar whose structure was deduced to be 2-N-seryl, 3-N-acetylglucuronamide in P. gingivalis and 2-N-glycyl, 3-N-acetylmannuronic acid in T. forsythia. The structures indicated the involvement of the Wbp pathway to produce 2,3-di-N-acetylglucuronic acid and a WbpS amidotransferase to produce the uronamide form of this sugar in P. gingivalis. The wbpS gene was identified as PGN_1234 as its deletion resulted in the inability to produce the uronamide. In addition, the P. gingivalis vimA mutant which lacks A-LPS was successfully complemented by the T. forsythia vimA gene; however, the linking sugar was altered to include glycine rather than serine. After removal of the acetyl group at C-2 by the putative deacetylase, VimE, VimA presumably transfers the amino acid to complete the biosynthesis. The data explain all the enzyme activities required for the biosynthesis of the linking sugar accounting for six A-LPS-specific genes. The linking sugar is therefore the key compound that enables the attachment of cargo proteins in P. gingivalis and T. forsythia. We propose to designate this novel linking sugar biosynthetic pathway the Wbp/Vim pathway. IMPORTANCE Porphyromonas gingivalis and Tannerella forsythia, two pathogens associated with severe gum disease, use the type IX secretion system (T9SS) to secrete and attach toxic arrays of virulence factor proteins to their cell surfaces. The proteins are tethered to the outer membrane via glycolipid anchors that have remained unidentified for more than 2 decades. In this study, the first sugar molecules (linking sugars) in these anchors are identified and found to be novel compounds. The novel biosynthetic pathway of these linking sugars is also elucidated. A diverse range of bacteria that do not have the T9SS were found to have the genes for this pathway, suggesting that they may synthesize similar linking sugars for utilization in different systems. Since the cell surface attachment of virulence factors is essential for virulence, these findings reveal new targets for the development of novel therapies.Paul D. VeithMikio ShojiRichard A. J. O’HairMichael G. LeemingShuai NieMichelle D. GlewGavin E. ReidKoji NakayamaEric C. ReynoldsAmerican Society for MicrobiologyarticlePorphyromonas gingivalisTannerella forsythiaglycoproteintype IX secretion systemlipopolysaccharideMicrobiologyQR1-502ENmBio, Vol 11, Iss 5 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Porphyromonas gingivalis Tannerella forsythia glycoprotein type IX secretion system lipopolysaccharide Microbiology QR1-502
spellingShingle	Porphyromonas gingivalis Tannerella forsythia glycoprotein type IX secretion system lipopolysaccharide Microbiology QR1-502 Paul D. Veith Mikio Shoji Richard A. J. O’Hair Michael G. Leeming Shuai Nie Michelle D. Glew Gavin E. Reid Koji Nakayama Eric C. Reynolds Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway
description	ABSTRACT Porphyromonas gingivalis and Tannerella forsythia use the type IX secretion system to secrete cargo proteins to the cell surface where they are anchored via glycolipids. In P. gingivalis, the glycolipid is anionic lipopolysaccharide (A-LPS), of partially known structure. Modified cargo proteins were deglycosylated using trifluoromethanesulfonic acid and digested with trypsin or proteinase K. The residual modifications were then extensively analyzed by tandem mass spectrometry. The C terminus of each cargo protein was amide-bonded to a linking sugar whose structure was deduced to be 2-N-seryl, 3-N-acetylglucuronamide in P. gingivalis and 2-N-glycyl, 3-N-acetylmannuronic acid in T. forsythia. The structures indicated the involvement of the Wbp pathway to produce 2,3-di-N-acetylglucuronic acid and a WbpS amidotransferase to produce the uronamide form of this sugar in P. gingivalis. The wbpS gene was identified as PGN_1234 as its deletion resulted in the inability to produce the uronamide. In addition, the P. gingivalis vimA mutant which lacks A-LPS was successfully complemented by the T. forsythia vimA gene; however, the linking sugar was altered to include glycine rather than serine. After removal of the acetyl group at C-2 by the putative deacetylase, VimE, VimA presumably transfers the amino acid to complete the biosynthesis. The data explain all the enzyme activities required for the biosynthesis of the linking sugar accounting for six A-LPS-specific genes. The linking sugar is therefore the key compound that enables the attachment of cargo proteins in P. gingivalis and T. forsythia. We propose to designate this novel linking sugar biosynthetic pathway the Wbp/Vim pathway. IMPORTANCE Porphyromonas gingivalis and Tannerella forsythia, two pathogens associated with severe gum disease, use the type IX secretion system (T9SS) to secrete and attach toxic arrays of virulence factor proteins to their cell surfaces. The proteins are tethered to the outer membrane via glycolipid anchors that have remained unidentified for more than 2 decades. In this study, the first sugar molecules (linking sugars) in these anchors are identified and found to be novel compounds. The novel biosynthetic pathway of these linking sugars is also elucidated. A diverse range of bacteria that do not have the T9SS were found to have the genes for this pathway, suggesting that they may synthesize similar linking sugars for utilization in different systems. Since the cell surface attachment of virulence factors is essential for virulence, these findings reveal new targets for the development of novel therapies.
format	article
author	Paul D. Veith Mikio Shoji Richard A. J. O’Hair Michael G. Leeming Shuai Nie Michelle D. Glew Gavin E. Reid Koji Nakayama Eric C. Reynolds
author_facet	Paul D. Veith Mikio Shoji Richard A. J. O’Hair Michael G. Leeming Shuai Nie Michelle D. Glew Gavin E. Reid Koji Nakayama Eric C. Reynolds
author_sort	Paul D. Veith
title	Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway
title_short	Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway
title_full	Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway
title_fullStr	Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway
title_full_unstemmed	Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway
title_sort	type ix secretion system cargo proteins are glycosylated at the c terminus with a novel linking sugar of the wbp/vim pathway
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/fb654876e30d46e7ac470725d551babe
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Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway

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