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 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.

Characterization of the O-Glycoproteome of Tannerella forsythia

“…These cargo proteins along with more than 30 others share a conserved C-terminal domain (CTD) signal to enable this secretion ( 20 – 22 ). The signal is cleaved ( 22 ) and the C terminus of the mature protein is conjugated to a polysaccharide via a novel linking sugar in a manner analogous to that for P. gingivalis , apparently via the PorU sortase ( 19 , 23 , 24 ). In T. forsythia , the linking sugar was identified as a 2-N-glycyl, 3-N-acetylmannuronic acid ( 24 ).…”

Prediction of Selected Biosynthetic Pathways for the Lipopolysaccharide Components in Porphyromonas gingivalis

“…Another region in the genome contains the three genes vimA , vimE , and vimF . The vimA and vimE genes encode additional enzymes that participate in 3-acetamido-2,3-dideoxy-2-(seryl)amino-α-D-glucuronamide biosynthesis [ 26 ], while VimF catalyzes an essential but undefined activity during A-type antigen biosynthesis [ 27 ] ( Figure 6 ).…”

“…A similar alignment of vimA , vimF, and vimE genes ( Figure 6 ) showed that this genetic region is also strictly conserved and that there are no insertions in the analyzed strains. Since the enzymes participate in the cargo modification by the type IX secretion system or O-type antigen biosynthesis [ 26 , 27 ], the strict conservation may be related to the biological function of their products in the pathogenesis of P. gingivalis .…”

“…Based on similarities with other systems, the A-type antigen genes were identified as gtfC [ 21 , 25 ], vimF [ 25 , 26 ], gtfE [ 25 ], gtfB [ 27 ], gtfF [ 25 ], and wbaP [ 28 ]; the O-type antigen genes were identified as wbaQ [ 28 ], gtfD [ 25 , 29 ], gtfE [ 25 ], and gtfB [ 25 ]; and the K-type polysaccharide genes were identified as pgn0223 / pg0106 [ 28 , 30 ], none/ pg0110 [ 13 ], pgn0232 / pg0118 [ 13 , 25 ], and pgn0233 / pg0119 [ 13 , 25 ] in P. gingivalis strains ATCC33277 and W83, respectively. Lipid A-core genes were identified as pgn0242 ( gtfG )/ pg0129 [ 22 , 31 ] and pgn1255 ( rfa )/ pg1155 [ 32 ] in strains ATCC33277 and W83, respectively.…”

Identification of the Type IX Secretion System Component, PorV (CHU_3238), Involved in Secretion and Localization of Proteins in Cytophaga hutchinsonii

“…The T9SS is involved in protein secretion and gliding motility and has been implicated in some bacterial diseases in many members of Bacteroidetes . It secretes cargo proteins with specific CTDs to the cell surface and anchors them with glycolipids ( Slakeski et al, 2011 ; Gorasia et al, 2015 ; Veith et al, 2020 ). C. hutchinsonii contains all the homologous proteins of the T9SS and most of the gliding-related genes.…”