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.

中文翻译：

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IX型分泌系统货物蛋白在C末端被糖基化，并带有Wbp / Vim途径的新型连接糖。

牙龈卟啉单胞菌和连翘属植物连翘使用IX型分泌系统将货物蛋白分泌到细胞表面，并通过糖脂将其锚定。在牙龈卟啉单胞菌中，糖脂是具有部分已知结构的阴离子脂多糖（A-LPS）。使用三氟甲磺酸将修饰的货物蛋白质去糖基化，并用胰蛋白酶或蛋白酶K消化。然后通过串联质谱法广泛分析残留的修饰。将每种货物蛋白的C末端酰胺键合至连接糖，其结构被推断为牙龈卟啉单胞菌中的2- N-丝氨酰，3- N-乙酰基葡萄糖醛酸酰胺和2- N-甘氨酰，3- N-乙酰甘露糖醛酸在连翘。结构表明，Wbp途径参与产生2,3-di- N-乙酰葡萄糖醛酸和WbpS酰胺转移酶的糖尿嘧啶形式的牙龈卟啉菌。该wbpS基因被确定为PGN_1234其缺失导致无法生产uronamide。此外，缺少A-LPS的牙龈卟啉单 胞菌vimA突变体已成功地由连翘连翘 vimA进行了补充。基因; 然而，连接糖被改变为包括甘氨酸而不是丝氨酸。通过假定的脱乙酰基酶VimE去除了C-2处的乙酰基后，VimA可能会转移氨基酸以完成生物合成。数据解释了连接糖的生物合成所需的所有酶活性，这解释了六个A-LPS特异性基因。因此，连接糖是使货物蛋白附着在牙龈卟啉单胞菌和连翘丁香中的关键化合物。我们建议指定这种新颖的连接糖生物合成途径的Wbp / Vim途径。