Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism.
Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism.
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DOI：
10.1371/journal.ppat.1005152
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发表时间：
2015-09
期刊：
影响因子：
6.7
通讯作者：
Reynolds EC
中科院分区：
文献类型：
--
作者：
Gorasia DG;Veith PD;Chen D;Seers CA;Mitchell HA;Chen YY;Glew MD;Dashper SG;Reynolds EC
牙龈卟啉单胞菌的IX型分泌系统（T9 SS）分泌具有保守的C-末端结构域（CTD）的蛋白质至细胞表面。C-末端信号对于这些蛋白质经由T9 SS跨外膜易位是必需的。在表面上，这些蛋白质的CTD在广泛糖基化之前被切割。据信，这些CTD蛋白上的修饰是阴离子脂多糖（A-LPS），其使得CTD蛋白能够附着到细胞表面。然而，CTD蛋白质修饰的确切位点和附着于细胞表面的机制尚不清楚。在这项研究中，我们的特点是两个wbaP（PG 1964）突变体，不合成A-LPS和积累CTD蛋白的澄清培养液（CCF）。CCF中CTD蛋白的CTD被切割，表明正常分泌，然而，CTD蛋白未被糖基化。从wbaP突变体的CCF纯化的CTD蛋白质的质谱分析揭示了在成熟CTD蛋白质的C-末端存在来自生长培养基的各种肽/氨基酸修饰。这表明修饰发生在野生型牙龈卟啉单胞菌中T9 SS底物的C末端。这通过来自野生型的CTD蛋白的分析得到证实，其中鉴定出648 Da接头连接在成熟CTD蛋白的C末端。重要的是，用蛋白酶K处理从CTD蛋白质释放648 Da接头，证明C-末端和修饰之间的肽键。总之，这提示了类似于分选酶A的机制，用于牙龈卟啉单胞菌中CTD蛋白的切割和修饰/附着。PG 0026被认为是CTD信号肽酶，现在被认为是牙龈卟啉单胞菌中的分选酶样蛋白。据我们所知，这是第一个生化证据表明在革兰氏阴性菌中存在分选酶样机制。由牙龈卟啉单胞菌引起的慢性牙周炎是一个严重的公共卫生问题。牙龈卟啉单胞菌通过IX型分泌系统（T9 SS）分泌包括牙龈卟啉菌蛋白酶的毒力因子。这些蛋白质含有C-末端信号，其允许它们通过T9 SS分泌，并且其在细胞表面被蛋白质PG 0026切割。在这里，我们确定了牙龈卟啉菌蛋白酶和其他蛋白质附着在这种细菌的细胞表面的机制。我们发现，在去除C-末端信号后，蛋白质通过肽连接被修饰为野生型中的阴离子脂多糖（A-LPS）的推定组分或来自缺乏A-LPS的突变体中的生长培养基的肽，这已经被建议将蛋白质锚到细胞表面。本研究的结果为A-LPS修饰这些蛋白质的确切位点提供了证据。此外，我们的结果还首次证明，该分泌系统的锚定机制涉及信号和锚（A-LPS）之间的取代，这可以通过称为分选酶的单一酶的作用来解释，我们认为该酶是PG 0026。这是革兰氏阴性菌中分选酶样机制的第一个证据。
The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria. Chronic periodontitis, associated with the pathogen Porphyromonas gingivalis, is a major public health problem. P. gingivalis secretes virulence factors including the gingipains via the type IX secretion system (T9SS). These proteins contain a C-terminal signal that allows their secretion through the T9SS and it is cleaved by the protein PG0026 at the cell surface. Here we identify a mechanism by which gingipains and other proteins attach to the cell surface of this bacterium. We found that after removal of the C-terminal signal, the proteins were modified via a peptide linkage to either a putative component of anionic lipopolysaccharide (A-LPS) in the wild type or peptides from the growth medium in mutants lacking A-LPS, which has been suggested to anchor the proteins to the cell surface. Results from this study provide evidence for the exact site of modification of these proteins with A-LPS. Furthermore, our results also demonstrate for the first time that the anchoring mechanism of this secretion system involves a substitution between the signal and the anchor (A-LPS) which can be explained by the action of a single enzyme known as a sortase, which we believe is PG0026. This is the first evidence for a sortase-like mechanism in Gram-negative bacteria.