PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.

牙龈卟啉单胞菌（Porphyromonas gingivalis）利用拟杆菌门特异性IX型分泌系统（type IX secretion system, T9SS）跨外膜（outer membrane, OM）分泌蛋白质，包括牙龈蛋白酶（gingipains）等毒力因子。分泌的蛋白质带有保守的羧基末端结构域，该结构域对IX型分泌至关重要，且会在分泌过程中被切割。在牙龈卟啉单胞菌中，IX型分泌系统底物会与阴离子脂多糖（anionic lipopolysaccharide, A-LPS）发生糖基化修饰，并锚定在外膜上。本研究通过对24株拟杆菌门基因组的比较分析，在牙龈卟啉单胞菌中鉴定出10个IX型分泌系统的潜在新型组分，其中之一为PG1058。对PG1058结构的计算机建模预测显示，其拥有全新的从N端到C端的架构，包含四肽重复（tetratricopeptide repeat, TPR）结构域、β-螺旋桨结构域、羧肽酶调节结构域样折叠（carboxypeptidase regulatory domain-like fold, CRD）以及类OmpA_C结构域的潜在肽聚糖结合结构域。牙龈卟啉单胞菌中pg1058基因的失活会导致菌落色素沉着与表面相关蛋白水解活性的丧失——这是IX型分泌系统突变株共有的典型表型。对亚细胞组分的免疫印迹（immunoblot）与液相色谱-串联质谱（LC-MS/MS）分析结果显示，IX型分泌系统底物在pg1058突变株的周质中发生积累；而全细胞酶联免疫吸附试验（ELISA）结果表明，Kgp牙龈蛋白酶并未出现在细胞表面，这证实了IX型分泌系统的功能受到扰动。免疫印迹、透射电子显微镜（TEM）以及全细胞ELISA分析表明，pg1058突变株可合成阴离子脂多糖且该物质可定位于细胞表面，但无法与IX型分泌系统底物蛋白结合。这提示PG1058对于IX型分泌系统底物的分泌至关重要，但并不参与阴离子脂多糖的转运过程。PG1058是一种预测的脂蛋白，通过对亚细胞组分开展全细胞ELISA、免疫印迹以及LC-MS/MS分析，确定其定位于外膜的周质侧。PG1058的结构预测与定位结果提示，其可能作为关键支架蛋白，连接IX型分泌系统的周质组分与外膜组分。