Protein Substrates of a Novel Secretion System Are Numerous in the Bacteroidetes Phylum and Have in Common a Cleavable C‑Terminal Secretion Signal, Extensive Post-Translational Modification, and Cell-Surface Attachment

The secretion of certain proteins
in Porphyromonas gingivalis is dependent on a C-terminal
domain (CTD). After secretion, the
CTD is cleaved prior to extensive modification of the mature protein,
probably with lipopolysaccharide, therefore enabling attachment to
the cell surface. In this study, bioinformatic analyses of the CTD
demonstrated the presence of three conserved sequence motifs. These
motifs were used to construct Hidden Markov Models (HMMs) that predicted
663 CTD-containing proteins in 21 fully sequenced species of the Bacteroidetes phylum, while no CTD-containing proteins were
predicted in species outside this phylum. Further HMM searching of Cytophaga hutchinsonii led to a total of 171 predicted CTD
proteins in that organism alone. Proteomic analyses of membrane fractions
and culture fluid derived from P. gingivalis and
four other species containing predicted CTDs (Parabacteroides
distasonis, Prevotella intermedia, Tannerella forsythia, and C. hutchinsonii) demonstrated that membrane localization, extensive post-translational
modification, and CTD-cleavage were conserved features of the secretion
system. The CTD cleavage site of 10 different proteins from 3 different
species was determined and found to be similar to the cleavage site
previously determined in P. gingivalis, suggesting
that homologues of the C-terminal signal peptidase (PG0026) are responsible
for the cleavage in these species.

牙龈卟啉单胞菌（Porphyromonas gingivalis）中部分蛋白质的分泌依赖于C端结构域（CTD）。该蛋白质完成分泌后，C端结构域会被切割，随后成熟蛋白会经历广泛的翻译后修饰，修饰类型大概率为脂多糖修饰，这一过程可使蛋白附着于细胞表面。本研究通过对C端结构域的生物信息学分析，发现其包含3个保守序列基序。研究人员利用这些基序构建了隐马尔可夫模型（Hidden Markov Models, HMMs），在21个已完成全基因组测序的拟杆菌门（Bacteroidetes）物种中，共预测得到663个含C端结构域的蛋白质；而在该门以外的物种中，未预测到任何含C端结构域的蛋白质。进一步对哈氏噬纤维菌（Cytophaga hutchinsonii）开展隐马尔可夫模型检索后，仅在该物种中就预测得到171个含C端结构域的蛋白质。研究人员对源自牙龈卟啉单胞菌以及另外4个预测含有C端结构域的物种——狄氏副拟杆菌（Parabacteroides distasonis）、中间普雷沃菌（Prevotella intermedia）、福赛斯坦纳菌（Tannerella forsythia）和哈氏噬纤维菌——的膜组分与培养液开展蛋白质组学分析，结果显示膜定位、广泛的翻译后修饰以及C端结构域切割均为该分泌系统的保守特征。研究人员确定了来自3个不同物种的10种不同蛋白质的C端结构域切割位点，发现其与此前在牙龈卟啉单胞菌中确定的切割位点相似，这表明C端信号肽酶（PG0026）的同源蛋白负责这些物种中的蛋白质切割过程。